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Ayalew S, Wegayehu T, Wondale B, Alemayehu DH, Kebede D, Agize H, Fisseha E, Desta T, Niway S, Piantadosi A, Mihret A. Plasma Mycobacterium tuberculosis cell-free DNA assay: a diagnostic tool for tuberculosis lymphadenitis. Infect Dis (Lond) 2025:1-13. [PMID: 40078121 DOI: 10.1080/23744235.2025.2478263] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2024] [Revised: 03/04/2025] [Accepted: 03/06/2025] [Indexed: 03/14/2025] Open
Abstract
BACKGROUND Bacterial confirmation in suspected tuberculosis lymphadenitis patients is challenging. This study evaluates plasma Mycobacterium tuberculosis cell-free DNA as a diagnostic tool for tuberculosis lymphadenitis. METHODS A quantitative PCR assay targeting IS6110, IS1081, and cyp141 genes was performed on plasma samples. The study included 95 tuberculosis lymphadenitis patients and 60 controls. Sensitivity of the plasma Mycobacterium tuberculosis cell-free DNA assay was assessed against fine needle aspiration GeneXpert Ultra, fine needle aspiration culture, and fine needle aspiration cytology, while specificity was determined using control groups. RESULTS Of the tuberculosis lymphadenitis cases, 71 (74.7%) were bacteriologically confirmed, and 24 (25.3%) were probable. In the control group, 50% had latent tuberculosis infection. The Mycobacterium tuberculosis cell-free DNA assay, targeting three genes, had an overall sensitivity of 65.3%, increasing to 70.4% for confirmed cases and 50% for probable cases, with specificity of 91.1%. Sensitivities for specific gene combinations were 62.1% for IS6110 and IS1081, 54.7% for IS6110 and cyp141, and 55.8% for IS1081 and cyp141. For individual genes, IS6110 showed 49.4% sensitivity (specificity: 93.3%), IS1081 had 51.6% (specificity: 96.0%), and cyp141 showed 28.4% (specificity: 96.7%). Combining positive results from all three genes in the cell-free DNA assay with fine needle aspiration culture and GeneXpert Ultra improved sensitivity to 76.8% and 85.3%, respectively. CONCLUSION This study demonstrated that Mycobacterium tuberculosis cell-free DNA can be detected in the plasma of over half of tuberculosis lymphadenitis patients. The plasma Mycobacterium tuberculosis cell-free DNA assay could serve as a valuable, less-invasive complement to existing fine needle aspiration diagnostics.
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Affiliation(s)
- Sosina Ayalew
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
- Department of Biology, College of Natural and Computational Sciences, Arba Minch University, Arba Minch, Ethiopia
| | - Teklu Wegayehu
- Department of Biology, College of Natural and Computational Sciences, Arba Minch University, Arba Minch, Ethiopia
| | - Biniam Wondale
- Department of Biology, College of Natural and Computational Sciences, Arba Minch University, Arba Minch, Ethiopia
| | | | - Dawit Kebede
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
| | - Haymanot Agize
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
| | - Emnet Fisseha
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
| | - Tigist Desta
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
| | - Sebsibe Niway
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
| | - Anne Piantadosi
- Department of Pathology and Department of Medicine, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA, USA
| | - Adane Mihret
- Armauer Hansen Research Institute, Addis Ababa, Ethiopia
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Liu Y, Tang Q, Tang S, Huang H, Kou L, Zhou Y, Ruan H, Yuan Y, He C, Ying B. Clinical evaluation of droplet digital PCR in suspected invasive pulmonary aspergillosis. Clin Chim Acta 2025; 569:120153. [PMID: 39862901 DOI: 10.1016/j.cca.2025.120153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2024] [Revised: 01/08/2025] [Accepted: 01/20/2025] [Indexed: 01/27/2025]
Abstract
Invasive pulmonary aspergillosis (IPA), the most common fungal infection, is associated with high mortality of affected patients. Traditional diagnostic methods exhibit limited sensitivity and specificity, raising big challenges for precise management of the patients. There is thus an urgent need to find out a timely and accurate diagnostic method in clinical practice. In this study, 163 patients suspected with IPA were enrolled. The medical data of the patients were retrieved from hospital information system. The 158 patients with complete data were classified into an IPA group with 122 cases (58 putative IPA, 19 probable IPA, and 45 possible IPA cases) and a non-IPA group with 36 cases. Cell-free DNA (cfDNA) of bronchoalveolar lavage fluid (BALF) or plasma samples was detected via a droplet digital PCR (ddPCR) assay targeting Aspergillus spp. Overall, this ddPCR assay demonstrated a higher sensitivity of 50.8 % for IPA diagnosis, compared with that of fungal culture (44.3 %) and smear test (10.7 %). Moreover, its sensitivity was higher in the IPA group (73.1 %) and putative IPA subgroup (88.2 %) when using BALF samples, compared with those using plasma samples (P < 0.01). It achieved a high specificity of 94.4 % for IPA diagnosis, with significant variations in cfDNA copy numbers across the subgroups (P < 0.05). In addition, the ddPCR results were associated with the prognosis of the patients at the discharge (P < 0.05). In conclusion, ddPCR assay demonstrated a good performance for IPA diagnosis when using BALF samples, especially for putative IPA. The ddPCR results could be integrated with clinical data to improve prognostic prediction.
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Affiliation(s)
- Yang Liu
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Qiuping Tang
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Sishi Tang
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Hengjian Huang
- West China Precision Medicine Industrial Technology Institute, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Lanxi Kou
- West China Precision Medicine Industrial Technology Institute, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Yi Zhou
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Hongxia Ruan
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Yu Yuan
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
| | - Chao He
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China.
| | - Binwu Ying
- Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China.
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Freimane L, Kivrāne A, Ulanova V, Vīksna A, Sevostjanovs E, Grīnberga S, Cīrule A, Krams A, Ranka R. Fluctuations in circulating cell-free mitochondrial and nuclear DNA copy numbers in blood plasma after anti-tuberculosis drug intake in patients with drug-susceptible tuberculosis. Tuberculosis (Edinb) 2025; 151:102611. [PMID: 39862444 DOI: 10.1016/j.tube.2025.102611] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Revised: 01/16/2025] [Accepted: 01/20/2025] [Indexed: 01/27/2025]
Abstract
Biomarker research characterising the effect of anti-tuberculosis (TB) chemotherapy on systemic body response is still limited. In this study, we aimed to investigate fluctuations in circulating cell-free mitochondrial DNA (ccf-mtDNA) and circulating cell-free nuclear DNA (ccf-nDNA) copy number (CN) in blood plasma of patients with drug-susceptible TB (DS-TB) and to decipher factors related to these fluctuations. The results showed considerable changes in ccf-mtDNA CN in plasma samples before drug intake and 2 and 6 h afterwards, with high inter patient variability at each time point. Multivariate linear regression revealed that the dynamics of ccf-mtDNA CN was influenced by patients' age, ethambutol pharmacokinetics, and body-mass index (BMI); ethambutol exposure emerged as the most significant factor. Very low ccf-nDNA CN in all three time points with little variation was observed; none factors were strongly associated with ccf-nDNA. In conclusion, our study revealed the effect of anti-TB chemotherapy, age and BMI on acute changes in circulating ccf-mtDNA CN in blood plasma and highlighted the systemic, mitochondria-related effects of ethambutol in patients with TB. Further studies with larger cohorts are needed to understand the biological relevance of ccf-DNA in patients with TB and to validate its application in TB treatment monitoring.
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Affiliation(s)
- Lauma Freimane
- Latvian Biomedical Research and Study Centre, Ratsupites street 1, k-1, Riga, LV-1067, Latvia
| | - Agnija Kivrāne
- Riga Stradiņš University, Pharmacogenetic and Precision Medicine Laboratory, Konsula street 21, Riga, LV-1007, Latvia
| | - Viktorija Ulanova
- Riga Stradiņš University, Pharmacogenetic and Precision Medicine Laboratory, Konsula street 21, Riga, LV-1007, Latvia
| | - Anda Vīksna
- Riga Stradiņš University, Pharmacogenetic and Precision Medicine Laboratory, Konsula street 21, Riga, LV-1007, Latvia; Riga East University Hospital, Centre of Tuberculosis and Lung Diseases, Upeslejas, Stopinu district, LV-2118, Latvia
| | - Eduards Sevostjanovs
- Latvian Institute of Organic Synthesis, Aizkraukles street 21, Riga, LV-1006, Latvia
| | - Solveiga Grīnberga
- Latvian Institute of Organic Synthesis, Aizkraukles street 21, Riga, LV-1006, Latvia
| | - Andra Cīrule
- Riga East University Hospital, Centre of Tuberculosis and Lung Diseases, Upeslejas, Stopinu district, LV-2118, Latvia
| | - Alvils Krams
- Riga East University Hospital, Centre of Tuberculosis and Lung Diseases, Upeslejas, Stopinu district, LV-2118, Latvia; University of Latvia, Raiņa bulvāris 19, Rīga, LV-1586, Latvia
| | - Renāte Ranka
- Latvian Biomedical Research and Study Centre, Ratsupites street 1, k-1, Riga, LV-1067, Latvia; Riga Stradiņš University, Pharmacogenetic and Precision Medicine Laboratory, Konsula street 21, Riga, LV-1007, Latvia.
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Jin Y, Conneely KN, Ma W, Naviaux RK, Siddique T, Allen EG, Guingrich S, Pascuzzi RM, Jin P. Whole-genome bisulfite sequencing of cell-free DNA unveils age-dependent and ALS-associated methylation alterations. Cell Biosci 2025; 15:26. [PMID: 39980027 PMCID: PMC11843967 DOI: 10.1186/s13578-025-01366-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Accepted: 02/11/2025] [Indexed: 02/22/2025] Open
Abstract
BACKGROUND Cell-free DNA (cfDNA) in plasma carries epigenetic signatures specific to tissue or cell of origin. Aberrant methylation patterns in circulating cfDNA have emerged as valuable tools for noninvasive cancer detection, prenatal diagnostics, and organ transplant assessment. Such epigenetic changes also hold significant promise for the diagnosis of neurodegenerative diseases, which often progresses slowly and has a lengthy asymptomatic period. However, genome-wide cfDNA methylation changes in neurodegenerative diseases remain poorly understood. RESULTS We used whole-genome bisulfite sequencing (WGBS) to profile age-dependent and ALS-associated methylation signatures in cfDNA from 30 individuals, including young and middle-aged controls, as well as ALS patients with matched controls. We identified 5,223 age-related differentially methylated loci (DMLs) (FDR < 0.05), with 51.6% showing hypomethylation in older individuals. Our results significantly overlapped with age-associated CpGs identified in a large blood-based epigenome-wide association study (EWAS). Comparing ALS patients to controls, we detected 1,045 differentially methylated regions (DMRs) in gene bodies, promoters, and intergenic regions. Notably, these DMRs were linked to key ALS-associated pathways, including endocytosis and cell adhesion. Integration with spinal cord transcriptomics revealed that 31% of DMR-associated genes exhibited differential expression in ALS patients compared to controls, with over 20 genes significantly correlating with disease duration. Furthermore, comparison with published single-nucleus RNA sequencing (snRNA-Seq) data of ALS demonstrated that cfDNA methylation changes reflects cell-type-specific gene dysregulation in the brain of ALS patients, particularly in excitatory neurons and astrocytes. Deconvolution of cfDNA methylation profiles suggested altered proportions of immune and liver-derived cfDNA in ALS patients. CONCLUSIONS cfDNA methylation is a powerful tool for assessing age-related changes and ALS-specific molecular dysregulation by revealing perturbed locus, genes, and the proportional contributions of different tissues/cells to the plasma. This technique holds promise for clinical application in biomarker discovery across a broad spectrum of neurodegenerative disorders.
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Affiliation(s)
- Yulin Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Karen N Conneely
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Wenjing Ma
- Department of Biostatistics, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Robert K Naviaux
- Departments of Medicine, Pediatrics, and Pathology, and the Mitochondrial and Metabolic Disease Center (MMDC), School of Medicine, University of California San Diego, San Diego, CA, 92103, USA
| | - Teepu Siddique
- Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA
| | - Emily G Allen
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Sandra Guingrich
- Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Robert M Pascuzzi
- Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Peng Jin
- Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, 30322, USA.
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5
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Peng L, Fang T, Dai L, Cai L. Diagnostic Value of Cross-priming Amplification Combined With CRISPR-Cas12b in Detecting Cell-free DNA in Tuberculous Pleural Effusion. Open Forum Infect Dis 2024; 11:ofae674. [PMID: 39660021 PMCID: PMC11630831 DOI: 10.1093/ofid/ofae674] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2024] [Accepted: 11/11/2024] [Indexed: 12/12/2024] Open
Abstract
Background Diagnosis of tuberculous pleural effusion (TPE) remains challenging. Studies have shown that detecting cell-free Mycobacterium tuberculosis (cf-TB) DNA in pleural effusion can improve TPE diagnosis. This study aimed to evaluate the diagnostic value of our recently developed TB One-Pot assay, which combines cross-priming amplification with CRISPR-Cas12b, in detecting cf-TB for TPE. Methods Pleural effusion samples were collected from inpatients with suspected TPE at Hangzhou Red Cross Hospital. After centrifugation, the precipitate was used for culture, Xpert, and pleural effusion cytologic testing, while the supernatant was used for biochemical and cf-TB assays, including TB One-Pot and the quantitative polymerase chain reaction method (cf-TB-PCR). Assessment of diagnostic performance was based on a comprehensive reference standard. Results A total of 115 patients were included: 88 TPE cases (diagnosed per the comprehensive reference standard) and 27 non-TPE cases. The sensitivity of TB One-Pot in detecting pleural cf-TB for diagnosing TPE was 64.8%, with an area under the curve (AUC) of 0.805, significantly superior to culture and Xpert (P < .05). When compared with cf-TB-PCR (sensitivity, 53.4%; AUC, 0.767) and the adenosine deaminase assay (sensitivity, 52.3%; AUC, 0.761), TB One-Pot demonstrated slightly higher sensitivity and AUC, but the differences were not statistically significant (P > .05). The specificity of TB One-Pot was 96.3%, while the specificity of the other tests was 100%, with no statistically significant differences (P > .05). Conclusions cf-TB provides direct evidence of the etiology of TPE. TB One-Pot for detecting cf-TB in diagnosing TPE outperforms existing TB laboratory tests and may represent a more effective approach for TPE diagnosis in resource-limited settings.
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Affiliation(s)
- Lijun Peng
- Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, China
| | - Tingting Fang
- Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, China
| | - Lingshan Dai
- Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, China
| | - Long Cai
- Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, China
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6
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Brown L, Colwill M, Poullis A. Gastrointestinal tuberculosis: Diagnostic approaches for this uncommon pathology. World J Clin Cases 2024; 12:5283-5287. [PMID: 39156088 PMCID: PMC11238685 DOI: 10.12998/wjcc.v12.i23.5283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 05/06/2024] [Accepted: 06/17/2024] [Indexed: 07/05/2024] Open
Abstract
A case report entitled "Primary gastroduodenal tuberculosis presenting as gastric outlet obstruction" recently published in the World Journal of Clinical Cases presented a rare cause of gastric outlet obstruction and highlighted the atypical manner in which gastrointestinal tuberculosis (TB) can present. The literature with regards to this rare pathology is limited to case reports and case series with the largest being published using data from between 2003 and 2013. However, since then the diagnostic tools available have significantly changed with more modern and increasingly accurate tests now available. This editorial reviews the current state of the art with regards to diagnosis in gastrointestinal TB.
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Affiliation(s)
- Lottie Brown
- Department of Gastroenterology, St George's University Hospital NHS Foundation Trust, London SW17 0QT, United Kingdom
| | - Michael Colwill
- Department of Gastroenterology, St George's University Hospital NHS Foundation Trust, London SW17 0QT, United Kingdom
| | - Andrew Poullis
- Department of Gastroenterology, St George's University Hospital NHS Foundation Trust, London SW17 0QT, United Kingdom
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7
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Tschan Y, Sasamalo M, Hiza H, Fellay J, Gagneux S, Reither K, Hella J, Portevin D. Diagnostic accuracy of a sequence-specific Mtb-DNA hybridization assay in urine: a case-control study including subclinical TB cases. Microbiol Spectr 2024; 12:e0042624. [PMID: 38717151 PMCID: PMC11237410 DOI: 10.1128/spectrum.00426-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2024] [Accepted: 04/19/2024] [Indexed: 06/06/2024] Open
Abstract
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains one of the deadliest infectious diseases globally. Timely diagnosis is a key step in the management of TB patients and in the prevention of further transmission events. Current diagnostic tools are limited in these regards. There is an urgent need for new accurate non-sputum-based diagnostic tools for the detection of symptomatic as well as subclinical TB. In this study, we recruited 52 symptomatic TB patients (sputum Xpert MTB/RIF positive) and 58 household contacts to assess the accuracy of a sequence-specific hybridization assay that detects the presence of Mtb cell-free DNA in urine. Using sputum Xpert MTB/RIF as a reference test, the magnetic bead-capture assay could discriminate active TB from healthy household contacts with an overall sensitivity of 72.1% [confidence interval (CI) 0.59-0.86] and specificity of 95.5% (CI 0.90-1.02) with a positive predictive value of 93.9% and negative predictive value of 78.2%. The detection of Mtb-specific DNA in urine suggested four asymptomatic TB infection cases that were confirmed in all instances either by concomitant Xpert MTB/RIF sputum testing or by follow-up investigation raising the specificity of the index test to 100%. We conclude that sequence-specific hybridization assays on urine specimens hold promise as non-invasive tests for the detection of subclinical TB. IMPORTANCE There is an urgent need for a non-sputum-based diagnostic tool allowing sensitive and specific detection of all forms of tuberculosis (TB) infections. In that context, we performed a case-control study to assess the accuracy of a molecular detection method enabling the identification of cell-free DNA from Mycobacterium tuberculosis that is shed in the urine of tuberculosis patients. We present accuracy data that would fulfill the target product profile for a non-sputum test. In addition, recent epidemiological data suggested that up to 50% of individuals secreting live bacilli do not present with symptoms at the time of screening. We report, here, that the investigated index test could also detect instances of asymptomatic TB infections among household contacts.
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Affiliation(s)
- Yves Tschan
- Swiss Tropical and Public Health Institute, Allschwil, Switzerland
- University of Basel, Basel, Switzerland
| | | | - Hellen Hiza
- Swiss Tropical and Public Health Institute, Allschwil, Switzerland
- University of Basel, Basel, Switzerland
- Ifakara Health Institute, Dar es Salaam, Tanzania
| | - Jacques Fellay
- School of Life Sciences, Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland
- Swiss Institute of Bioinformatics, Lausanne, Switzerland
- Precision Medicine Unit, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Sébastien Gagneux
- Swiss Tropical and Public Health Institute, Allschwil, Switzerland
- University of Basel, Basel, Switzerland
| | - Klaus Reither
- Swiss Tropical and Public Health Institute, Allschwil, Switzerland
- University of Basel, Basel, Switzerland
| | - Jerry Hella
- Ifakara Health Institute, Dar es Salaam, Tanzania
| | - Damien Portevin
- Swiss Tropical and Public Health Institute, Allschwil, Switzerland
- University of Basel, Basel, Switzerland
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Yang Y, Hua C, Liu Y, Yang C, Mi Y, Qiu W. Droplet digital PCR aids in the diagnosis of children with fever of unknown origin --A typical case report. Heliyon 2024; 10:e30961. [PMID: 38778949 PMCID: PMC11109792 DOI: 10.1016/j.heliyon.2024.e30961] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2023] [Revised: 05/08/2024] [Accepted: 05/08/2024] [Indexed: 05/25/2024] Open
Abstract
Many clinical conditions can cause fever of unknown origin (FUO) in children, but the etiological diagnosis remains challenging despite the variety of inspection methods available at present. This study aims to investigate the effectiveness of droplet digital polymerase chain reaction (ddPCR) in identifying pathogens in children with FUO as a novel application. A 7-month-old boy failed to obtain etiology evidence for his disease through various tests. After collecting peripheral blood for ddPCR analysis, Staphylococcus aureus and Escherichia coli were detected, and Sanger sequencing confirmed the pathogens. During the disease, the child developed septic arthritis and osteomyelitis in the femur. Despite the patient's fever being removed, his limb activity improving, and inflammatory biomarkers decreasing, avascular necrosis of the femoral head remained after targeted antibiotic treatment and surgery. If the patient had undergone ddPCR analysis at an early stage, it may be possible to avoid sequelae. ddPCR helps identify pathogens in the diagnosis of children with FUO and could be a promising complementary tool.
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Affiliation(s)
- Ying Yang
- Department of Infectious Diseases, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, 3333 Binsheng Road, Binjiang District, Hangzhou, Zhejiang, 310052, China
| | - Chunzhen Hua
- Department of Infectious Diseases, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, 3333 Binsheng Road, Binjiang District, Hangzhou, Zhejiang, 310052, China
| | - Yan Liu
- Department of Expanded Program on Immunization, Hangzhou Center for Disease Control and Prevention, Hangzhou, Zhejiang, 310021, China
| | - Cheng Yang
- Clinical Laboratory Center, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, 310052, China
| | - Yumei Mi
- Department of Infectious Diseases, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, 3333 Binsheng Road, Binjiang District, Hangzhou, Zhejiang, 310052, China
| | - Wei Qiu
- Department of Radiology, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, 310052, China
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9
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Li L, Henkle E, Youngquist BM, Seo S, Hamed K, Melnick D, Lyon CJ, Jiang L, Zelazny AM, Hu TY, Winthrop KL, Ning B. Serum Cell-Free DNA-based Detection of Mycobacterium avium Complex Infection. Am J Respir Crit Care Med 2024; 209:1246-1254. [PMID: 38190702 PMCID: PMC11146540 DOI: 10.1164/rccm.202303-0401oc] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Accepted: 01/04/2024] [Indexed: 01/10/2024] Open
Abstract
Rationale: Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) pulmonary disease (PD), which exhibits increasing global incidence. Current microbiologic methods routinely used in clinical practice lack sensitivity and have long latencies, leading to delays in diagnosis and treatment initiation and evaluation. A clustered regularly interspaced short palindromic repeats (CRISPR)-based assay that measures MAC cell-free DNA (cfDNA) concentrations in serum could provide a rapid means to detect MAC infection and monitor response to antimicrobial treatment. Objectives: To develop and optimize a CRISPR MAC assay for MAC infection detection and to evaluate its diagnostic and prognostic performance in two MAC disease cohorts. Methods: MAC cfDNA serum concentrations were measured in individuals with diagnoses of MAC disease or who had bronchiectasis or chronic obstructive pulmonary disease diagnoses without histories of NTM PD or NTM-positive sputum cultures. Diagnostic performance was analyzed using pretreatment serum from two cohorts. Serum MAC cfDNA changes during MAC PD treatment were evaluated in a subset of patients with MAC PD who received macrolide-based multidrug regimens. Measurements and Main Results: The CRISPR MAC assay detected MAC cfDNA in MAC PD with 97.6% (91.6-99.7%) sensitivity and 97.6% (91.5-99.7%) specificity overall. Serum MAC cfDNA concentrations markedly decreased after MAC-directed treatment initiation in patients with MAC PD who demonstrated MAC culture conversion. Conclusions: This study provides preliminary evidence for the utility of a serum-based CRISPR MAC assay to rapidly detect MAC infection and monitor the response to treatment.
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Affiliation(s)
- Lin Li
- Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China
- Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, and
| | | | - Brady M. Youngquist
- Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, and
| | - Seungyeon Seo
- Department of Laboratory Medicine, NIH Clinical Center, NIH, Bethesda, MD; and
| | | | | | - Christopher J. Lyon
- Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, and
| | - Li Jiang
- Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China
| | - Adrian M. Zelazny
- Department of Laboratory Medicine, NIH Clinical Center, NIH, Bethesda, MD; and
| | - Tony Y. Hu
- Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, and
- Department of Biomedical Engineering, School of Science and Engineering, Tulane University, New Orleans, LA
| | - Kevin L. Winthrop
- Division of Infectious Diseases, Schools of Medicine and Public Health, Oregon Health & Science University, Portland, OR
| | - Bo Ning
- Center for Cellular and Molecular Diagnostics, Department of Biochemistry and Molecular Biology, School of Medicine, and
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10
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Li G, Cannon K, Sisniega C, Fergie J. Cell-free DNA blood test for the diagnosis of pediatric tuberculous meningitis. J Clin Tuberc Other Mycobact Dis 2024; 35:100421. [PMID: 38420617 PMCID: PMC10899014 DOI: 10.1016/j.jctube.2024.100421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/02/2024] Open
Abstract
Pediatric tuberculous meningitis (TBM) is a severe form of tuberculosis that may present in children. The current diagnostic methods may have a limited impact on initial clinical decision-making. We present three children with tuberculous meningitis who had Mycobacterium tuberculosis complex cell-free DNA (cfDNA) detected in their blood within three days of sampling. Our cases described here illustrate for the first time the potential role of cfDNA blood tests in the rapid diagnosis of TBM.
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Affiliation(s)
- Guyu Li
- Division of Pediatric Infectious Diseases, Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, United States
| | - Kendall Cannon
- Driscoll Children's Hospital/ Texas A&M College of Medicine, Corpus Christi, TX 78411, United States
| | - Carlos Sisniega
- Division of Pediatric Cardiology, UCLA Mattel Children's Hospital, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, United States
| | - Jaime Fergie
- Department of Infectious Disease, Driscoll Children's Hospital/ Texas A&M College of Medicine, Corpus Christi, TX 78411, United States
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11
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David D, Das M, Mani Chandra H. A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples. Mol Biol Rep 2024; 51:504. [PMID: 38616219 DOI: 10.1007/s11033-024-09470-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Accepted: 03/22/2024] [Indexed: 04/16/2024]
Abstract
BACKGROUND Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. METHODS Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. RESULTS The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. CONCLUSION The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.
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Affiliation(s)
- Diana David
- Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, 632106, India
- Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, Tamil Nadu, 632115, India
| | - Madhusmita Das
- Molecular Biology and Immunology, Schieffelin Institute of Health Research and Leprosy Centre, Karigiri, Vellore, Tamil Nadu, 632106, India
| | - Harish Mani Chandra
- Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, Tamil Nadu, 632115, India.
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Salazar MP, da Costa Lima Suassuna Monteiro JF, Veloso Carvalho-Silva WH, Nunes Diniz GT, Werkhauser RP, Lapa Montenegro LM, Schindler HC. Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection. Biotechniques 2024; 76:235-244. [PMID: 38602382 DOI: 10.2144/btn-2023-0080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2023] [Accepted: 03/22/2024] [Indexed: 04/12/2024] Open
Abstract
Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/μl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.
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Affiliation(s)
- Marcela Pereira Salazar
- Laboratory of Immunoepidemiology, Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
- Contributed equally to this work and are considered as co-first authors
| | - Juliana Figueiredo da Costa Lima Suassuna Monteiro
- Laboratory of Immunoepidemiology, Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
- Contributed equally to this work and are considered as co-first authors
| | | | - George Tadeu Nunes Diniz
- Laboratory of Quantitative Methods, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
| | - Roberto Pereira Werkhauser
- Laboratory of Immunoepidemiology, Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
| | - Lílian Maria Lapa Montenegro
- Laboratory of Immunoepidemiology, Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
| | - Haiana Charifker Schindler
- Laboratory of Immunoepidemiology, Department of Immunology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Pernambuco, 50740-465, Brazil
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13
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Rees CE, Swift BM, Haldar P. State-of-the-art detection of Mycobacterium tuberculosis in blood during tuberculosis infection using phage technology. Int J Infect Dis 2024; 141S:106991. [PMID: 38447755 DOI: 10.1016/j.ijid.2024.106991] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Revised: 02/27/2024] [Accepted: 02/28/2024] [Indexed: 03/08/2024] Open
Abstract
Tuberculosis (TB), an aerosol-transmitted infection caused by Mycobacterium tuberculosis (Mtb), remains the commonest cause of death globally, from an infectious bacterial disease. Nine years on from the launch of the World Health Organization (WHO)'s END-TB strategy, disease incidence rates are stubbornly unchanged [1]. While this represents, in part, a reversal of improving trends caused by the COVID-19 pandemic, it also reflects the fragility and inadequacy of healthcare systems to sustain TB control [2]. Although multifactorial, a key reason for this is the ineffectiveness of existing clinical tools to meet the two key objectives of the END-TB strategy-(i) early diagnosis and treatment of TB disease (to limit onward transmission); and (ii) disease prevention through screening for asymptomatic TB infection (TBI). Meeting both objectives will rely on the development of new biomarkers with high accuracy, but the global nature of the TB problem also requires that new tests are rapid, low cost and can be measured in patients by sampling from universally accessible sites. In this review, we will present the accumulating evidence for circulating Mtb in both TB disease and asymptomatic TBI and discuss the potential utility of novel bacteriophage-based technology for blood-based detection of Mtb.
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Affiliation(s)
| | - Benjamin Mc Swift
- Royal Veterinary College, Department of Pathobiology and Population Sciences, Herts, UK
| | - Pranabashis Haldar
- NIHR Leicester Biomedical Research Centre, Department of Respiratory Sciences, University of Leicester, Leicester, UK.
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14
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Jain R, Gupta G, Mitra DK, Guleria R. Diagnosis of extra pulmonary tuberculosis: An update on novel diagnostic approaches. Respir Med 2024; 225:107601. [PMID: 38513873 DOI: 10.1016/j.rmed.2024.107601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2023] [Revised: 02/29/2024] [Accepted: 03/12/2024] [Indexed: 03/23/2024]
Abstract
Tuberculosis (TB) remains a major global public health problem worldwide. Though Pulmonary TB (PTB) is mostly discussed, one in five cases of TB present are extrapulmonary TB (EPTB) that manifests conspicuous diagnostic and management challenges with respect to the site of infection. The diagnosis of EPTB is often delayed or even missed due to insidious clinical presentation, pauci-bacillary nature of the disease, and lack of laboratory facilities in the resource limited settings. Culture, the classical gold standard for the diagnosis of tuberculosis, suffers from increased technical and logistical constraints in EPTB cases. Other than culture, several other tests are available but their feasibility and effciacy for the detection of EPTB is still the matter of interest. We need more specific and precise test/s for the various forms of EPTB diagnosis which can easily be applied in the routine TB control program is required. A test that can contribute remarkably towards improving EPTB case detection reducing the morbidity and mortality is the utmost requirement. In this review we described the scenario of molecular and other noval methods available for laboratory diagnosis of EPTB, and also discussed the challenges linked with each diagnostic method. This review will make the readers aware of new emerging diagnostic techniques in the field of EPTB diagnosis. They can make an informed decision to choose the appropriate one according to the test availability, their clinical settings and financial considerations.
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Affiliation(s)
- Rashi Jain
- Department of Pulmonary Critical Care and Sleep Medicine, All India Institute of Medical Sciences, New Delhi, 110029, India; Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, 110029, India
| | - Gopika Gupta
- Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, 110029, India
| | - D K Mitra
- Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, 110029, India
| | - Randeep Guleria
- Department of Pulmonary Critical Care and Sleep Medicine, All India Institute of Medical Sciences, New Delhi, 110029, India; Institute of Internal Medicine & Respiratory and Sleep Medicine, Medanta-The Medicity, Gurugram, Haryana, 122033, India.
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15
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Misra A, Powell EA. Preanalytical Challenges of Molecular Microbiology Tests. Clin Lab Med 2024; 44:33-43. [PMID: 38280796 DOI: 10.1016/j.cll.2023.10.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2024]
Abstract
As infectious disease diagnostics increasingly incorporates molecular techniques, there are unique preanalytical concerns that must be considered. First, noninvasive specimen types that may be inadequate for culture-based diagnostics may be acceptable when using molecular tests. Second, specimen containers must be evaluated for the presence of substances that may interfere with amplification or sequencing reactions. Finally, the capacity of transport, storage, and processing conditions to maintain nucleic acid integrity and avoid contamination must be assessed. This review explores these issues and the effects they may have on result quality.
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Affiliation(s)
- Anisha Misra
- Department of Laboratory Medicine, Cleveland Clinic, Robert J. Tomsich Pathology and Laboratory Medicine Institute, 10300 Carnegie Avenue LL-1, Cleveland, OH 44195, USA
| | - Eleanor A Powell
- Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 3188 Bellevue Avenue, Cincinnati, OH 45219, USA.
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Ferdosnejad K, Zamani MS, Soroush E, Fateh A, Siadat SD, Tarashi S. Tuberculosis and lung cancer: metabolic pathways play a key role. NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS 2024; 43:1262-1281. [PMID: 38305273 DOI: 10.1080/15257770.2024.2308522] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 01/09/2024] [Accepted: 01/14/2024] [Indexed: 02/03/2024]
Abstract
Despite the fact that some cases of tuberculosis (TB) are undiagnosed and untreated, it remains a serious global public health issue. In the diagnosis, treatment, and control of latent and active TB, there may be a lack of effectiveness. An understanding of metabolic pathways can be fundamental to treat latent TB infection and active TB disease. Rather than targeting Mycobacterium tuberculosis, the control strategies aim to strengthen host responses to infection and reduce chronic inflammation by effectively enhancing host resistance to infection. The pathogenesis and progression of TB are linked to several metabolites and metabolic pathways, and they are potential targets for host-directed therapies. Additionally, metabolic pathways can contribute to the progression of lung cancer in patients with latent or active TB. A comprehensive metabolic pathway analysis is conducted to highlight lung cancer development in latent and active TB. The current study aimed to emphasize the association between metabolic pathways of tumor development in patients with latent and active TB. Health control programs around the world are compromised by TB and lung cancer due to their special epidemiological and clinical characteristics. Therefore, presenting the importance of lung cancer progression through metabolic pathways occurring upon TB infection can open new doors to improving control of TB infection and active TB disease while stressing that further evaluations are required to uncover this correlation.
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Affiliation(s)
| | | | - Erfan Soroush
- Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
| | - Abolfazl Fateh
- Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
- Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
| | - Seyed Davar Siadat
- Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
- Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
| | - Samira Tarashi
- Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
- Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
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Chen S, Wang C, Zou Y, Zong Z, Xue Y, Jia J, Dong L, Zhao L, Chen L, Liu L, Chen W, Huang H. Tuberculosis-targeted next-generation sequencing and machine learning: An ultrasensitive diagnostic strategy for paucibacillary pulmonary tuberculosis and tuberculous meningitis. Clin Chim Acta 2024; 553:117697. [PMID: 38145644 DOI: 10.1016/j.cca.2023.117697] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Revised: 12/03/2023] [Accepted: 12/04/2023] [Indexed: 12/27/2023]
Abstract
BACKGROUND Existing diagnostic approaches for paucibacillary tuberculosis (TB) are limited by the low sensitivity of testing methods and difficulty in obtaining suitable samples. METHODS An ultrasensitive TB diagnostic strategy was established, integrating efficient and specific TB targeted next-generation sequencing and machine learning models, and validated in clinical cohorts to test plasma cfDNA, cerebrospinal fluid (CSF) DNA collected from tuberculous meningitis (TBM) and pediatric pulmonary TB (PPTB) patients. RESULTS In the detection of 227 samples, application of the specific thresholds of CSF DNA (AUC = 0.974) and plasma cfDNA (AUC = 0.908) yielded sensitivity of 97.01 % and the specificity of 95.65 % in CSF samples and sensitivity of 82.61 % and specificity of 86.36 % in plasma samples, respectively. In the analysis of 44 paired samples from TBM patients, our strategy had a high concordance of 90.91 % (40/44) in plasma cfDNA and CSF DNA with both sensitivity of 95.45 % (42/44). In the PPTB patient, the sensitivity of the TB diagnostic strategy yielded higher sensitivity on plasma specimen than Xpert assay on gastric lavage (28.57 % VS. 15.38 %). CONCLUSIONS Our TB diagnostic strategy provides greater detection sensitivity for paucibacillary TB, while plasma cfDNA as an easily collected specimen, could be an appropriate sample type for PTB and TBM diagnosis.
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Affiliation(s)
- Suting Chen
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
| | - Congli Wang
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yijun Zou
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhaojing Zong
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China; Department of Respiratory and Critical Care Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi 563001, China
| | - Yi Xue
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
| | - Junnan Jia
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
| | - Lingling Dong
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
| | - Liping Zhao
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
| | - Lu Chen
- Beijing Macroµ-test Bio-Tech Co., Ltd., Beijing 101300, China
| | - Licheng Liu
- Beijing Macroµ-test Bio-Tech Co., Ltd., Beijing 101300, China
| | - Weijun Chen
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Hairong Huang
- National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing 101149, China.
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18
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Tseng YH, Pan SW, Feng JY, Su WJ, Huang CYF, Chen YM. Detecting circulating microbial cell-free DNA by next-generation sequencing in patients with Mycobacterium avium complex-lung disease: A pilot study. Tzu Chi Med J 2024; 36:67-75. [PMID: 38406566 PMCID: PMC10887338 DOI: 10.4103/tcmj.tcmj_191_23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Revised: 08/22/2023] [Accepted: 09/20/2023] [Indexed: 02/27/2024] Open
Abstract
Objectives Determining a diagnosis for non-Tuberculous mycobacterium (NTM)-lung disease (LD) remains difficult. The value of circulating cell-free DNA (cfDNA) secreted from microbes has been established in the detection of pathogens in septic patients. However, it is unknown whether NTM-derived cfDNA is detectable in plasma from patients with NTM-LD and whether this is associated with the disease status of NTM-LD, especially in patients with Mycobacterium avium complex (MAC)-LD. Materials and Methods In this pilot study, from 2018 to 2019, we enrolled adult patients with MAC-LD at Taipei Veterans General Hospital in Taiwan for the detection of circulating cfDNA. We performed cfDNA extraction from plasma, next-generation sequencing (NGS) for nonhuman cfDNA, and sequence matching to a microbial database and then assessed the association between pathogen cfDNA and MAC-LD. Results Two (40%) plasma samples from MAC-LD patients had detectable MAC-specific cfDNA, namely one instance of DNA polymerase III alpha subunit and one instance of ATP-binding cassette transporters permease. The plasma samples from the three other MAC-LD cases and the one tuberculosis control were negative for either NTM-derived cfDNA or tuberculosis-related cfDNA. In addition to MAC-specific cfDNA, Ralstonia solanacearum, Staphylococcus aureus, and Pasteurella multocida were the most observed bacteria in our patients. The two patients with MAC-cfDNA positivity yielded higher radiographic scores (P = 0.076) and presented a higher number of nonhuman reads than those without MAC-cfDNA positivity (P = 0.083). Conclusion Using NGS method, we demonstrated MAC-cfDNA was detectable in patients with MAC-LD. Further large-scale research is warranted to assess the clinical value of detecting MAC-specific cfDNA in MAC-LD patients.
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Affiliation(s)
- Yen-Han Tseng
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Program in Molecular Medicine, School of Life Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Sheng-Wei Pan
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Jia-Yih Feng
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Wei-Juin Su
- Division of Chest Medicine, Department of Internal Medicine, China Medical University Hospital, Taipei Branch, Taipei, Taiwan
| | - Chi-Ying F Huang
- Program in Molecular Medicine, School of Life Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Institute of Biopharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yuh-Min Chen
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
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Kontsevaya I, Heyckendorf J, Koops F, Hillemann D, Goldmann T, Upton CM, De Jager V, Diacon A, Lange C. Transrenal Mycobacterium tuberculosis DNA in pulmonary tuberculosis patients during the first 14 days of treatment. Microbiol Spectr 2023; 11:e0234823. [PMID: 37882572 PMCID: PMC10714760 DOI: 10.1128/spectrum.02348-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 09/11/2023] [Indexed: 10/27/2023] Open
Abstract
IMPORTANCE This study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.
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Affiliation(s)
- Irina Kontsevaya
- Division of Clinical Infectious Diseases, Research Center Borstel, Leibniz Lung Center, Borstel, Germany
- German Center for Infection Research, Borstel, Germany
- Respiratory Medicine and International Health, University of Lübeck, Lübeck, Germany
- Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, United Kingdom
| | - Jan Heyckendorf
- Department of Internal Medicine I, University Medical Center Schleswig-Holstein, Kiel, Germany
| | - Frauke Koops
- Division of Clinical Infectious Diseases, Research Center Borstel, Leibniz Lung Center, Borstel, Germany
- German Center for Infection Research, Borstel, Germany
| | - Doris Hillemann
- National Reference Center, Research Center Borstel, Borstel, Germany
| | - Torsten Goldmann
- Division of Histology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany
- Airway Research Center North, Member of the German Center for Lung Research, Großhansdorf, Germany
| | | | | | | | - Christoph Lange
- Division of Clinical Infectious Diseases, Research Center Borstel, Leibniz Lung Center, Borstel, Germany
- German Center for Infection Research, Borstel, Germany
- Respiratory Medicine and International Health, University of Lübeck, Lübeck, Germany
- Baylor College of Medicine, Houston, Texas, USA
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Adhit KK, Wanjari A, Menon S, K S. Liquid Biopsy: An Evolving Paradigm for Non-invasive Disease Diagnosis and Monitoring in Medicine. Cureus 2023; 15:e50176. [PMID: 38192931 PMCID: PMC10772356 DOI: 10.7759/cureus.50176] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Accepted: 12/08/2023] [Indexed: 01/10/2024] Open
Abstract
Liquid biopsy stands as an innovative instrument in the realm of precision medicine, enabling non-invasive disease diagnosis and the early detection of cancer. Liquid biopsy helps in the extraction of circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and cell-free DNA (cfDNA) from blood samples and other body fluids, thereby facilitating disease diagnosis and prediction of high-risk patients. Various techniques such as advanced sequencing methods and biomarker-based cell capture have led to the isolation and study of the different biomarkers such as ctDNA, cfDNA, and CTCs. These biopsies also have immense potential in the early detection and diagnosis of various diseases across all medical specialties, prediction and screening of high-risk cases, and detection of different immune response patterns in response to infectious diseases, and also help in predicting treatment outcomes. Although liquid biopsy has the potential to disrupt the field of medical diagnosis, it is met by various challenges such as limited tumor-derived components, less specificity, and inadequate advancement in methods to isolate biomarkers. Despite all these challenges, liquid biopsies provide the potential to become a minimally invasive method of diagnosis that would facilitate real-time monitoring of patients, which differentiates them from traditional tissue biopsies. This article aims to provide a complete overview of the current technologies, different biomarkers, and body fluids that can be used in liquid biopsy and its clinical applications and the potential impact that liquid biopsy holds in the field of precision medicine, facilitating early diagnosis and prompt management of various diseases and cancers.
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Affiliation(s)
- Kanishk K Adhit
- Medicine, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Anil Wanjari
- Medicine, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Sharanya Menon
- Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
| | - Siddhaarth K
- Medicine, Jawaharlal Nehru Medical College, Datta Meghe Institute of Higher Education and Research, Wardha, IND
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Kanaujia R, Sharma V, Biswal M, Singh S, Ray P, Angrup A. Microbial cell-free DNA detection: Minimally invasive diagnosis of infectious diseases. Indian J Med Microbiol 2023; 46:100433. [PMID: 37945127 DOI: 10.1016/j.ijmmb.2023.100433] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2023] [Revised: 07/07/2023] [Accepted: 07/19/2023] [Indexed: 11/12/2023]
Abstract
BACKGROUND Detection of infectious diseases, especially among immunocompromised and patients on prolonged anti-microbial treatment, remains challenging, limited by conventional techniques with low sensitivity and long-turnaround time. Molecular detection by polymerase chain reaction (PCR) also has limited utility as it requires a targeted approach with prior suspicion of the infecting organism. Advancements in sequencing methodologies, specifically next-generation sequencing (NGS), have presented a promising opportunity to identify pathogens in cases where conventional techniques may be inadequate. However, the direct application of these techniques for diagnosing invasive infections is still limited by the need for invasive sampling, highlighting the pressing need to develop and implement non-invasive or minimally invasive approaches to improve the diagnosis of invasive infections. OBJECTIVES The objectives of this article are to explore the notable features, clinical utility, and constraints associated with the detection of microbial circulating cell-free DNA (mcfDNA) as a minimally invasive diagnostic tool for infectious diseases. CONTENT The mcfDNA detection provides an opportunity to identify micro-organisms in the blood of a patient. It is especially beneficial in immunocompromised patients where invasive sampling is not possible or where repeated cultures are negative. This review will discuss the applications and constraints of detecting mcfDNA for diagnosing infections and the various platforms available for its detection.
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Affiliation(s)
| | - Vikas Sharma
- Department of Medical Microbiology, PGIMER, Chandigarh, India
| | - Manisha Biswal
- Department of Medical Microbiology, PGIMER, Chandigarh, India
| | - Shreya Singh
- Department of Medical Microbiology, AIMS, Mohali, India
| | - Pallab Ray
- Department of Medical Microbiology, PGIMER, Chandigarh, India
| | - Archana Angrup
- Department of Medical Microbiology, PGIMER, Chandigarh, India.
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22
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Singh UB, Angitha KP, Bhatnagar A, Sharma S, Bir R, Singh K, Nabeta P, Ruhwald M, Kabra SK, Lodha R. GeneXpert Ultra in Urine Samples for Diagnosis of Extra-Pulmonary Tuberculosis. Curr Microbiol 2023; 80:361. [PMID: 37796343 DOI: 10.1007/s00284-023-03503-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2023] [Accepted: 09/18/2023] [Indexed: 10/06/2023]
Abstract
Extra-pulmonary tuberculosis (EPTB) continues to be difficult to diagnose. Novel biomarkers in biological specimens offer promise. Detection of Mycobacterium tuberculosis (Mtb) DNA in urine could prove useful in diagnosis of EPTB, possibly due to disseminated disease or micro-abscesses reported in kidneys. The current study was designed to detect Mtb DNA in stored urine samples from patients with EPTB. Diagnosis of EPTB was reached using Microbiological Reference Standards (MRS) on samples from the disease site using WHO Recommended Diagnostics (WRD), [smear microscopy, liquid culture (MGIT-960)] and GX (molecular WRD, mWRD) and Comprehensive reference standards [CRS, clinical presentation, microbiological reference standards, radiology, histopathology]. GX-Ultra was performed on urine samples stored in -80oC deep freezer, retrospectively. Of 70 patients, 51 (72.9%) were classified as confirmed TB, 11 (15.7%) unconfirmed TB, and 8 (11.4%) unlikely TB. GX-Ultra in urine samples demonstrated sensitivity of 52.9% and specificity of 57.9% against MRS, and higher sensitivity of 56.5% and specificity of 100% against CRS. The sensitivity and specificity of GX-Ultra in urine was 53.6% and 75% for pus sample subset and 52.2% and 53.3% for fluid sample subset. Urine being non-invasive and easy to collect, detection of Mtb DNA using mWRD in urine samples is promising for diagnosis of EPTB.
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Affiliation(s)
- Urvashi B Singh
- Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
| | - K P Angitha
- Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
| | - Anuj Bhatnagar
- Department of Tuberculosis and Chest Diseases, Rajan Babu Institute for Pulmonary Medicine and Tuberculosis, New Delhi, India
| | - Sangeeta Sharma
- Department of Pediatrics, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
| | - Raunak Bir
- Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
| | - Kiran Singh
- Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
| | - Pamela Nabeta
- The global alliance for diagnostics, FIND, Geneva, Switzerland
| | - Morten Ruhwald
- The global alliance for diagnostics, FIND, Geneva, Switzerland
| | - Sushil K Kabra
- Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India
| | - Rakesh Lodha
- Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India
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Sharma P, Gupta RK, Anthwal D, Dass M, Yadav R, Behera A, Sethi S, Singhal R, Dhooria S, Aggarwal AN, Haldar S. Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis. Tuberculosis (Edinb) 2023; 142:102369. [PMID: 37536090 DOI: 10.1016/j.tube.2023.102369] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/13/2023] [Accepted: 06/25/2023] [Indexed: 08/05/2023]
Abstract
Pleural tuberculosis (pTB) is a grave clinical challenge. A novel cell-free M. tuberculosis DNA (cfM.tb-DNA) probe-based-qPCR assay was developed for the diagnosis of pTB. Total cell-free DNA was extracted from pleural fluid (PF) and paired plasma samples and cfM.tb-DNA was quantified by probe-based qPCR targeting devR (109-bp) gene of M. tuberculosis in patients with pleural effusion. Patient categorization was done using 'Composite-Reference-Standard' formulated for the study. Assay cut-offs were determined from samples in the 'Development set' (n = 17; 'Definite & Probable' pTB; n = 9 and 'Non-TB'; n = 8) by ROC-curve analysis and applied to 'Validation set' (n = 112; 'Definite' pTB; n = 8, 'Probable' pTB; n = 34, 'Possible' pTB; n = 28 and 'Non-TB'; n = 42). cfM.tb-DNA qPCR had a sensitivity of 62.5% (95%CI; 24.4,91.4) in 'Definite' pTB category and 59.5% (95%CI; 43.2,74.3) in 'Definite & Probable' pTB category with 95.2% (95%CI; 83.8,99.4) specificity using PF. In plasma (n = 85), the assay had a sub-optimal sensitivity of 7.6% (95%CI; 0.95,25.1) with 88.2% (95%CI; 72.5,96.7) specificity in 'Definite & Probable' pTB group. Xpert MTB/RIF assay detected only six-samples in the 'Validation set'. Logistic regression analysis indicated that PF-cfM.tb-DNA qPCR provided incremental advantage over existing pTB diagnostic algorithms. To the best of our knowledge, this is the first report describing the utility of cfM.tb-DNA for pTB diagnosis in India.
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Affiliation(s)
- Pratibha Sharma
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Rakesh Kumar Gupta
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Divya Anthwal
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Manisha Dass
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Rakesh Yadav
- Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Ashish Behera
- Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Sunil Sethi
- Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Ritu Singhal
- Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
| | - Sahajal Dhooria
- Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Ashutosh Nath Aggarwal
- Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India
| | - Sagarika Haldar
- Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
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Bányász B, Antal J, Dénes B. False Positives in Brucellosis Serology: Wrong Bait and Wrong Pond? Trop Med Infect Dis 2023; 8:tropicalmed8050274. [PMID: 37235322 DOI: 10.3390/tropicalmed8050274] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 05/07/2023] [Accepted: 05/09/2023] [Indexed: 05/28/2023] Open
Abstract
This review summarizes the status of resolving the problem of false positive serologic results (FPSR) in Brucella serology, compiles our knowledge on the molecular background of the problem, and highlights some prospects for its resolution. The molecular basis of the FPSRs is reviewed through analyzing the components of the cell wall of Gram-negative bacteria, especially the surface lipopolysaccharide (LPS) with details related to brucellae. After evaluating the efforts that have been made to solve target specificity problems of serologic tests, the following conclusions can be drawn: (i) resolving the FPSR problem requires a deeper understanding than we currently possess, both of Brucella immunology and of the current serology tests; (ii) the practical solutions will be as expensive as the related research; and (iii) the root cause of FPSRs is the application of the same type of antigen (S-type LPS) in the currently approved tests. Thus, new approaches are necessary to resolve the problems stemming from FPSR. Such approaches suggested by this paper are: (i) the application of antigens from R-type bacteria; or (ii) the further development of specific brucellin-based skin tests; or (iii) the application of microbial cell-free DNA as analyte, whose approach is detailed in this paper.
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Affiliation(s)
- Borbála Bányász
- Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest, 1143 Budapest, Hungary
- Laboratory of Immunology, Veterinary Diagnostic Directorate, National Food Chain Safety Office, 1143 Budapest, Hungary
| | - József Antal
- Omixon Biocomputing Ltd., 1117 Budapest, Hungary
| | - Béla Dénes
- Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest, 1143 Budapest, Hungary
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25
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Cao Y, Jiang T, Lin Y, Fang X, Ding P, Song H, Li P, Li Y. Time-series prediction and detection of potential pathogens in bloodstream infection using mcfDNA sequencing. Front Cell Infect Microbiol 2023; 13:1144625. [PMID: 37249984 PMCID: PMC10213887 DOI: 10.3389/fcimb.2023.1144625] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2023] [Accepted: 04/18/2023] [Indexed: 05/31/2023] Open
Abstract
Introduction Next-generation sequencing of microbial cell free DNA (mcfDNA-seq) has emerged as a promising diagnostic method for blood stream infection (BSI) and offers the potential to detect pathogens before blood culture. However, its application is limited by a lack of clinical validation. Methods We conducted sequential mcfDNA-seq on blood samples from ICU participants at high risk of BSI due to pneumonia, or intravascular catheterization; and explored whether mcfDNA-seq could diagnose and detect pathogens in advance of blood culture positivity. Blood culture results were used as evaluation criteria. Results A total of 111 blood samples were collected during the seven days preceding and on the day of onset of 16 BSI episodes from 13 participants. The diagnostic and total predictive sensitivity of mcfDNA-seq were 90% and 87.5%, respectively. The proportion of pathogenic bacteria was relatively high in terms of both diagnosis and prediction. The reads per million of etiologic agents trended upwards in the days approaching the onset of BSI. Discussion Our work found that mcfDNA-seq has high diagnostic sensitivity and could be used to identify pathogens before the onset of BSI, which could help expand the clinical application of mcfDNA-seq.
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Affiliation(s)
- Yinghao Cao
- Department of Clinical Laboratory Medicine, School of Medicine, South China University of Technology, Guangzhou, China
- Department of Clinical Laboratory Medicine, The Sixth Medical Center of People's Liberation Army (PLA) General Hospital of Beijing, Beijing, China
| | - Tingting Jiang
- Department of Epidemiology and Biostatistics, School of Public Health, An Hui Medical University, Hefei, China
- Biosecurity Department, Chinese People's Liberation Army (PLA) Center for Disease Control and Prevention, Beijing, China
| | - Yanfeng Lin
- Biosecurity Department, Chinese People's Liberation Army (PLA) Center for Disease Control and Prevention, Beijing, China
| | - Xiaofeng Fang
- Department of Clinical Laboratory Medicine, School of Medicine, South China University of Technology, Guangzhou, China
- Department of Clinical Laboratory Medicine, The Sixth Medical Center of People's Liberation Army (PLA) General Hospital of Beijing, Beijing, China
| | - Peipei Ding
- Department of Clinical Laboratory Medicine, The Sixth Medical Center of People's Liberation Army (PLA) General Hospital of Beijing, Beijing, China
| | - Hongbin Song
- Department of Epidemiology and Biostatistics, School of Public Health, An Hui Medical University, Hefei, China
- Biosecurity Department, Chinese People's Liberation Army (PLA) Center for Disease Control and Prevention, Beijing, China
| | - Peng Li
- Biosecurity Department, Chinese People's Liberation Army (PLA) Center for Disease Control and Prevention, Beijing, China
| | - Yanjun Li
- Department of Clinical Laboratory Medicine, School of Medicine, South China University of Technology, Guangzhou, China
- Department of Clinical Laboratory Medicine, The Sixth Medical Center of People's Liberation Army (PLA) General Hospital of Beijing, Beijing, China
- School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China
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26
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Bararia A, Chakraborty P, Roy P, Chattopadhay BK, Das A, Chatterjee A, Sikdar N. Emerging role of non-invasive and liquid biopsy biomarkers in pancreatic cancer. World J Gastroenterol 2023; 29:2241-2260. [PMID: 37124888 PMCID: PMC10134423 DOI: 10.3748/wjg.v29.i15.2241] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Revised: 02/02/2023] [Accepted: 03/15/2023] [Indexed: 04/14/2023] Open
Abstract
A global increase in the incidence of pancreatic cancer (PanCa) presents a major concern and health burden. The traditional tissue-based diagnostic techniques provided a major way forward for molecular diagnostics; however, they face limitations based on diagnosis-associated difficulties and concerns surrounding tissue availability in the clinical setting. Late disease development with asymptomatic behavior is a drawback in the case of existing diagnostic procedures. The capability of cell free markers in discriminating PanCa from autoimmune pancreatitis and chronic pancreatitis along with other precancerous lesions can be a boon to clinicians. Early-stage diagnosis of PanCa can be achieved only if these biomarkers specifically discriminate the non-carcinogenic disease stage from malignancy with respect to tumor stages. In this review, we comprehensively described the non-invasive disease detection approaches and why these approaches are gaining popularity for their early-stage diagnostic capability and associated clinical feasibility.
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Affiliation(s)
- Akash Bararia
- Human Genetics Unit, Indian Statistical Institute, Kolkata 700108, India
| | - Prosenjeet Chakraborty
- Department of Molecular Biosciences, SVYASA School of Yoga and Naturopathy, Bangalore 560105, India
| | - Paromita Roy
- Department of Pathology, Tata Medical Center, Kolkata 700160, India
| | | | - Amlan Das
- Department of Biochemistry, Royal Global University, Assam 781035, India
| | - Aniruddha Chatterjee
- Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin 9061, New Zealand
- School of Health Sciences and Technology, University of Petroleum and Energy Studies, Dehradun 248007, India
| | - Nilabja Sikdar
- Human Genetics Unit, Indian Statistical Institute, Kolkata 700108, India
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Thakku SG, Lirette J, Murugesan K, Chen J, Theron G, Banaei N, Blainey PC, Gomez J, Wong SY, Hung DT. Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13. Nat Commun 2023; 14:1803. [PMID: 37002219 PMCID: PMC10064635 DOI: 10.1038/s41467-023-37183-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Accepted: 03/06/2023] [Indexed: 04/03/2023] Open
Abstract
Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.
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Affiliation(s)
| | | | - Kanagavel Murugesan
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Julie Chen
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Grant Theron
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research and SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Niaz Banaei
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA
- Clinical Microbiology Laboratory, Stanford Health Care, Palo Alto, CA, USA
| | - Paul C Blainey
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA
| | - James Gomez
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Sharon Y Wong
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Deborah T Hung
- Broad Institute of MIT and Harvard, Cambridge, MA, USA.
- Department of Genetics, Harvard Medical School, Boston, MA, USA.
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.
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28
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Cobra HADAB, Mozella AP, da Palma IM, Salim R, Leal AC. Cell-free Deoxyribonucleic Acid: A Potential Biomarker of Chronic Periprosthetic Knee Joint Infection. J Arthroplasty 2022; 37:2455-2459. [PMID: 35840076 DOI: 10.1016/j.arth.2022.07.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2022] [Revised: 07/05/2022] [Accepted: 07/06/2022] [Indexed: 02/02/2023] Open
Abstract
BACKGROUND The correct diagnosis of a chronic periprosthetic joint infection (PJI) is a major challenge in clinical practice, with the "gold standard" for diagnosis yet to be established. Synovial fluid analysis has been proven to be a useful tool for that purpose. Cell-free DNA (cf-DNA) levels have been shown to be increased in several conditions such as cancer, trauma, and sepsis. Therefore, this study was designed to evaluate the potential of synovial fluid cf-DNA quantification for the diagnosis of chronic periprosthetic infections following total knee arthroplasty. METHODS A prospective study with patients undergoing total knee arthroplasty revision surgery for any indication was performed. PJI diagnosis was defined according to the Second International Consensus Meeting on Musculoskeletal Infection (2018) criteria. The study cohort consisted of 26 patients classified as infected and 40 as noninfected. Synovial fluid cf-DNA direct quantification by fluorescent staining was made. Sensitivity, specificity, and receiver operating characteristic curve were calculated. RESULTS The cf-DNA levels were significantly higher in patients who had PJIs (122.5 ± 57.2 versus 4.6 ± 2.8 ng/μL, P < .0001). With a cutoff of 15 ng/μL, the area under the receiver operating characteristic, sensitivity, and specificity of cf-DNA were 0.978, 96.2%, and 100%, respectively. CONCLUSION The present study has shown that cf-DNA is increased in synovial fluid of patients who have chronic PJIs. It is a promising biomarker for knee PJI diagnosis and further studies are needed to confirm its utility.
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Affiliation(s)
- Hugo A de A B Cobra
- Center for Surgery of Knee, National Institute of Traumatology and Orthopaedics, Rio de Janeiro, Brazil
| | - Alan P Mozella
- Center for Surgery of Knee, National Institute of Traumatology and Orthopaedics, Rio de Janeiro, Brazil
| | - Idemar M da Palma
- Rios D'or Hospital, Rio de Janeiro, Brazil; Montese Medical Center, Rio de Janeiro, Brazil
| | - Rodrigo Salim
- Department of Orthopaedics and Anaesthesiology, Ribeirão Preto Medical School, University of São Paulo, São Paulo, Brazil
| | - Ana C Leal
- Teaching and Research Division, National Institute of Traumatology and Orthopaedics, Rio de Janeiro, Brazil
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Pan SW, Syed RR, Catanzaro DG, Ho ML, Shu CC, Tsai TY, Tseng YH, Feng JY, Chen YM, Su WJ, Catanzaro A, Rodwell TC. Circulating mitochondrial cell-free DNA dynamics in patients with mycobacterial pulmonary infections: Potential for a novel biomarker of disease. Front Immunol 2022; 13:1040947. [PMID: 36466831 PMCID: PMC9709461 DOI: 10.3389/fimmu.2022.1040947] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Accepted: 10/31/2022] [Indexed: 11/17/2022] Open
Abstract
ObjectivesHuman mitochondrial cell-free DNA (Mt-cfDNA) may serve as a useful biomarker for infectious processes. We investigated Mt-cfDNA dynamics in patients with pulmonary mycobacterial infections to determine if this novel biomarker could be used to differentiate disease states and severity.MethodsPatients with pulmonary tuberculosis (PTB), latent tuberculosis infection (LTBI), and nontuberculous mycobacterial-lung disease (NTM-LD) were enrolled at a tertiary care hospital in Taiwan between June 2018 and August 2021. Human Mt-cfDNA and nuclear-cfDNA (Nu-cfDNA) copy numbers were estimated by quantitative polymerase chain reaction. Variables associated with PTB and 2-month sputum culture-positivity, indicating poor treatment response, were assessed using logistic regression.ResultsAmong 97 patients with PTB, 64 with LTBI, and 51 with NTM-LD, Mt-cfDNA levels were higher in patients with PTB than in LTBI (p=0.001) or NTM-LD (p=0.006). In the Mycobacterium tuberculosis-infected population, Mt-cfDNA levels were highest in smear-positive PTB patients, followed by smear-negative PTB (p<0.001), and were lowest in LTBI persons (p=0.009). A Mt-cfDNA, but not Nu-cfDNA, level higher than the median helped differentiate culture-positive PTB from culture-negative PTB and LTBI (adjusted OR 2.430 [95% CI 1.139–5.186], p=0.022) and differentiate PTB from NTM-LD (adjusted OR 4.007 [1.382–12.031], p=0.011). Mt-cfDNA levels decreased after 2 months of treatment in PTB patients (p=0.010). A cutoff Mt-cfDNA level greater than 62.62 x 106 copies/μL-plasma was associated with a 10-fold risk of 2-month culture-positivity (adjusted OR 9.691 [1.046–89.813], p=0.046).ConclusionElevated Mt-cfDNA levels were associated with PTB disease and failed sputum conversion at 2 months in PTB patients, and decreased after treatment.
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Affiliation(s)
- Sheng-Wei Pan
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California San Diego, La Jolla, CA, United States
| | - Rehan R. Syed
- Division of Infectious Diseases and Global Public Health, University of California San Diego, La Jolla, CA, United States
| | - Donald G. Catanzaro
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, United States
| | - Mei-Lin Ho
- Department of Chemistry, Soochow University, Taipei, Taiwan
- Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, United States
| | - Chin-Chung Shu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Tsung-Yeh Tsai
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yen-Han Tseng
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Jia-Yih Feng
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- *Correspondence: Jia-Yih Feng,
| | - Yuh-Min Chen
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Wei-Juin Su
- Division of Chest Medicine, China Medical University Hospital, Taipei Branch, Taipei, Taiwan
| | - Antonino Catanzaro
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California San Diego, La Jolla, CA, United States
| | - Timothy C. Rodwell
- Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California San Diego, La Jolla, CA, United States
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30
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Chang A, Mzava O, Djomnang LAK, Lenz JS, Burnham P, Kaplinsky P, Andama A, Connelly J, Bachman CM, Cattamanchi A, Steadman A, De Vlaminck I. Metagenomic DNA sequencing to quantify Mycobacterium tuberculosis DNA and diagnose tuberculosis. Sci Rep 2022; 12:16972. [PMID: 36216964 PMCID: PMC9551046 DOI: 10.1038/s41598-022-21244-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Accepted: 09/26/2022] [Indexed: 12/29/2022] Open
Abstract
Tuberculosis (TB) remains a significant cause of mortality worldwide. Metagenomic next-generation sequencing has the potential to reveal biomarkers of active disease, identify coinfection, and improve detection for sputum-scarce or culture-negative cases. We conducted a large-scale comparative study of 428 plasma, urine, and oral swab samples from 334 individuals from TB endemic and non-endemic regions to evaluate the utility of a shotgun metagenomic DNA sequencing assay for tuberculosis diagnosis. We found that the composition of the control population had a strong impact on the measured performance of the diagnostic test: the use of a control population composed of individuals from a TB non-endemic region led to a test with nearly 100% specificity and sensitivity, whereas a control group composed of individuals from TB endemic regions exhibited a high background of nontuberculous mycobacterial DNA, limiting the diagnostic performance of the test. Using mathematical modeling and quantitative comparisons to matched qPCR data, we found that the burden of Mycobacterium tuberculosis DNA constitutes a very small fraction (0.04 or less) of the total abundance of DNA originating from mycobacteria in samples from TB endemic regions. Our findings suggest that the utility of a minimally invasive metagenomic sequencing assay for pulmonary tuberculosis diagnostics is limited by the low burden of M. tuberculosis and an overwhelming biological background of nontuberculous mycobacterial DNA.
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Affiliation(s)
- Adrienne Chang
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Omary Mzava
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Liz-Audrey Kounatse Djomnang
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Joan Sesing Lenz
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Philip Burnham
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Peter Kaplinsky
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
| | - Alfred Andama
- grid.11194.3c0000 0004 0620 0548Department of Internal Medicine, Makerere University College of Health Sciences, Kampala, Uganda
| | | | | | - Adithya Cattamanchi
- grid.266102.10000 0001 2297 6811Center for Tuberculosis and Division of Pulmonary and Critical Care Medicine, University of California San Francisco, San Francisco, CA USA
| | | | - Iwijn De Vlaminck
- grid.5386.8000000041936877XNancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY USA
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Hong D, Wang P, Zhang J, Li K, Ye B, Li G, Zhou J, Tong Z, Ke L, Shi S, Li W. Plasma metagenomic next-generation sequencing of microbial cell-free DNA detects pathogens in patients with suspected infected pancreatic necrosis. BMC Infect Dis 2022; 22:675. [PMID: 35931956 PMCID: PMC9356476 DOI: 10.1186/s12879-022-07662-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Accepted: 07/30/2022] [Indexed: 11/17/2022] Open
Abstract
Background Infected pancreatic necrosis (IPN) is a life-threatening complication of acute pancreatitis (AP). Timely diagnosis of IPN could facilitate appropriate treatment, but there is a lack of reliable non-invasive screening tests. In this study, we aimed to evaluate the diagnostic value of plasma metagenomic next-generation sequencing (mNGS) based on circulating microbial cell-free DNA in patients with suspected IPN. Methods From October 2020 to October 2021, 44 suspected IPN patients who underwent plasma mNGS were reviewed. Confirmatory diagnosis of IPN within two weeks after the index blood sampling was considered the reference standard. The confirmation of IPN relied on the microbiological results of drains obtained from the necrotic collections. The distribution of the pathogens identified by plasma mNGS was analyzed. Positive percent agreement (PPA) and negative percent agreement (NPA) were evaluated based on the conformity between the overall mNGS results and culture results of IPN drains. In addition, the clinical outcomes were compared between mNGS positive and negative patients. Results Across all the study samples, thirteen species of bacteria and five species of fungi were detected by mNGS. The positivity rate of plasma mNGS was 54.55% (24/44). Of the 24 mNGS positive cases, twenty (83.33%, 95% CI, 68.42–98.24%) were consistent with the culture results of IPN drains. The PPA and NPA of plasma mNGS for IPN were 80.0% (20/25; 95% CI, 64.32–95.68%) and 89.47% (17/19; 95% CI, 75.67–100%), respectively. Compared with the mNGS negative group, patients in the positive group had more new-onset septic shock [12 (50.0%) vs. 4 (20.0%), p = 0.039]. Conclusion IPN relevant pathogens can be identified by plasma mNGS, potentially facilitating appropriate treatment. The clinical application of mNGS in this cohort appears feasible. Supplementary Information The online version contains supplementary material available at 10.1186/s12879-022-07662-2.
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Affiliation(s)
- Donghuang Hong
- The First School of Clinical Medicine, Southern Medical University, Guangzhou, China.,Department of Critical Care Medicine, Fujian Provincial Hospital, No.134 East Street, Fuzhou, 350001, Fujian, China
| | - Peng Wang
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Jingzhu Zhang
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Kaiwei Li
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Bo Ye
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Gang Li
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Jing Zhou
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Zhihui Tong
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China
| | - Lu Ke
- Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China.,National Institute of Healthcare Data Science, Nanjing University, Nanjing, China
| | - Songjing Shi
- Department of Critical Care Medicine, Fujian Provincial Hospital, No.134 East Street, Fuzhou, 350001, Fujian, China.
| | - Weiqin Li
- The First School of Clinical Medicine, Southern Medical University, Guangzhou, China. .,Center of Severe Acute Pancreatitis (CSAP), Department of Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, No. 305 Zhongshan East Road, Nanjing, 210002, Jiangsu, China. .,National Institute of Healthcare Data Science, Nanjing University, Nanjing, China.
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Huang Z, LaCourse SM, Kay AW, Stern J, Escudero JN, Youngquist BM, Zheng W, Vambe D, Dlamini M, Mtetwa G, Cranmer LM, Njuguna I, Wamalwa DC, Maleche-Obimbo E, Catanzaro DG, Lyon CJ, John-Stewart G, DiNardo A, Mandalakas AM, Ning B, Hu TY. CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study. THE LANCET. MICROBE 2022; 3:e482-e492. [PMID: 35659882 PMCID: PMC9300929 DOI: 10.1016/s2666-5247(22)00087-8] [Citation(s) in RCA: 47] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 03/17/2022] [Accepted: 03/23/2022] [Indexed: 11/28/2022]
Abstract
BACKGROUND Tuberculosis remains a leading cause of global mortality, especially for adults and children living with HIV (CLHIV) underdiagnosed by sputum-based assays. Non-sputum-based assays are needed to improve tuberculosis diagnosis and tuberculosis treatment monitoring. Our aim in this study was to determine whether ultrasensitive detection of Mycobacterium tuberculosis cell-free DNA (Mtb-cfDNA) in blood can diagnose tuberculosis and evaluate tuberculosis treatment responses. METHODS In this molecular diagnostics study we analysed archived serum from two patient populations evaluated for tuberculosis in Eswatini and Kenya to detect Mtb-cfDNA, analysing serum from all individuals who had both sufficient serum volumes and clear diagnostic results. An optimised CRISPR-mediated tuberculosis (CRISPR-TB) assay was used to detect Mtb-cfDNA in serum at enrolment from adults and children with presumptive tuberculosis and their asymptomatic household contacts, and at enrolment and during tuberculosis treatment from a cohort of symptomatic CLHIV at high risk for tuberculosis, who provided longitudinal serum at enrolment and during tuberculosis treatment. FINDINGS CRISPR-TB identified microbiologically and clinically confirmed tuberculosis cases in the predominantly HIV-negative Eswatini adult cohort with 96% sensitivity (27 [96%] of 28, 95% CI 80-100) and 94% specificity (16 [94%] of 17, 71-100), and with 83% sensitivity (5 [83%] of 6, 36-100) and 95% specificity (21 [95%] of 22, 77-100) in the paediatric cohort, including all six cases of extrapulmonary tuberculosis. In the Kenyan CLHIV cohort, CRISPR-TB detected all (13 [100%] of 13, 75-100) confirmed tuberculosis cases and 85% (39 [85%] of 46, 71-94) of unconfirmed tuberculosis cases diagnosed by non-microbiological clinical findings. CLHIV who were CRISPR-TB positive at enrolment had a 2·4-times higher risk of mortality by 6 months after enrolment. Mtb-cfDNA signal decreased after tuberculosis treatment initiation, with near or complete Mtb-cfDNA clearance by 6 months after tuberculosis treatment initiation. INTERPRETATION CRISPR-mediated detection of circulating Mtb-cfDNA shows promise to increase the identification of paediatric tuberculosis and HIV-associated tuberculosis, and potential for early diagnosis and rapid monitoring of tuberculosis treatment responses. FUNDING US Department of Defense, National Institute of Child Health and Human Development, National Institute of Allergy and Infectious Diseases, University of Washington Center for AIDS Research, and the Weatherhead Presidential Endowment fund.
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Affiliation(s)
- Zhen Huang
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA; State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi, China
| | - Sylvia M LaCourse
- Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA
| | - Alexander W Kay
- Global TB Program, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Baylor Children's Foundation-Eswatini, Mbabane, Eswatini
| | - Joshua Stern
- Department of Global Health, University of Washington, Seattle, WA, USA
| | - Jaclyn N Escudero
- Department of Global Health, University of Washington, Seattle, WA, USA
| | - Brady M Youngquist
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA
| | - Wenshu Zheng
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA
| | - Debrah Vambe
- Eswatini National Tuberculosis Control Programme, Ministry of Health, Manzini, Eswatini
| | - Muyalo Dlamini
- National TB Reference Laboratory, Eswatini Health Laboratory Services, Ministry of Health, Mbabane, Eswatini
| | - Godwin Mtetwa
- Global TB Program, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA; Baylor Children's Foundation-Eswatini, Mbabane, Eswatini
| | - Lisa M Cranmer
- Department of Pediatrics, Division of Infectious Diseases, Emory University, Atlanta, GA, USA; Children's Healthcare of Atlanta, Atlanta, GA, USA; Department of Epidemiology, Emory Rollins School of Public Health, Atlanta, GA, USA
| | - Irene Njuguna
- Department of Global Health, University of Washington, Seattle, WA, USA; Research and Programmes, Kenyatta National Hospital, Nairobi, Kenya
| | - Dalton C Wamalwa
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Paediatrics and Child Health, University of Nairobi, Nairobi, Kenya
| | - Elizabeth Maleche-Obimbo
- Department of Global Health, University of Washington, Seattle, WA, USA; Department of Paediatrics and Child Health, University of Nairobi, Nairobi, Kenya
| | - Donald G Catanzaro
- Department of Biological Sciences, University of Arkansas, Fayetteville, AR, USA
| | - Christopher J Lyon
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA
| | - Grace John-Stewart
- Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA; Department of Global Health, University of Washington, Seattle, WA, USA; Department of Epidemiology, University of Washington, Seattle, WA, USA; Department of Pediatrics, University of Washington, Seattle, WA, USA
| | - Andrew DiNardo
- Global TB Program, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
| | - Anna M Mandalakas
- Global TB Program, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA
| | - Bo Ning
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA
| | - Tony Y Hu
- Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, New Orleans, LA, USA; Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA, USA.
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Abdulgader SM, Okunola AO, Ndlangalavu G, Reeve BW, Allwood BW, Koegelenberg CF, Warren RM, Theron G. Diagnosing Tuberculosis: What Do New Technologies Allow Us to (Not) Do? Respiration 2022; 101:797-813. [PMID: 35760050 PMCID: PMC9533455 DOI: 10.1159/000525142] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Accepted: 05/10/2022] [Indexed: 12/11/2022] Open
Abstract
New tuberculosis (TB) diagnostics are at a crossroads: their development, evaluation, and implementation is severely damaged by resource diversion due to COVID-19. Yet several technologies, especially those with potential for non-invasive non-sputum-based testing, hold promise for efficiently triaging and rapidly confirming TB near point-of-care. Such tests are, however, progressing through the pipeline slowly and will take years to reach patients and health workers. Compellingly, such tests will create new opportunities for difficult-to-diagnose populations, including primary care attendees (all-comers in high burden settings irrespective of reason for presentation) and community members (with early stage disease or risk factors like HIV), many of whom cannot easily produce sputum. Critically, all upcoming technologies have limitations that implementers and health workers need to be cognizant of to ensure optimal deployment without undermining confidence in a technology that still offers improvements over the status quo. In this state-of-the-art review, we critically appraise such technologies for active pulmonary TB diagnosis. We highlight strengths, limitations, outstanding research questions, and how current and future tests could be used in the presence of these limitations and uncertainties. Among triage tests, CRP (for which commercial near point-of-care devices exist) and computer-aided detection software with digital chest X-ray hold promise, together with late-stage blood-based assays that detect host and/or microbial biomarkers; however, aside from a handful of prototypes, the latter category has a shortage of promising late-stage alternatives. Furthermore, positive results from new triage tests may have utility in people without TB; however, their utility for informing diagnostic pathways for other diseases is under-researched (most sick people tested for TB do not have TB). For confirmatory tests, few true point-of-care options will be available soon; however, combining novel approaches like tongue swabs with established tests like Ultra have short-term promise but first require optimizations to specimen collection and processing procedures. Concerningly, no technologies yet have compelling evidence of meeting the World Health Organization optimal target product profile performance criteria, especially for important operational criteria crucial for field deployment. This is alarming as the target product profile criteria are themselves almost a decade old and require urgent revision, especially to cater for technologies made prominent by the COVID-19 diagnostic response (e.g., at-home testing and connectivity solutions). Throughout the review, we underscore the importance of how target populations and settings affect test performance and how the criteria by which these tests should be judged vary by use case, including in active case finding. Lastly, we advocate for health workers and researchers to themselves be vocal proponents of the uptake of both new tests and those - already available tests that remain suboptimally utilized.
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Affiliation(s)
- Shima M. Abdulgader
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Anna O. Okunola
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Gcobisa Ndlangalavu
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Byron W.P. Reeve
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Brian W. Allwood
- Division of Pulmonology, Department of Medicine, Tygerberg Hospital, Stellenbosch University, Cape Town, South Africa
| | - Coenraad F.N. Koegelenberg
- Division of Pulmonology, Department of Medicine, Tygerberg Hospital, Stellenbosch University, Cape Town, South Africa
| | - Rob M. Warren
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
| | - Grant Theron
- DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
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Khimova E, Gonzalo X, Popova Y, Eliseev P, Andrey M, Nikolayevskyy V, Broda A, Drobniewski F. Urine biomarkers of pulmonary tuberculosis. Expert Rev Respir Med 2022; 16:615-621. [PMID: 35702997 DOI: 10.1080/17476348.2022.2090341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Abstract
INTRODUCTION Sputum-based tuberculosis diagnosis does not address the needs of certain categories of patients. Active development of a noninvasive urine-based diagnosis could provide an alternative approach. We reviewed publications covering more than 30 urine biomarkers proposed as significant for TB diagnosis. Analytical approaches were heterogeneous in design and methods; few studies on diagnostic outcome prediction described a formal specificity and sensitivity analysis. AREAS COVERED This review describes studies of non-sputum diagnostic approaches of pulmonary TB based on urine using specific TB biomarkers. The search was performed until December 2021, using terms [Tuberculosis] + [urine] + [biomarkers] in PubMed and Cochrane databases. Publications concerning LAM urine diagnostics were excluded as they have been described elsewhere. EXPERT OPINION Microbiological culture of sputum is considered to be the 'gold standard' diagnostic for pulmonary TB but the methodology is slow due to the slow growth of the TB bacteria. Urine provides a large volume of sample. Investigators have evaluated urine for either TB pathogen biomarkers or host biomarkers with some success as the review demonstrates. Detection sensitivity remains a significant problem. In future, combination of host and pathogen biomarkers could increase the sensitivity and specificity of TB diagnosis.
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Affiliation(s)
- Elena Khimova
- Department of Phthisiopulmonology, Northern State Medical University, Arkhangelsk, Russia
| | - Ximena Gonzalo
- Department of Infectious Diseases, Imperial College London, London, UK
| | - Yulia Popova
- Department of Phthisiopulmonology, Northern State Medical University, Arkhangelsk, Russia
| | - Platon Eliseev
- Department of Phthisiopulmonology, Northern State Medical University, Arkhangelsk, Russia
| | - Maryandyshev Andrey
- Department of Phthisiopulmonology, Northern State Medical University, Arkhangelsk, Russia
| | | | - Agnieszka Broda
- Department of Infectious Diseases, Imperial College London, London, UK
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Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study. Tuberculosis (Edinb) 2022; 135:102213. [DOI: 10.1016/j.tube.2022.102213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2021] [Revised: 05/21/2022] [Accepted: 05/22/2022] [Indexed: 11/24/2022]
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Accuracy of Pneumocystis jirovecii Plasma Cell-Free DNA PCR for Noninvasive Diagnosis of Pneumocystis Pneumonia. J Clin Microbiol 2022; 60:e0010122. [PMID: 35387472 DOI: 10.1128/jcm.00101-22] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii is a serious infection in immunocompromised hosts which requires prompt diagnosis and treatment. The recommended specimen for diagnosis of PCP is bronchoalveolar lavage (BAL) fluid, which is invasive and may not be possible in unstable patients. The aim of this study was to evaluate the accuracy of noninvasive P. jirovecii plasma cell-free DNA (cfDNA) PCR using recently optimized preanalytical and analytical methods. Adult patients undergoing clinical testing for PCP with direct fluorescent antibody stain (DFA), respiratory PCR, and/or β-d-glucan were included in this study. Sensitivity and specificity P. jirovecii plasma cfDNA PCR was determined in PCP suspects categorized as proven and probable. A total of 149 patients were included in this study, of which 10 had proven and 27 had probable PCP. Most patients (95.9%, 143/149) were immunocompromised, including hematological malignancies (30.1%), bone marrow transplant (11.2%), solid organ transplantation (47.6%), and HIV/AIDS (4.2%). P. jirovecii plasma cfDNA PCR showed sensitivity and specificity of 100% (10/10; 95% confidence interval [CI], 69.2 to 100) and 93.4% (127/136; 95% CI, 87.8 to 96.9), and 48.6% (18/37; 95% CI, 31.9 to 65.6) and 99.1% (108/109; 95% CI, 94.9 to 100) in proven and proven/probable cases, respectively. P. jirovecii cell-free DNA PCR was similar in sensitivity but with substantially improved specificity over β-d-glucan (sensitivity, 60.0% [18/30; 95% CI, 40.6 to 77.3]); specificity, 66.7% [22/33; 95% CI, 48.2 to 82.0]) in patients with proven/probable PCP. Plasma cfDNA PCR offers a noninvasive testing option for early and accurate diagnosis of PCP, particularly in patients who cannot tolerate bronchoscopy.
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Mor P, Dahiya B, Parshad S, Gulati P, Mehta PK. Recent updates in diagnosis of abdominal tuberculosis with emphasis on nucleic acid amplification tests. Expert Rev Gastroenterol Hepatol 2022; 16:33-49. [PMID: 34923892 DOI: 10.1080/17474124.2022.2021068] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
INTRODUCTION Abdominal tuberculosis (TB) is a common epitome of extrapulmonary TB (EPTB), wherein peritoneal and intestinal TB are the most prevalent forms. Diagnosis of abdominal TB is a daunting challenge owing to variable anatomical locations, paucibacillary nature of specimens and atypical clinical presentations that mimic other abdominal diseases, such as Crohn's disease and malignancies. In this review, we made a comprehensive study on the diagnosis of abdominal TB. AREA COVERED Various modalities employed for abdominal TB diagnosis include clinical features, imaging, bacteriological tests (smear/culture), histopathological/cytological observations, interferon-gamma release assays and nucleic acid amplification tests (NAATs). Among NAATs, loop-mediated isothermal amplification assay, PCR, multiplex-PCR, nested PCR, real-time PCR and GeneXpert® MTB/RIF were discussed. Identification of circulating Mycobacterium tuberculosis cell-free DNA by real-time PCR within ascitic fluids is another useful approach. EXPERT OPINION Several novel molecular/immunological methods, such as GeneXpert Ultra, aptamer-linked immobilized sorbent assay, immuno-PCR (I-PCR) and nanoparticle-based I-PCR have recently been developed for detecting pulmonary TB and several EPTB types, which may also be explored for abdominal TB diagnosis. Precise and prompt diagnosis of abdominal TB may initiate an early therapy so as to reduce the complications, i.e. abdominal pain, ascites, abdominal distension, intestinal obstruction/perforation, etc., and avoid surgical involvement.Plain Language SummaryAbdominal tuberculosis (TB) is a manifestation of extrapulmonary TB (EPTB), where peritoneal and intestinal TB are two major forms. Diagnosis of abdominal TB is difficult owing to low bacterial load present in clinical samples and non-specific clinical presentations as it mimics other diseases such as inflammatory bowel diseases, abdominal malignancies, etc. Bacteriological tests (smear/culture) almost fail owing to poor sensitivities and it is not always possible to get representative tissue samples for histopathological and cytological observations. In recent years, molecular tests i.e. nucleic acid amplification tests (NAATs), such as PCR/multiplex-PCR (M-PCR), nested PCR and GeneXpert are widely employed. Markedly, PCR/M-PCR and nested PCR exhibited reasonable good sensitivities/specificities, while GeneXpert revealed low sensitivity in most of the studies but high specificity, thus it could assist in differential diagnosis of intestinal TB and Crohn's disease. Further, novel molecular/immunological tests employed for pulmonary TB and other EPTB types were described and those tests can also be utilized to diagnose abdominal TB. Reliable and rapid diagnosis of abdominal TB would initiate an early start of anti-tubercular therapy and reduce the severe complications.
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Affiliation(s)
- Preeti Mor
- Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
| | - Bhawna Dahiya
- Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
| | - Sanjeev Parshad
- Department of General Surgery, Pt. B.D. Sharma University of Health Sciences, Rohtak, India
| | - Pooja Gulati
- Department of Microbiology, Maharshi Dayanand University, Rohtak, India
| | - Promod K Mehta
- Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
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Dixon RV, Skaria E, Lau WM, Manning P, Birch-Machin MA, Moghimi SM, Ng KW. Microneedle-based devices for point-of-care infectious disease diagnostics. Acta Pharm Sin B 2021; 11:2344-2361. [PMID: 34150486 PMCID: PMC8206489 DOI: 10.1016/j.apsb.2021.02.010] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 11/25/2020] [Accepted: 01/28/2021] [Indexed: 02/08/2023] Open
Abstract
Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.
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Key Words
- AC, alternating current
- APCs, antigen-presenting cells
- ASSURED, affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users
- Biomarker detection
- Biosensor
- CMOS, complementary metal-oxide semiconductor
- COVID, coronavirus disease
- COVID-19
- CSF, cerebrospinal fluid
- CT, computerised tomography
- CV, cyclic voltammetry
- DC, direct current
- DNA, deoxyribonucleic acid
- DPV, differential pulse voltammetry
- EBV, Epstein–Barr virus
- EDC/NHS, 1-ethyl-3-(3-dimethylaminoproply) carbodiimide/N-hydroxysuccinimide
- ELISA, enzyme-linked immunosorbent assay
- GOx, glucose oxidase
- HIV, human immunodeficiency virus
- HPLC, high performance liquid chromatography
- HRP, horseradish peroxidase
- IP, iontophoresis
- ISF, interstitial fluid
- IgG, immunoglobulin G
- Infectious disease
- JEV, Japanese encephalitis virus
- MN, microneedle
- Microneedle
- NA, nucleic acid
- OBMT, one-touch-activated blood multidiagnostic tool
- OPD, o-phenylenediamine
- PCB, printed circuit board
- PCR, polymerase chain reaction
- PDMS, polydimethylsiloxane
- PEDOT, poly(3,4-ethylenedioxythiophene)
- PNA, peptide nucleic acid
- PP, polyphenol
- PPD, poly(o-phenylenediamine)
- PoC, point-of-care
- Point-of-care diagnostics (PoC)
- SALT, skin-associated lymphoid tissue
- SAM, self-assembled monolayer
- SEM, scanning electron microscope
- SERS, surface-enhanced Raman spectroscopy
- SWV, square wave voltammetry
- Skin
- TB, tuberculosis
- UV, ultraviolet
- VEGF, vascular endothelial growth factor
- WHO, World Health Organisation
- cfDNA, cell-free deoxyribonucleic acid
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Affiliation(s)
- Rachael V. Dixon
- School of Pharmacy, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
| | - Eldhose Skaria
- Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK
| | - Wing Man Lau
- School of Pharmacy, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
| | - Philip Manning
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
| | - Mark A. Birch-Machin
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
| | - S. Moein Moghimi
- School of Pharmacy, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
| | - Keng Wooi Ng
- School of Pharmacy, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
- Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK
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39
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Progress toward Developing Sensitive Non-Sputum-Based Tuberculosis Diagnostic Tests: the Promise of Urine Cell-Free DNA. J Clin Microbiol 2021; 59:e0070621. [PMID: 33980646 DOI: 10.1128/jcm.00706-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A highly accurate, non-sputum-based test for tuberculosis (TB) detection is a key priority for the field of TB diagnostics. A recent study in the Journal of Clinical Microbiology by Oreskovic and colleagues (J Clin Microbiol 59:e00074-21, 2021, https://doi.org/10.1128/JCM.00074-21) reports the performance of an optimized urine cell-free DNA (cfDNA) test using sequence-specific purification combined with short-target PCR to improve the accuracy of TB detection. Their retrospective clinical study utilized frozen urine samples (n = 73) from study participants diagnosed with active pulmonary TB in South Africa and compared results to non-TB patients in South Africa and the United States in an early-phase validation study. Overall, this cfDNA technique detected TB with a sensitivity of 83.7% (95% CI: 71.0 to 91.5) and specificity of 100% (95% CI: 86.2 to 100), which meet the World Health Organization's published performance criteria. Sensitivity was 73.3% in people without HIV (95% CI: 48.1 to 89.1) and 76% in people with smear-negative TB (95% CI: 56.5 to 88.5). In this commentary, we discuss the results of this optimized urine TB cfDNA assay within the larger context of TB diagnostics and pose additional questions for further research.
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Oreskovic A, Panpradist N, Marangu D, Ngwane MW, Magcaba ZP, Ngcobo S, Ngcobo Z, Horne DJ, Wilson DPK, Shapiro AE, Drain PK, Lutz BR. Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA. J Clin Microbiol 2021; 59:e0007421. [PMID: 33789959 PMCID: PMC8373247 DOI: 10.1128/jcm.00074-21] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 03/19/2021] [Indexed: 01/17/2023] Open
Abstract
Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (<100 bp) and low concentration of TB-specific fragments. We aimed to improve the diagnostic sensitivity of TB urine cfDNA by increasing recovery of short fragments during sample preparation. We developed a highly sensitive sequence-specific purification method that uses hybridization probes immobilized on magnetic beads to capture short TB cfDNA (50 bp) with 91.8% average efficiency. Combined with short-target PCR, the assay limit of detection was ≤5 copies of cfDNA in 10 ml urine. In a clinical cohort study in South Africa, our urine cfDNA assay had 83.7% sensitivity (95% CI: 71.0 to 91.5%) and 100% specificity (95% CI: 86.2 to 100%) for diagnosis of active pulmonary TB when using sputum Xpert MTB/RIF as the reference standard. The detected cfDNA concentration was 0.14 to 2,804 copies/ml (median 14.6 copies/ml) and was inversely correlated with CD4 count and days to culture positivity. Sensitivity was nonsignificantly higher in HIV-positive (88.2%) compared to HIV-negative patients (73.3%), and was not dependent on CD4 count. Sensitivity remained high in sputum smear-negative (76.0%) and urine lipoarabinomannan (LAM)-negative (76.5%) patients. With improved sample preparation, urine cfDNA is a viable biomarker for TB diagnosis. Our assay has the highest reported accuracy of any TB urine cfDNA test to date and has the potential to enable rapid non-sputum-based TB diagnosis across key underserved patient populations.
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Affiliation(s)
- Amy Oreskovic
- Department of Bioengineering, University of Washington, Seattle, Washington, USA
| | - Nuttada Panpradist
- Department of Bioengineering, University of Washington, Seattle, Washington, USA
| | - Diana Marangu
- Department of Paediatrics and Child Health, University of Nairobi, Nairobi, Kenya
| | - M. William Ngwane
- Umkhuseli Innovation and Research Management, Pietermaritzburg, South Africa
| | - Zanele P. Magcaba
- Umkhuseli Innovation and Research Management, Pietermaritzburg, South Africa
| | - Sindiswa Ngcobo
- Umkhuseli Innovation and Research Management, Pietermaritzburg, South Africa
| | - Zinhle Ngcobo
- Umkhuseli Innovation and Research Management, Pietermaritzburg, South Africa
| | - David J. Horne
- Department of Medicine, University of Washington, Seattle, Washington, USA
- Department of Global Health, University of Washington, Seattle, Washington, USA
| | - Douglas P. K. Wilson
- Umkhuseli Innovation and Research Management, Pietermaritzburg, South Africa
- Edendale Hospital, University of KwaZulu-Natal, Pietermaritzburg, South Africa
| | - Adrienne E. Shapiro
- Department of Medicine, University of Washington, Seattle, Washington, USA
- Department of Global Health, University of Washington, Seattle, Washington, USA
| | - Paul K. Drain
- Department of Medicine, University of Washington, Seattle, Washington, USA
- Department of Global Health, University of Washington, Seattle, Washington, USA
| | - Barry R. Lutz
- Department of Bioengineering, University of Washington, Seattle, Washington, USA
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Pan SW, Su WJ, Chan YJ, Chuang FY, Feng JY, Chen YM. Mycobacterium tuberculosis-derived circulating cell-free DNA in patients with pulmonary tuberculosis and persons with latent tuberculosis infection. PLoS One 2021; 16:e0253879. [PMID: 34166477 PMCID: PMC8224927 DOI: 10.1371/journal.pone.0253879] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Accepted: 06/14/2021] [Indexed: 01/15/2023] Open
Abstract
Objectives The timely diagnosis of pulmonary tuberculosis (PTB) is challenging. Although pathogen-derived circulating cell-free DNA (cfDNA) has been detected in humans, the significance of Mycobacterium tuberculosis (MTB)-cfDNA detection in patients with PTB remains unclear. Methods This study enrolled patients with PTB and persons with latent tuberculosis infection (LTBI) as the study and control groups, respectively, from 2018 to 2020. We measured interferon-γ levels and calculated blood monocyte-to-lymphocyte ratio (MLR). We conducted plasma cfDNA extraction, quantitative polymerase chain reaction (qPCR), and droplet digital PCR targeting the IS6110 gene of MTB. We calculated the sensitivity and specificity of using MTB-cfDNA to identify PTB and analyzed the factors associated with PTB diagnosis and MTB-cfDNA positivity. Results We enrolled 24 patients with PTB and 57 LTBI controls. The sensitivity of using MTB-cfDNA to identify PTB was 54.2%(13/24) in total and 46.2%(6/13) in smear-negative cases. Two LTBI controls (3.5%) tested positive for MTB-cfDNA, indicating a specificity of 96.5%(55/57). By using MTB-cfDNA positivity and an MLR ≥0.42 to identify PTB, sensitivity increased to 79.2%(19/24). Among patients with PTB, MTB-specific interferon-γ levels were higher in MTB-cfDNA positive participants than in those who tested negative (7.0 ±2.7 vs 2.7±3.0 IU/mL, p<0.001). MTB-cfDNA levels declined after 2 months of anti-tuberculosis therapy (p<0.001). Conclusion The sensitivity of using MTB-cfDNA to identify PTB in participants was 54.2%, which increased to 79.2% after incorporating an MLR ≥0.42 into the analysis. MTB-cfDNA positivity was associated with MTB-specific immune response, and MTB-cfDNA levels declined after treatment. The clinical value of MTB-cfDNA in PTB management necessitates further investigation.
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Affiliation(s)
- Sheng-Wei Pan
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Institute of Public Health, National Yang Ming Chiao Tung University, Taipei, Taiwan
- * E-mail: (JYF); (SWP)
| | - Wei-Juin Su
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yu-Jiun Chan
- Institute of Public Health, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- Division of Microbiology, Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
| | - Fan-Yi Chuang
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Jia-Yih Feng
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- * E-mail: (JYF); (SWP)
| | - Yuh-Min Chen
- Department of Chest Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
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Hiza H, Hella J, Arbués A, Magani B, Sasamalo M, Gagneux S, Reither K, Portevin D. Case-control diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood. Sci Rep 2021; 11:13190. [PMID: 34162973 PMCID: PMC8222251 DOI: 10.1038/s41598-021-92596-z] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Accepted: 06/07/2021] [Indexed: 11/09/2022] Open
Abstract
CD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24 h turnaround time. We recruited 479 GeneXpert positive cases and 108 symptomatic but GeneXpert negative controls from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing IFN-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus. Significantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84-0.91). The assay performance was not significantly affected by HIV status. To conclude, we successfully implemented TAM-TB immunoassay routine testing with a 24 h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test.
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Affiliation(s)
- Hellen Hiza
- Ifakara Health Institute, Bagamoyo, Tanzania.,Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Jerry Hella
- Ifakara Health Institute, Bagamoyo, Tanzania.,Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Ainhoa Arbués
- Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Beatrice Magani
- Ifakara Health Institute, Bagamoyo, Tanzania.,Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Mohamed Sasamalo
- Ifakara Health Institute, Bagamoyo, Tanzania.,Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Sebastien Gagneux
- Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Klaus Reither
- Swiss Tropical and Public Health Institute, Basel, Switzerland.,University of Basel, Basel, Switzerland
| | - Damien Portevin
- Swiss Tropical and Public Health Institute, Basel, Switzerland. .,University of Basel, Basel, Switzerland.
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Pollock NR, MacIntyre AT, Blauwkamp TA, Blair L, Ho C, Calderon R, Franke MF. Detection of Mycobacterium tuberculosis cell-free DNA to diagnose TB in pediatric and adult patients. Int J Tuberc Lung Dis 2021; 25:403-405. [PMID: 33977910 DOI: 10.5588/ijtld.21.0055] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Affiliation(s)
- N R Pollock
- Department of Laboratory Medicine, Boston Children´s Hospital and Harvard Medical School, Boston, MA
| | | | | | - L Blair
- Karius Inc, Redwood City, CA, USA
| | - C Ho
- Karius Inc, Redwood City, CA, USA
| | - R Calderon
- Socios En Salud Sucursal Peru, Lima, Peru, Programa Acadêmico de Tuberculose, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, RJ, Brazil
| | - M F Franke
- Department of Global Health and Social Medicine, Harvard Medical School, Boston, MA, USA
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44
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Kamra E, Mehta PK. Current updates in diagnosis of male urogenital tuberculosis. Expert Rev Anti Infect Ther 2021; 19:1175-1190. [PMID: 33688791 DOI: 10.1080/14787210.2021.1902305] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Introduction: Urogenital tuberculosis (UGTB) is a common manifestation of extrapulmonary TB (EPTB), which affects both men and women in a ratio of 2:1. Similar to other EPTB types, diagnosis of UGTB is quite challenging owing to atypical clinical presentation and paucibacillary nature of specimens. This review is primarily focused on the current updates developed in the diagnosis of male UGTB.Area covered: Smear/culture, imaging, histopathology, and interferon-γ release assays are the main modalities employed for detecting male UGTB cases. Moreover, we described the utility of nucleic acid amplification tests (NAATs), including loop-mediated isothermal amplification, PCR, nested-PCR, and GeneXpert (MTB/RIF) assays. The possibility of using other novel modalities, such as immuno-PCR (I-PCR), aptamer-linked immobilized sorbent assay (ALISA), and identification of circulating cell-free DNA (cfDNA) by NAATs were also discussed.Expert opinion: The current methods used for the diagnosis of male UGTB are not adequate. Therefore, the latest molecular/immunological tools, i.e. Xpert Ultra, Truenat MTBTM, I-PCR, ALISA, and cfDNA detection employed for the diagnosis of other EPTB forms and pulmonary TB may also be exploited for UGTB diagnosis. Reliable and timely diagnosis of male UGTB may initiate an early start of anti-tubercular therapy that would reduce infertility and other complications associated with disease.
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Affiliation(s)
- Ekta Kamra
- Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
| | - Promod K Mehta
- Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
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45
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Papaiakovou M, Littlewood DTJ, Gasser RB, Anderson RM. How qPCR complements the WHO roadmap (2021-2030) for soil-transmitted helminths. Trends Parasitol 2021; 37:698-708. [PMID: 33931342 DOI: 10.1016/j.pt.2021.04.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 04/02/2021] [Accepted: 04/08/2021] [Indexed: 12/19/2022]
Abstract
Complementing the launch of the World Health Organization (WHO) roadmap (2021-2030) we explore key elements needing attention before recruitment of qPCR as the main diagnostics tool to confirm reduction or elimination of soil-transmitted helminth (STH) transmission in both control and elimination programmes. Given the performance limitations of conventional methods, a proposed harmonised qPCR will provide a diagnostic tool, with the sensitivity and specificity required to monitor low-intensity infections, following mass drug administration (MDA). Technical and logistical challenges associated with introducing qPCR as a stand-alone tool are highlighted, and a decision-making scheme on how qPCR can support surveillance, resistance detection, and elimination is presented. An accurate point-of-care (POC) diagnostic test needs to be developed to support STH control in the field, and STH biorepositories need to be established and maintained to ensure that reference materials are available for research and validation.
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Affiliation(s)
- Marina Papaiakovou
- Department of Infectious Disease Epidemiology, St Mary's Campus, Imperial College London, London, UK; London Centre for Neglected Tropical Disease Research (LCNTDR), Imperial College London, London, UK.
| | - D Timothy J Littlewood
- Science Directorate, Natural History Museum, London, UK; London Centre for Neglected Tropical Disease Research (LCNTDR), Imperial College London, London, UK
| | - Robin B Gasser
- Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia
| | - Roy M Anderson
- Department of Infectious Disease Epidemiology, St Mary's Campus, Imperial College London, London, UK; London Centre for Neglected Tropical Disease Research (LCNTDR), Imperial College London, London, UK
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46
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Anoopkumar AN, Aneesh EM. Assessing the importance of Molecular and Genetic perspectives in Prophesying the KFD transmission risk provinces in the Western Ghats, Kerala, INDIA in context with spatial distribution, Extensive genetic Diversity, and phylogeography. Comp Immunol Microbiol Infect Dis 2021; 76:101652. [PMID: 33910066 DOI: 10.1016/j.cimid.2021.101652] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Accepted: 04/09/2021] [Indexed: 12/15/2022]
Abstract
The deadly effects of KFD have been pointed in southern India; however, the infecting regions have been getting larger in recent epochs. People who live or work in regions where KFDV infected tick vectors are present are severely prone to procuring the infection. Being aware of tick vectors and infectious agents' geospatial location is vital to direct sustenance approaches to prevent and manage such infectious diseases as KFD. The present investigation has focussed on the spatial distribution, Extensive genetic Diversity, and phylogeography to forecast the probable KFD disease risk provinces in the Western Ghats. The statistical analysis for diversity indices and community comparison has been performed by using SPSS version 24.0.0 and R software version 3.4.2. The nucleotide sequences of the respective ticks and KFDV were retrieved from NCBI. The first strand of this investigation revealed that, around the world, the Indian province was found to exhibit a maximum range of diversity for tick vectors. The next strands prophesied the KFD transmission risk areas in the Western Ghats region, India, with computational spatial analysis and phylogeography. The final strand exposed the genetic diversity of the KFD virus and the tick vectors in terms of their spatial distribution worldwide.
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Affiliation(s)
- A N Anoopkumar
- Communicable Disease Research Laboratory (CDRL), Department of Zoology, St. Joseph's College, Irinjalakuda, University of Calicut, Kerala, India.
| | - Embalil Mathachan Aneesh
- Communicable Disease Research Laboratory (CDRL), Department of Zoology, St. Joseph's College, Irinjalakuda, University of Calicut, Kerala, India.
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47
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Oreskovic A, Lutz BR. Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples. PLoS One 2021; 16:e0247851. [PMID: 33635932 PMCID: PMC7909704 DOI: 10.1371/journal.pone.0247851] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2020] [Accepted: 02/12/2021] [Indexed: 12/19/2022] Open
Abstract
Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6-117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25-150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.
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Affiliation(s)
- Amy Oreskovic
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
| | - Barry R. Lutz
- Department of Bioengineering, University of Washington, Seattle, Washington, United States of America
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48
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Senchyna F, Hogan CA, Murugesan K, Moreno A, Ho DY, Subramanian A, Schwenk HT, Budvytiene I, Costa HA, Gombar S, Banaei N. Clinical Accuracy and Impact of Plasma Cell-Free DNA Fungal PCR Panel for Non-Invasive Diagnosis of Fungal Infection. Clin Infect Dis 2021; 73:1677-1684. [PMID: 33606010 DOI: 10.1093/cid/ciab158] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Indexed: 01/13/2023] Open
Abstract
BACKGROUND Invasive fungal infection (IFI) is a growing cause of morbidity and mortality in oncology and transplant patients. Diagnosis of IFI is often delayed due to need for invasive biopsy and low sensitivity of conventional diagnostic methods. Fungal cell-free DNA (cfDNA) detection in plasma is a novel testing modality for the non-invasive diagnosis of IFI. METHODS A novel bioinformatic pipeline was created to interrogate fungal genomes and identify multicopy sequences for cfDNA PCR targeting. A real-time PCR panel was developed for 12 genera and species most commonly causing IFI. Sensitivity and specificity of the fungal PCR panel were determined using plasma samples from patients with IFI and non-IFI controls. Clinical impact of fungal PCR panel was evaluated prospectively based on the treating team's interpretation of the results. RESULTS Overall, the sensitivity and specificity were 56.5% (65/115, 95% confidence interval [CI], 47.4%-65.2%) and 99.5% (2064/2075; 95% CI, 99.0%-99.7%), respectively. In the subset of patients with an optimized plasma volume (2mL), sensitivity was 69.6% (48/69; 95% CI, 57.9%-79.2%). Sensitivity was 91.7% (11/12; 95% CI, 62.5%-100%) for detection of Mucorales agents, 56.3% (9/16; 95% CI, 33.2%-76.9%) for Aspergillus species, and 84.6% (11/13; 95% CI, 56.5%-96.9%) for Candida albicans. In a prospective evaluation of 226 patients with suspected IFI, cfDNA testing was positive in 47 (20.8%) patients and resulted in a positive impact on clinical management in 20/47 (42.6%). CONCLUSIONS The fungal cfDNA PCR panel offers a non-invasive approach to early diagnosis of IFI, providing actionable results for personalized care.
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Affiliation(s)
- Fiona Senchyna
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Catherine A Hogan
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.,Clinical Microbiology Laboratory, Stanford University Medical Center, Palo Alto, CA, USA
| | - Kanagavel Murugesan
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Angel Moreno
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Dora Y Ho
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Aruna Subramanian
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Hayden T Schwenk
- Division of Infectious Diseases, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
| | - Indre Budvytiene
- Clinical Microbiology Laboratory, Stanford University Medical Center, Palo Alto, CA, USA
| | - Helio A Costa
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.,Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, CA, USA
| | - Saurabh Gombar
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
| | - Niaz Banaei
- Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.,Clinical Microbiology Laboratory, Stanford University Medical Center, Palo Alto, CA, USA.,Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA
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49
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Banaei N, Musser KA, Salfinger M, Somoskovi A, Zelazny AM. Novel Assays/Applications for Patients Suspected of Mycobacterial Diseases. Clin Lab Med 2020; 40:535-552. [DOI: 10.1016/j.cll.2020.08.010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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50
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Sudbury EL, Clifford V, Messina NL, Song R, Curtis N. Mycobacterium tuberculosis-specific cytokine biomarkers to differentiate active TB and LTBI: A systematic review. J Infect 2020; 81:873-881. [PMID: 33007340 DOI: 10.1016/j.jinf.2020.09.032] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Revised: 08/21/2020] [Accepted: 09/18/2020] [Indexed: 12/12/2022]
Abstract
OBJECTIVES New tests are needed to overcome the limitations of existing immunodiagnostic tests for tuberculosis (TB) infection, including their inability to differentiate between active TB and latent TB infection (LTBI). This review aimed to identify the most promising cytokine biomarkers for use as stage-specific markers of TB infection. METHODS A systematic review was done using electronic databases to identify studies that have investigated Mycobacterium tuberculosis (MTB)-specific cytokine responses as diagnostic tools to differentiate between LTBI and active TB. RESULTS The 56 studies included in this systematic review measured the MTB-specific responses of 100 cytokines, the most frequently studied of which were IFN-γ, IL-2, TNF-α, IP-10, IL-10 and IL-13. Ten studies assessed combinations of cytokines, most commonly IL-2 and IFN-γ. For most cytokines, findings were heterogenous between studies. The variation in results likely relates to differences in the study design and laboratory methods, as well as participant and environmental factors. CONCLUSIONS Although several cytokines show promise as stage-specific markers of TB infection, this review highlights the need for further well-designed studies, in both adult and paediatric populations, to establish which cytokine(s) will be of most use in a new generation of immunodiagnostic tests.
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Affiliation(s)
- Eva L Sudbury
- Department of Paediatrics, The University of Melbourne, The Royal Children's Hospital Melbourne, Parkville, Australia; Infectious Diseases Research Group, Murdoch Children's Research Institute, Parkville, Australia.
| | - Vanessa Clifford
- Department of Paediatrics, The University of Melbourne, The Royal Children's Hospital Melbourne, Parkville, Australia; Infectious Diseases Research Group, Murdoch Children's Research Institute, Parkville, Australia; Infectious Diseases Unit, The Royal Children's Hospital, Parkville, Australia.
| | - Nicole L Messina
- Department of Paediatrics, The University of Melbourne, The Royal Children's Hospital Melbourne, Parkville, Australia; Infectious Diseases Research Group, Murdoch Children's Research Institute, Parkville, Australia.
| | - Rinn Song
- Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the National Institute for Health Research Oxford Biomedical Research Centre, Oxford, UK; Division of Infectious Diseases, Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
| | - Nigel Curtis
- Department of Paediatrics, The University of Melbourne, The Royal Children's Hospital Melbourne, Parkville, Australia; Infectious Diseases Research Group, Murdoch Children's Research Institute, Parkville, Australia; Infectious Diseases Unit, The Royal Children's Hospital, Parkville, Australia.
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