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1
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Kuba Y, Nidaira M, Maeshiro N, Komase K, Kamiya H, Kyan H. Analysis of Suspected Measles Cases with Discrepant Measles-Specific IgM and rRT-PCR Test Results, Japan. Emerg Infect Dis 2024; 30:926-933. [PMID: 38579738 PMCID: PMC11060445 DOI: 10.3201/eid3005.231757] [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: 04/07/2024] Open
Abstract
We investigated clinically suspected measles cases that had discrepant real-time reverse transcription PCR (rRT-PCR) and measles-specific IgM test results to determine diagnoses. We performed rRT-PCR and measles-specific IgM testing on samples from 541 suspected measles cases. Of the 24 IgM-positive and rRT-PCR--negative cases, 20 were among children who received a measles-containing vaccine within the previous 6 months; most had low IgG relative avidity indexes (RAIs). The other 4 cases were among adults who had an unknown previous measles history, unknown vaccination status, and high RAIs. We detected viral nucleic acid for viruses other than measles in 15 (62.5%) of the 24 cases with discrepant rRT-PCR and IgM test results. Measles vaccination, measles history, and contact history should be considered in suspected measles cases with discrepant rRT-PCR and IgM test results. If in doubt, measles IgG avidity and PCR testing for other febrile exanthematous viruses can help confirm or refute the diagnosis.
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Affiliation(s)
| | - Minoru Nidaira
- Okinawa Prefectural Institute of Health and Environment, Okinawa, Japan (Y. Kuba, M. Nidaira, N. Maeshiro, H. Kyan)
- National Institute of Infectious Diseases, Tokyo, Japan (K. Komase, H. Kamiya)
| | - Noriyuki Maeshiro
- Okinawa Prefectural Institute of Health and Environment, Okinawa, Japan (Y. Kuba, M. Nidaira, N. Maeshiro, H. Kyan)
- National Institute of Infectious Diseases, Tokyo, Japan (K. Komase, H. Kamiya)
| | - Katsuhiro Komase
- Okinawa Prefectural Institute of Health and Environment, Okinawa, Japan (Y. Kuba, M. Nidaira, N. Maeshiro, H. Kyan)
- National Institute of Infectious Diseases, Tokyo, Japan (K. Komase, H. Kamiya)
| | - Hajime Kamiya
- Okinawa Prefectural Institute of Health and Environment, Okinawa, Japan (Y. Kuba, M. Nidaira, N. Maeshiro, H. Kyan)
- National Institute of Infectious Diseases, Tokyo, Japan (K. Komase, H. Kamiya)
| | - Hisako Kyan
- Okinawa Prefectural Institute of Health and Environment, Okinawa, Japan (Y. Kuba, M. Nidaira, N. Maeshiro, H. Kyan)
- National Institute of Infectious Diseases, Tokyo, Japan (K. Komase, H. Kamiya)
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2
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Zhang Z, Liu D, Hu J, Sun W, Liu K, Li J, Xu H, Liu J, He L, Jiang D, Gu M, Hu S, Wang X, Liu X, Liu X. Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus. Arch Virol 2019; 164:1111-1119. [PMID: 30790106 DOI: 10.1007/s00705-019-04180-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2018] [Accepted: 01/18/2019] [Indexed: 02/08/2023]
Abstract
H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.
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Affiliation(s)
- Zhujun Zhang
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Dong Liu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Jiao Hu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Wenqiang Sun
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Kaituo Liu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Juan Li
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Haixu Xu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Jing Liu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Lihong He
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Daxiu Jiang
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Min Gu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Shunlin Hu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Xiaoquan Wang
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Xiaowen Liu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China.,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China.,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China
| | - Xiufan Liu
- Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, China. .,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China. .,Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-food Safety and Quality, Ministry of Agriculture of China (26116120), Yangzhou University, Yangzhou, China.
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3
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Tan SK, Shen P, Lefterova MI, Sahoo MK, Fung E, Odegaard JI, Davis RW, Pinsky BA, Scharfe C. Transplant Virus Detection Using Multiplex Targeted Sequencing. J Appl Lab Med 2017; 2:757-769. [PMID: 31245786 DOI: 10.1373/jalm.2017.024521] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Background Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation. Methods We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients. Results For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR. Conclusions Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.
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Affiliation(s)
- Susanna K Tan
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA
| | - Peidong Shen
- Stanford Genome Technology Center, Stanford University, Palo Alto, CA
| | - Martina I Lefterova
- Department of Pathology, Stanford University School of Medicine, Palo Alto, CA
| | - Malaya K Sahoo
- Department of Pathology, Stanford University School of Medicine, Palo Alto, CA
| | - Eula Fung
- Stanford Genome Technology Center, Stanford University, Palo Alto, CA
| | - Justin I Odegaard
- Department of Pathology, Stanford University School of Medicine, Palo Alto, CA
| | - Ronald W Davis
- Stanford Genome Technology Center, Stanford University, Palo Alto, CA
| | - Benjamin A Pinsky
- Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA.,Department of Pathology, Stanford University School of Medicine, Palo Alto, CA
| | - Curt Scharfe
- Department of Genetics, Yale University School of Medicine, New Haven, CT
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4
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Abstract
Human roseoloviruses include three different species, human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), genetically related to human cytomegalovirus. They exhibit a wide cell tropism in vivo and, like other herpesviruses, induce a lifelong latent infection in humans. In about 1% of the general population, HHV-6 DNA is covalently integrated into the subtelomeric region of cell chromosomes (ciHHV-6). Many active infections, corresponding to primary infections, reactivations, or exogenous reinfections, are asymptomatic. They also may cause serious diseases, particularly in immunocompromised individuals, including hematopoietic stem-cell transplant (HSCT) and solid-organ transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients. This opportunistic pathogenic role is formally established for HHV-6 infection and less clear for HHV-7. It mainly concerns the central-nervous system, bone marrow, lungs, gastrointestinal tract, skin, and liver. As the best example, HHV-6 causes both exanthema subitum, a benign disease associated with primary infection, and severe encephalitis associated with virus reactivations in HSCT recipients. Diagnosis using serologic and direct antigen-detection methods currently exhibits limitations. The most prominent technique is the quantification of viral DNA in blood, other body fluids, and organs by means of real-time polymerase-chain reaction (PCR). The antiviral compounds ganciclovir, foscarnet, and cidofovir are effective against active infections, but there is currently no consensus regarding the indications of treatment or specifics of drug administration. Numerous questions about HHV-6A, HHV-6B, HHV-7 are still pending, concerning in particular clinical impact and therapeutic options in immunocompromised patients.
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5
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Agut H, Bonnafous P, Gautheret-Dejean A. Update on infections with human herpesviruses 6A, 6B, and 7. Med Mal Infect 2016; 47:83-91. [PMID: 27773488 DOI: 10.1016/j.medmal.2016.09.004] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Accepted: 09/19/2016] [Indexed: 11/19/2022]
Abstract
Human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7) are genetically related to cytomegalovirus. They belong to the Roseolovirus genus and to the Betaherpesvirinae subfamily. They infect T cells, monocytes-macrophages, epithelial cells, and central nervous system cells. These viruses are ubiquitous and are responsible for lifelong chronic infections, most often asymptomatic, in the vast majority of the general adult population. HHV-6B is responsible for exanthema subitum, which is a benign disease of infants. HHV-6A and HHV-6B also cause opportunistic infections in immunocompromised patients: encephalitis, hepatitis, bone marrow suppression, colitis, and pneumonitis. Their etiological role in chronic diseases such as multiple sclerosis, cardiomyopathy, and thyroiditis is still controversial. The pathogenicity of HHV-7 is less clear and seems to be much more restricted. Chromosomal integration of HHV-6A and HHV-6B is transmissible from parents to offspring and observed in about 1% of the general population. This integration raises the question of potential associated diseases and can be a confounding factor for the diagnosis of active infections by both viruses. The diagnosis of HHV-6A, HHV-6B, and HHV-7 infections is rather based on gene amplification (PCR), which allows for the detection and quantification of the viral genome, than on serology, which is mainly indicated in case of primary infection. Ganciclovir, foscarnet, and cidofovir inhibit the replication of HHV-6A, HHV-6B, and HHV-7. Severe infections may thus be treated but these therapeutic indications are still poorly defined.
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Affiliation(s)
- H Agut
- Service de virologie, CERVI, hôpitaux universitaires La Pitié Salpêtrière-Charles-Foix, Assistance publique-Hôpitaux de Paris, 83, boulevard de l'Hôpital, 75651 Paris cedex 13, France; Inserm, CIMI-Paris UMR 1135, Équipe 1 PVI, Sorbonne universités, UPMC université Paris 6, 75013 Paris, France.
| | - P Bonnafous
- Inserm, CIMI-Paris UMR 1135, Équipe 1 PVI, Sorbonne universités, UPMC université Paris 6, 75013 Paris, France.
| | - A Gautheret-Dejean
- Service de virologie, CERVI, hôpitaux universitaires La Pitié Salpêtrière-Charles-Foix, Assistance publique-Hôpitaux de Paris, 83, boulevard de l'Hôpital, 75651 Paris cedex 13, France; Inserm, CIMI-Paris UMR 1135, Équipe 1 PVI, Sorbonne universités, UPMC université Paris 6, 75013 Paris, France; Faculté de pharmacie, université Paris-Descartes, 75006 Paris, France.
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6
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Prevalence of herpesviruses at onset of idiopathic nephrotic syndrome. Pediatr Nephrol 2014; 29:2325-31. [PMID: 24899237 DOI: 10.1007/s00467-014-2860-1] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2013] [Revised: 04/15/2014] [Accepted: 05/14/2014] [Indexed: 01/15/2023]
Abstract
BACKGROUND Idiopathic nephrotic syndrome (INS) is likely a primary immune disorder, but viruses might also be involved in the mechanisms of the disease. Here, we investigate the link between herpesvirus infection and the first manifestation of INS in children. METHODS A prospective, multicentre, and population-based case-control study called NEPHROVIR included 164 patients, aged 6 months to 15 years old, newly diagnosed with INS, and 233 controls matched for gender, age, and period of sample. The analysis was done on 124 patients and 196 controls. Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and human herpesvirus-7 (HHV-7) DNA prevalence at diagnosis were assessed from whole peripheral blood samples, as well as EBV and CMV viral load and seroprevalence. RESULTS EBV DNA was significantly more prevalent in cases than in controls (50.8 vs 29.1 %; OR = 2.6; p = 0.0002), with no difference in viral load. A significant difference was also found for CMV (11.3 vs 3.6 %; p = 0.02) and HHV-7 (83 vs 72 %; p = 0.02) DNA prevalence between cases and controls. There were significantly more EBV and CMV recent infections or reactivations based on VCA-IgM and CMV IgM in cases than controls, while there were no differences in IgG seroprevalence. CONCLUSION The prevalence of positive EBV DNA detection and recent infection or reactivation is higher in children at onset of INS compared to a population matched for age, gender, and time of sampling.
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7
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Nahdi I, Abdelwahed RB, Boukoum H, Bressollette-Bodin C, Attia S, Yahia SB, Fisson S, Khairallah M, Aouni M. Herpesvirus detection and cytokine levels (IL-10, IL-6, and IFN-γ) in ocular fluid from Tunisian immunocompetent patients with uveitis. J Med Virol 2013; 85:2079-86. [DOI: 10.1002/jmv.23708] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/17/2013] [Indexed: 12/14/2022]
Affiliation(s)
- Imen Nahdi
- Laboratory of Contagious Diseases and Biologically Active Substances, LR99-ES27; Faculty of Pharmacy, Monastir University; Monastir Tunisia
| | - Rym Ben Abdelwahed
- Laboratory of Contagious Diseases and Biologically Active Substances, LR99-ES27; Faculty of Pharmacy, Monastir University; Monastir Tunisia
- National Institute of the Health and the Medical Research (INSERM), UMRS 872; Research Center of the Cordeliers, Pierre et Marie Curie-Paris 6 University; Paris France
| | - Hannen Boukoum
- Laboratory of Contagious Diseases and Biologically Active Substances, LR99-ES27; Faculty of Pharmacy, Monastir University; Monastir Tunisia
| | | | - Sonia Attia
- Ophthalmology Department; Fattouma Bourguiba University Hospital; Tunisia
| | - Salim Ben Yahia
- Ophthalmology Department; Fattouma Bourguiba University Hospital; Tunisia
| | - Sylvain Fisson
- National Institute of the Health and the Medical Research (INSERM), UMRS 872; Research Center of the Cordeliers, Pierre et Marie Curie-Paris 6 University; Paris France
- Genethon, UMRS 951; University of Evry Val d'Essonne; Evry France
| | - Moncef Khairallah
- Ophthalmology Department; Fattouma Bourguiba University Hospital; Tunisia
| | - Mahjoub Aouni
- Laboratory of Contagious Diseases and Biologically Active Substances, LR99-ES27; Faculty of Pharmacy, Monastir University; Monastir Tunisia
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Oakes B, Hoagland-Henefield M, Komaroff AL, Erickson JL, Huber BT. Human Endogenous Retrovirus-K18 Superantigen Expression and Human Herpesvirus-6 and Human Herpesvirus-7 Viral Loads in Chronic Fatigue Patients. Clin Infect Dis 2013; 56:1394-400. [DOI: 10.1093/cid/cit086] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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9
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Razonable RR. Human herpesviruses 6, 7 and 8 in solid organ transplant recipients. Am J Transplant 2013; 13 Suppl 3:67-77; quiz 77-8. [PMID: 23347215 DOI: 10.1111/ajt.12008] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2012] [Revised: 07/05/2012] [Accepted: 07/05/2012] [Indexed: 01/25/2023]
Abstract
Human herpesviruses (HHV) 6 and 7 are ubiquitous infections that reactivate commonly in transplant recipients. However, clinical diseases due to these viruses are reported only in 1% of solid organ transplant recipients. Fever, rash and bone marrow suppression are the most common manifestations, but symptoms of tissue invasive disease may be observed. Treatment of HHV-6 and HHV-7 disease includes antiviral therapy and cautious reduction in immunosuppression. HHV-8 is an oncogenic gamma-herpesvirus that causes Kaposi's sarcoma, Castleman's disease and primary effusion lymphomas in transplant recipients. Nonmalignant diseases such as bone marrow suppression and multiorgan failure have also been associated with HHV-8. Reduction in immunosuppression is the first line treatment of HHV-8 infection. Other alternatives for treatment, especially for HHV-8 diseases not responsive to immuno-minimization strategies, are surgery and chemotherapy. Sirolimus has been shown to be a beneficial component for the treatment of Kaposi's sarcoma and the role of antivirals for HHV-8 infection is being investigated.
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Affiliation(s)
- R R Razonable
- Division of Infectious Diseases, Department of Medicine, College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
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10
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Abstract
Molecular diagnostic techniques for viral testing have undergone rapid development in recent years. They are becoming more widely used than the classical virological assays in the majority of clinical virology laboratories, and now represent a new method for the diagnosis of human viral infections. Recently, new techniques based on multiplex RT‐PCR amplification followed by microarray analysis have been developed and evaluated. On the basis of amplification of viral genome‐specific fragments by multiplex RT‐PCR and their subsequent detection via hybridization with microorganism‐specific binding probes on solid surfaces, they allow simultaneous detection and identification of multiple viruses in a single clinical sample. The management of viral central nervous system and respiratory tract infections currently represents the two main applications of the microarrays in routine virological practice. Microarrays have shown reliable results in comparison with those of referenced (RT)‐PCR assays, and appear to be of major interest for the detection of a broad range of respiratory and neurotropic viruses, assessment of the pathogenicity of newly discovered or neglected viruses, and identification of multiple viral infections in clinical samples. Despite several limitations observed during the different studies performed, this new technology might improve the clinical management of patients by enlarging the range of the viruses detected, in particular in cases of severe infections leading to patient hospitalization in the intensive‐care unit. They might also help in the prevention of nosocomial transmission in hospital departments by contributing to the development of new epidemiological surveillance systems for viral infections.
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Affiliation(s)
- N Lěvěque
- Clinical and Molecular Virology Unit, University Hospital; Clinical and Molecular Virology Unit, University Hospital.
| | - F Renois
- Clinical and Molecular Virology Unit, University Hospital; Faculty of Medicine, EA-4684 CardioVir, SFR-CAP santé, Reims, France
| | - L Andréoletti
- Clinical and Molecular Virology Unit, University Hospital; Faculty of Medicine, EA-4684 CardioVir, SFR-CAP santé, Reims, France
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Yun T, Ni Z, Hua J, Ye W, Chen L, Zhang S, Zhang Y, Zhang C. Development of a one-step real-time RT-PCR assay using a minor-groove-binding probe for the detection of duck Tembusu virus. J Virol Methods 2012; 181:148-54. [DOI: 10.1016/j.jviromet.2012.01.019] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2011] [Revised: 01/16/2012] [Accepted: 01/24/2012] [Indexed: 11/15/2022]
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12
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Quantitation of human herpesvirus-6A, -6B and -7 DNAs in whole blood, mononuclear and polymorphonuclear cell fractions from healthy blood donors. J Clin Virol 2012; 53:151-5. [DOI: 10.1016/j.jcv.2011.10.017] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2011] [Revised: 10/17/2011] [Accepted: 10/26/2011] [Indexed: 01/22/2023]
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13
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Caïola D, Karras A, Flandre P, Boutolleau D, Scieux C, Agut H, Legendre C, Gautheret-Dejean A. Confirmation of the low clinical effect of human herpesvirus-6 and -7 infections after renal transplantation. J Med Virol 2012; 84:450-6. [DOI: 10.1002/jmv.23206] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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14
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Takao S, Shigemoto N, Shimazu Y, Tanizawa Y, Fukuda S, Matsuo T. Detection of Exanthematic Viruses Using a TaqMan Real-Time PCR Assay Panel in Patients with Clinically Diagnosed or Suspected Measles. Jpn J Infect Dis 2012; 65:444-8. [DOI: 10.7883/yoken.65.444] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
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Rapid virological diagnosis of central nervous system infections by use of a multiplex reverse transcription-PCR DNA microarray. J Clin Microbiol 2011; 49:3874-9. [PMID: 21918017 DOI: 10.1128/jcm.01214-11] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Viruses are the main etiological cause of central nervous system (CNS) infections. A rapid molecular diagnosis is recommended to improve the therapeutic management of patients. The aim of this study was to evaluate the performances of a DNA microarray, the Clart Entherpex kit (Genomica, Coslada, Spain), allowing the rapid and simultaneous detection of 9 DNA and RNA neurotropic viruses: herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and the human enteroviruses (HEVs). This evaluation was performed with 28 samples from the European proficiency panels (Quality Control for Molecular Diagnostics [QCMD]; Glasgow, Scotland) and then with 78 cerebrospinal fluid (CSF) specimens. The majority of the QCMD results obtained by the DNA microarray were similar to those recorded by the overall QCMD participants. The main discrepant results were observed for low concentrations of HSV-2 and HEVs. From the clinical samples, the kit detected 27 of the 28 herpesvirus CNS infections and all of the 30 HEV-positive CSF samples. No false-positive result was observed among the 20 virus-negative CSF samples. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 98.3, 100, 95.2, and 100%, respectively, when the results were compared to those of commercially available PCR assays. Interestingly, HHV-7 was detected in 11 (37%) of the 30 HEV-positive CSF samples from children suffering from aseptic meningitis causing significantly longer lengths of stay at the hospital than infection with HEVs alone (2.4 versus 1.4 days; P = 0.038). In conclusion, this preliminary study showed that this DNA microarray could be a valuable molecular diagnostic tool for single and mixed DNA and RNA virus infections of the CNS.
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17
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Kaklikkaya I, Kaklikkaya N, Birincioglu I, Buruk K, Turan N. Detection of human herpesvirus 6 DNA but not human herpesvirus 7 or 8 DNA in atherosclerotic and nonatherosclerotic vascular tissues. Heart Surg Forum 2011; 13:E345-9. [PMID: 20961841 DOI: 10.1532/hsf98.20101023] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
INTRODUCTION Various viral infections are thought to play a role in the development of atherosclerosis. A number of studies suggest that certain viruses from the Herpesviridae family in particular may lead to atherosclerosis. METHODS We investigated the presence of human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8) DNA in carotid, iliac, and coronary artery specimens obtained from a group of adult autopsy cases by means of polymerase chain reaction (PCR) analysis and nested PCR techniques. A 28-subject study group with at least type IV atherosclerosis and a 25-subject control group with no visible atherosclerosis were enrolled. RESULTS HHV-6 DNA was found in the carotid artery specimen of 1 subject with atherosclerosis, in an iliac artery specimen of another subject, and in the iliac artery specimen of one of the control subjects. HHV-7 or HHV-8 DNA was not found in either the atherosclerosis or control cases. CONCLUSIONS This study is the first to demonstrate the presence of HHV-6 in atherosclerotic vascular tissues. HHV-7 and HHV-8 were not found in atherosclerotic tissues; however, further research on broader study groups and with different protocols is needed to determine whether these viruses play a role in the formation of atherosclerosis.
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Affiliation(s)
- Islam Kaklikkaya
- Department of Cardiovascular Surgery, Karadeniz Technical University, Trabzon, Turkey.
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18
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Abstract
In routine molecular diagnostics, detection of herpesviruses has made a major impact. Infection with herpesviruses is indicated by demonstrating the presence of the virus in selected specimens. Rapid and reliable detection of herpesvirus DNA helps to decrease the lethality as well as the sequelae of herpesvirus infection in patients at risk. This chapter discusses specimen types and both laboratory-developed and commercially available assays useful for molecular detection of herpesviruses. To meet the need for reliable laboratory results, it is advisable to employ maximum automated and standardized kits based on reagents and standards of reproducible high quality. In the routine diagnostic laboratory, introduction of IVD/CE and/or FDA-labeled tests is preferred.
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Affiliation(s)
- Harald H Kessler
- Molecular Diagnostics Laboratory, IHMEM, Medical University of Graz, Graz, Austria
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Hudnall SD, Chen T, Allison P, Tyring SK, Heath A. Herpesvirus prevalence and viral load in healthy blood donors by quantitative real-time polymerase chain reaction. Transfusion 2008; 48:1180-7. [PMID: 18422852 DOI: 10.1111/j.1537-2995.2008.01685.x] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND After primary infection, human herpesviruses (HHVs) maintain long-term latent persistence, often punctuated years later by sporadic episodes of symptomatic lytic activation. Also, blood-borne herpesvirus from healthy persistently infected blood donors can lead to active primary infection of immunocompromised transfusion recipients. STUDY DESIGN AND METHODS Utilizing a set of newly developed real-time polymerase chain reaction assays for detection and quantification of all eight human herpesviruses, the prevalence and viral DNA load of white cell-enriched blood from 100 randomly selected blood donors from the southeast Texas region are reported. RESULTS Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), and HHV-8 DNA were not detected in any donor sample. In contrast, Epstein-Barr virus (EBV) (72%) and HHV-7 (65%) were commonly detected, HHV-6 (30%) was often detected (Type B only), and cytomegalovirus (CMV; 1%) was rarely detected. Median viral loads of positive samples (per milliliter of blood) ranged from 4278 for HHV-6 to less than 46 for EBV. CONCLUSIONS These results suggest that the potential for transfusion-mediated transmission of herpesviruses from healthy adult blood donors is high for EBV and HHV-7; moderately high for HHV-6; uncommon for CMV; and rare for HSV-1, HSV-2, VZV, and HHV-8. Perhaps the most remarkable finding in this study was the detection of a single donor sample with greater than 6.1 x 10(7) HHV-6 Type B genome equivalents per mL blood. Given that this extraordinarily high level of HHV-6 DNA was obtained from a healthy adult blood donor, this phenomenon is likely unrelated to active infection or immunodeficiency.
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Affiliation(s)
- S David Hudnall
- Department of Pathology and Laboratory Medicine, University of Texas Medical Branch, Galveston, TX 77555-0741, USA.
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Lu YY, Yan JY, Feng Y, Xu CP, Shi W, Mao HY. Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR. Lett Appl Microbiol 2007; 46:20-5. [PMID: 17944840 PMCID: PMC7197896 DOI: 10.1111/j.1472-765x.2007.02253.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Aims: Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assay based on a TaqMan‐minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5. Methods and Results: Conserved regions in the haemagglutinin genes of avian influenza viruses subtype H5 served as targets for the primers and TaqMan‐MGB probe design. Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions. A plasmid containing the haemagglutinin gene was constructed and in vitro transcribed for a quantitative assay of copy numbers of the target gene. The results revealed that the optimal concentration of primers and probe was 640 and 480 nmol l−1, respectively. The threshold of 100 copies of target molecules could be detected. The linear range for detection was determined as 102 to 108 molecules in reaction. Conclusions: It took less than 3 h to complete the detection from viral RNA extraction, with good sensitivity and repeatability. Significance and Impact of the Study: Real‐time RT‐PCR assay with MGB probe was an effective means for quick and quantitative laboratory detection and monitoring of H5 avian influenza viruses.
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Affiliation(s)
- Y Y Lu
- Institute of Virology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China.
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Achour A, Boutolleau D, Slim A, Agut H, Gautheret-Dejean A. Human herpesvirus-6 (HHV-6) DNA in plasma reflects the presence of infected blood cells rather than circulating viral particles. J Clin Virol 2007; 38:280-5. [PMID: 17339132 DOI: 10.1016/j.jcv.2006.12.019] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2006] [Accepted: 12/22/2006] [Indexed: 10/23/2022]
Abstract
BACKGROUND The presence of HHV-6 DNA in plasma or serum is considered a good marker of active infection. However, it is ignored whether this DNA corresponds to virus particles produced by lymphoid tissue infection or virus-free DNA released from infected circulating blood cells. OBJECTIVES To investigate whether HHV-6 DNA in whole plasma is nonencapsidated and its amount is correlated to cellular and human herpesvirus-7 (HHV-7) DNA loads in plasma subfractions as well as in corresponding peripheral blood mononuclear cells (PBMCs). STUDY DESIGN Whole plasma samples from immunocompromised patients were submitted to a DNase-resistance test. Plasma samples from a second group of patients were split up into three subfractions: P1 (pellet of clarification), P2 (pellet of ultracentrifugation), and S (supernatant of ultracentrifugation). HHV-6, HHV-7, and cellular DNA loads were determined in each fraction and PBMCs using specific real-time PCR. RESULTS Among 14 whole plasma samples, the majority of HHV-6 DNA detected was unprotected against DNase, i.e. nonencapsidated. The study of 35 other plasma samples revealed that cellular DNA was present in all subfractions from all samples whereas HHV-6 DNA was detected in 13 P1, 12 P2, 10 S fractions, and HHV-7 DNA in only one P1 fraction. Accordingly, median HHV-6 DNA load was significantly higher in P1 than in P2 and S fractions. The detection of HHV-6 DNA in plasma subfractions was statistically associated with a higher HHV-6 viral load in PBMCs (p<or=0.0003). CONCLUSIONS Taken together, these data tend to favour the hypothesis of a release of HHV-6 and cellular DNA into plasma following the lysis of infected PBMCs. HHV-6 DNA in plasma does not necessarily reflect the amount of virus produced by the active infection of distant lymphoid tissue and organs.
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Affiliation(s)
- Abla Achour
- Laboratoire de Virologie, Université Pierre et Marie Curie-Paris6 EA 2387, Groupe Hospitalier Pitié-Salpêtrière, Université Paris 6, Paris, France
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Abstract
The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.
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Affiliation(s)
| | | | - T. Lion
- Corresponding author. Tel.: +43 1 40470 489; fax: +43 1 40470 437.
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Abstract
BACKGROUND Conflicting results on the association of pityriasis rosea and human herpesvirus 7 infection have been reported by different investigators. AIM To review the level of evidence for such an association. METHODS Medline was searched with unlimited data entry and 13 reports were retrieved. The data were analyzed for a causative association according to the criteria of Fredericks and Relman, which take into consideration latent infection and the reactivation of viruses characteristic of herpesviruses, and the roles of sequence-based detection methods. RESULTS None of the criteria was substantiated by the findings of most investigators. Factors leading to the discrepancies of the results were discussed. CONCLUSION There is currently insufficient evidence that human herpesvirus 7 infection is causally related to pityriasis rosea.
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Affiliation(s)
- Antonio A T Chuh
- Department of Medicine, University of Hong Kong and Queen Mary Hospital, Pokfulam, Hong Kong SAR, China.
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Zhao JR, Bai YJ, Zhang QH, Wan Y, Li D, Yan XJ. Detection of hepatitis B virus DNA by real-time PCR using TaqMan-MGB probe technology. World J Gastroenterol 2005; 11:508-10. [PMID: 15641135 PMCID: PMC4250800 DOI: 10.3748/wjg.v11.i4.508] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To develop a real-time PCR for detecting hepatitis B virus (HBV) DNA based on TaqMan technology using a new MGB probe.
METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.
RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r>0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.
CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.
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Affiliation(s)
- Jin-Rong Zhao
- Institute of Genetic Diagnosis, Fourth Military Medical University, 17 Changle West Road, Xi'an 710032, Shaanxi Province, China.
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Boutolleau D, Bonduelle O, Sabard A, Devers L, Agut H, Gautheret-Dejean A. Detection of human herpesvirus 7 DNA in peripheral blood reflects mainly CD4+ cell count in patients infected with HIV. J Med Virol 2005; 76:223-8. [PMID: 15834866 DOI: 10.1002/jmv.20345] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The opportunistic behavior and the potential interactions of human herpesvirus 7 (HHV-7) with human immunodeficiency virus (HIV)-1 in HIV-1-infected patients were investigated in comparison with HHV-6, another human roseolovirus. Roseolovirus DNAs were detected and quantified in peripheral blood mononuclear cells (PBMCs) from 198 HIV-seronegative healthy blood donors, 38 HIV-1-infected patients classified as long-term non-progressors, and 99 HIV-1-infected patients classified as progressors. The rate of HHV-7 DNA detection was higher in healthy donors (78%) than in long-term non-progressors (47%; P = 0.0003) or in progressors (52%; P < 0.0001). HHV-7 cell load was higher in healthy donors (median: 212 EqCop/10(6) PBMCs) and in long-term non-progressors (median: 105 EqCop/10(6) PBMCs) than in progressors (median: 48 EqCop/10(6) PBMCs; P < 0.0001 and P = 0.015, respectively). Among progressors, HHV-7 detection was correlated positively with the CD4(+) T-lymphocyte count (P = 0.028). Neither HHV-7 detection rate nor cell load was correlated with the HIV-1 plasma load. As a whole, HHV-6 detection rate and cell load were lower than the HHV-7 counterparts, albeit exhibiting similar differences between healthy donors, long-term non-progressors, and progressors. In conclusion, HHV-7 infection does not appear to be stimulated by HIV-1 infection, nor interact with it. Rather, HHV-7 detection rate and cell load reflect CD4(+) T-lymphocyte count, with higher values in healthy donors and long-term non-progressors than in progressors.
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Affiliation(s)
- David Boutolleau
- Laboratoire de Virologie, UPRES EA 2387, Groupe Hospitalier Pitié-Salpêtrière, Paris, France.
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Mackay IM, Arden KE, Nitsche A. Real-time Fluorescent PCR Techniques to Study Microbial-Host Interactions. METHODS IN MICROBIOLOGY 2004; 34:255-330. [PMID: 38620210 PMCID: PMC7148886 DOI: 10.1016/s0580-9517(04)34010-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
This chapter describes how real-time polymerase chain reaction (PCR) performs and how it may be used to detect microbial pathogens and the relationship they form with their host. Research and diagnostic microbiology laboratories contain a mix of traditional and leading-edge, in-house and commercial assays for the detection of microbes and the effects they impart upon target tissues, organs, and systems. The PCR has undergone significant change over the last decade, to the extent that only a small proportion of scientists have been able or willing to keep abreast of the latest offerings. The chapter reviews these changes. It discusses the second-generation of PCR technology-kinetic or real-time PCR, a tool gaining widespread acceptance in many scientific disciplines but especially in the microbiology laboratory.
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Affiliation(s)
- Ian M Mackay
- Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Qld, Australia
- Clinical Medical Virology Centre, University of Queensland, Brisbane, Qld, Australia
| | - Katherine E Arden
- Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Qld, Australia
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27
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Abstract
Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.
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Affiliation(s)
- I M Mackay
- Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre and Department of Paediatrics, Royal Children's Hospital, Brisbane, Queensland, Australia.
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Boutolleau D, Bonduelle O, Agut H, Gautheret-Dejean A. Is human herpesvirus-7 a marker or a competitor for HIV progression? AIDS 2004; 18:358-9. [PMID: 15075568 DOI: 10.1097/00002030-200401230-00040] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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Agut H, Fillet A, Gautheret-Dejean A, Boutolleau D. Détection, quantification et analyse des génomes viraux dans les infections à herpèsvirus humains 6 et 7 (HHV-6, HHV-7). ACTA ACUST UNITED AC 2003. [PMCID: PMC7147852 DOI: 10.1016/s0923-2532(03)00065-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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Safronetz D, Humar A, Tipples GA. Differentiation and quantitation of human herpesviruses 6A, 6B and 7 by real-time PCR. J Virol Methods 2003; 112:99-105. [PMID: 12951217 DOI: 10.1016/s0166-0934(03)00196-4] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The beta-herpesviruses cause considerable morbidity in immunocompromised individuals, such as transplant patients. Most notably within this group is human cytomegalovirus, although HHV-6 and -7 are a growing concern. Identifying HHV-6 and -7 as the cause of post-transplant illness can be challenging due to high seroprevalence and latency properties associated with these human herpesviruses. We have developed a sensitive and specific real-time PCR assay, which can differentiate reliably and quantify HHV-6A, -6B and -7. Using two sets of hybridization probes specific for HHV-6A or -6B and HHV-7, the assay reliably differentiates the three viruses using melting curve analysis. The lower limit of detection for all three viruses was determined to be ten viral genomes. This real-time PCR assay will be useful for differentiation and quantitation of HHV-6A, -6B and -7, especially for monitoring transplant patients.
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Affiliation(s)
- D Safronetz
- Department of Medical Microbiology, University of Manitoba, Man., Winnipeg, Canada
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