False positive detection of serum cryptococcal antigens due to insufficient sample dilution: A case series

Table 1 Case reports of false positive cryptococcal antigen

Ref.	Age (yr)/gender	Country	Underlying disease (including Immunosupressive disease or drug use)	Sample source/Detection method/Reagent company	Basis of diagnosis	Possible causes	Outcome
Matsumoto et al[24], 2019	58/F	United States	SLE with secondary immune thrombocytopenic purpura	CSF/LA/CALAS® Meridian Bioscience Inc., Cincinnati, Ohio	(1) Reexamination of serum LA suggested negative; (2) Both CSF ink staining and culture results were negative; and (3) Head MRI showed abnormal signals in the left superior frontal cortex, consistent with subacute ischemia; Cardiac hypertrophy suggested Libman-Sack endocarditis. The final diagnosis was "thromboembolic cerebrovascular transient ischemia"	Nonspecific interference with the autoantibodies of SLE circulation in patients	Survived
Augeret al[25], 2019	26/M	United States	None	CSF/LA	Blood and CSF culture identified Df-2 infection	Common antigenic surface components may exist in DF-2
Volozhantsev et al[26], 2020	33/M	United States	Aplastic anemia; after bone marrow transplantation	Serum/LA/ IM Inc, American Microscan	The autopsy confirmed Trichosporon asahiti infection	Similar structures of polysaccharides may exist in Trichosporon asahiti	Died
Zhu et al[27], 2018	29/F	United States	Non-Hodgkin's lymphoma	CSF/LA/CALAS, Meridian Diagnostics, Cincinnati, Ohio, and CRYPTO-LA, International Biological Laboratories, Cranbury, New Jersey	CSF culture indicated Stomatococcus infection	Stomatococcus infection may cross-react with LA	Died
This case 1	53/M	China	None	Serum/LFA/ IMMY Immuno-Mycologics, Norman, Oklahoma, United States	(1) Anticryptococcal treatment failed; and (2) Lung puncture tissue culture suggested aspergillus	Insufficient sample dilution	Survived
Case 2	67/F	China	Bronchial asthma	Serum/LFA/ IMMY Immuno-Mycologics, Norman, Oklahoma, United States	Lung biopsy and left supraclavicular lymph node biopsy indicated lung adenocarcinoma	Insufficient sample dilution	Survived
Case 3	67/M	China	Post-orthotopic liver transplantation	Serum/LFA/ IMMY Immuno-Mycologics, Norman, Oklahoma, United States	(1) The local hospital reexamination of CRAG was negative; and (2) Anti-bacterial therapy was effective	Insufficient sample dilution	Survived

SLE: Systemic lupus erythematosus; CSF: Cerebrospinal fluid; LA: Latex agglutination; MRI: Magnetic resonance imaging; CRAG: Cryptococcal antigen.

Table 2 Possible causes of false positive results in three cryptococcal antigen detection techniques

Detection method	LA	LFA	ELISA
Sample	Cerebrospinal fluid/Serum	Cerebrospinal fluid/Serum/Plasma /Whole blood	Cerebrospinal fluid/Serum
Possible causes of false positive	Serum of rheumatoid factor, agarose dehydration, hydroxyethyl starch, and containing Fe3+/dL > 200 mg was present, Circular slides are not properly washed, the inactivation of Streptomyces protease in the kit and some nonspecific reactions in patients with HIV infection occur	LFA has antigenic cross-reaction with aspergillus which may lead to false positive results1; Sample dilution is insufficient2	Samples are Infected with other microbial infections, such as Trichosporon1; Reagents and samples to be tested are contaminated2

¹Lateral flow assay (LFA) should not be used as a screening test for the general population, but only should be performed when clinical evidence suggests the possibility of cryptococcosis.

²LFA may also have potential interference factors, including samples pretreated with 2-mercaptoethanol or those containing vaginal ointment, caffeine, ascorbic acid, itraconazole, amphotericin B, acetaminophen or acetylsalicylic acid. However, the above factors have not been systematically evaluated.

LA: Latex agglutination; LFA: Lateral flow assay; ELISA: Enzyme linked immunosorbent assay; HIV: Human immunodeficiency virus.