Serological methods for diagnosis of fungal infections: How relevant are they?

Author:

Professor Immaculata Xess (IN)

Abstract:

Introduction:
Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes (1). Serological detection of fungus-specific antibodies or antigens is also rapid and has potential for diagnosis and screening for IFI, although antibody presence does not always correlate with disease. Serological techniques are important non- culture-based tools for diagnosing IFIs especially for laboratories without availability to molecular tests
This talk puts into perspective current serological assays for diagnosing IFIs and the promises and pitfalls associated with them.

Serological test available for detecting IFI
(1,3)-β-d-glucan (BDG): a polysaccharide fungal cell wall component. BDG is a pan-fungal antigen present in Candida spp., Pneumocystis jivoveci, Aspergillus spp., Acremonium spp., Fusarium spp. exceptions being Cryptococcus spp., Mucrorales, and the yeast phase of Blastomyces dematitidis) [47]
The only FDA-approved BDG assay is the Fungitell Assay (Associates of Cape Cod, MA, USA). It has been shown to be useful for diagnosing intra-abdominal candidiasis and blood culture negative cases of pneumophila pneumonia [48]. In a meta-analysis study, serum BDG sensitivity and specificity for IC were 75–80% and 60–80%, respectively [48, 49]. Till date, there are many BDG assays available besides Fungitell, i.e., Fungitec-G, Beta Glucan-BGStar, Beta-Glucan test (Mauha, Japan) and each assay has varying cutoff values, sensitivity and specificity depending on the fungal strain involved, patient population and assay platform used. Another disadvantage of BDG is that it is a non-specific pan-fungal biomarker with low sensitivity values and high false positive results due to cross-reactivity. Racil et al. [58]
Galactomannan (GM) assay A useful diagnostic tool is the testing of Aspergillus species’ cell wall constituents, such as galactomannan. The measurement of cell wall components of Aspergillus species, such as galactomannan, is a useful diagnostic tool. GM is the main antigen detected in cases of IA and can be readily detected in bronchioalveolar lavage (BAL) and cerebrospinal fluid (CSF) etc. [68]. This assay’s overall sensitivity and specificity values ranged from 67 to 100 percent and 86 to 100 percent, respectively [69, 70] Currently, there is only one FDA-approved assay for the detection of Aspergillus GM (Platelia Aspergillus enzyme immunoassay (EIA); Bio-Rad, Marnes-la-Coquette, France) in patients’ serum and bronchoalveolar lavage (BAL) specimens.
Another improvement has been the release of a novel GM-EIA assay developed by IMMY. In the Bio-Rad GM EIA, a single rat monoclonal antibody referred to as EB-A2 has been used that binds to the fungal galactomannan. The new IMMY GM-EIA assay (just like the IMMY lateral flow kit) involves using two monoclonal antibodies, where one binds to a similar GM epitope as does EB-A2 and the other binds a novel target. In study by White et al. [14], they evaluated the newly released IMMYGM-EIA in a retrospective case–control study. The team discovered that the IMMY GM-EIA displayed a sensitivity of 71% and a specificity of 98%, respectively, with a positive threshold of 0.5.
Lateral flow device assay (LFA): new advances The development of an LFA-based test for the detection of Cryptococcal antigen has been a landmark achievement and has revolutionised the diagnosis of cryptococcal infection, especially in resource-limited settings. Cryptococcus neoformans is a dimorphic fungus with its different morphotypes enabling this opportunist pathogen to better adapt and exhibit different levels of pathogenicity in various hosts.
CrAg can also be detected by latex agglutination tests and enzyme immunoassay (EIA) with more than 99% sensitivity. However, there is still a requirement to use a point-of-care (POC) based test (POC is testing that is performed near or at the site of a patient with the result leading to a possible change in the care of the patient). POC testing is advantageous for rural and distant areas since it can be done without laboratory equipment or facilities [80]. This led to the development of CrAg-LFA. CrAg-LFA is a POC test employing dipstick with monoclonal antibodies that can detect the capsular antigen in the four serotypes (A, B, C, and D) of the pathogenic Cryptococcus neoformans species complex and the C. gattii species complex. The turn-around time is less than ten minutes, and you don’t need any complicated tools or highly skilled workers. The primary benefit of this test is its capacity to detect extremely low levels of circulating CrAg during the prodromal phase (around 22 days before symptoms), enabling prompt treatment and thus reducing the overall mortality rates [81, 82]. Many companies have been in the development of CrAg-LFA, of which CrAg LFA from Immuno-Mycologics, Inc. (IMMY; Norman, OK) is the only FDA approved one.
Another dipstick CrAg LFA, called Dynamiker Cryptococcal Antigen LFA, was introduced in 2014 (Tianjin Co., Ltd.). It is a dipstick sandwich immunochromatographic assay for the detection of capsular polysaccharide antigens of Cryptococcus species complex (Cryptococcus neoformans and Cryptococcus gattii) in human serum and cerebral spinal fluid (CSF) as shown in Fig. 1. Kwizera et al. [83] assessed the Dynamiker CrAg-diagnostic LFA’s performance in blood, plasma, and CSF samples from symptomatic and asymptomatic HIV patients in comparison to the IMMY CrAg-LFA (reference standard).
Liu et al. [86] evaluated the diagnostic performance of FungiXpert LFA and the IMMY CrAg LFA using eight cerebrospinal fluid (CSF) and 119 serum/plasma samples. Compared to IMMY CrAg LFA, the FungiXpert LFA demonstrated 99.1% sensitivity and 98.9% specificity in the qualitative test.
However, there is still a requirement to use a point-of-care (POC) based test (POC is testing that is performed near or at the site of a patient with the result leading to a possible change in the care of the patient). POC testing is advantageous for rural and distant areas since it can be done without laboratory equipment or facilities [80]. This led to the development of CrAg-LFA. CrAg-LFA is a POC test employing dipstick with monoclonal antibodies that can detect the capsular antigen in the four serotypes (A, B, C, and D) of the pathogenic Cryptococcus neoformans species complex and the C. gattii species complex. The turn-around time is less than ten minutes, and you don’t need any complicated tools or highly skilled workers. The primary benefit of this test is its capacity to detect extremely low levels of circulating CrAg during the prodromal phase (around 22 days before symptoms), enabling prompt treatment and thus reducing the overall mortality rates [81, 82]. Many companies have been in the development of CrAg-LFA, of which CrAg LFA from Immuno-Mycologics, Inc. (IMMY; Norman, OK) is the only FDA approved one.

It has been reported that the reliability of CrAg LFA falls out recently, thereby hindering an effective treatment of cryptococcal infections and causing a waste of time. Shi et al. [85] evaluated four commercially available LFAs with a set of well-defined C. gattii/C. neoformans species complexes. In this study, all seven pathogenic Cryptococcus species were detected by the IMMY CrAg LFA and FungiXpert Cryptococcal Capsular Polysaccharide Detection K-Set LFA (FungiXpert, Era Biology, Tianjin, China). However, Cryptococcus bacillisporus and some Cryptococcus tetragattii strains could not be detected by the Biosynex LFA. This implies the importance of the consideration of the revised cryptococcal taxonomy in the product setup and validation. Furthermore, Liu et al. [86] evaluated the diagnostic performance of FungiXpert LFA and the IMMY CrAg LFA using eight cerebrospinal fluid (CSF) and 119 serum/plasma samples. Compared to IMMY CrAg LFA, the FungiXpert LFA demonstrated 99.1% sensitivity and 98.9% specificity in the qualitative test. The Intraclass Correlation Coefficient of the semi-quantitative results of CrAg titer tests via the two assays was 0.976. This indicates that FungiXpert LFA is also a rapid screening method for the effective and practical diagnosis and treatment of cryptococcosis.

Conclusion:
GMI was higher in the patient while neutropenia was present, but as neutropenia resolved, GMI decreased. It indicates that a negative GM test is due to increased neutrophil counts in 10 days or less, rather than the patient no longer having IA. Furthermore, a negative GM result in a patient on antifungal prophylaxis or in an immunocompromised non-neutropenic patient does not rule out the possibility of IA.

Abstract Number: 22

Conference Year: 2024

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