Neglected Human Systemic Mycoses Diagnosis

Neglected Human Systemic Mycoses Diagnosis: Comparison

Please note this is a comparison between Version 1 by Clayton Luiz Borges and Version 2 by Peter Tang.

Systemic mycoses have been viewed as neglected diseases and they are responsible for deaths and disabilities around the world. Rapid, low-cost, simple, highly-specific and sensitive diagnostic tests are critical components of patient care, disease control and active surveillance. However, the diagnosis of fungal infections represents a great challenge because of the decline in the expertise needed for identifying fungi, and a reduced number of instruments and assays specific to fungal identification.

systemic mycoses
diagnosis
new targets
in silico approaches

1. Introduction

The diagnosis of systemic mycoses can often be a challenge; however, it is very important to ensure the most appropriate treatment and clinical follow-up to monitor treatment effectiveness and side effects [1]. In this sense, a fast and accurate diagnosis could reduce the empirical antifungal therapies, impact on evolutionary selection pressure and contribute to resistance emergence management [2]. The main challenges are the lack of sensitive and specific methods for early diagnosis, the lack of standardization of serological and molecular tests, the wide antigenic variability of the clinical isolates, and the fastidious and slow-growing nature of some fungal species [1]. In addition, in low-prevalence areas, the positive predictive values of some nonculture-based tests could be significantly lower than in endemic areas.

Overall, histopathologic, direct and culture examinations from clinical samples are often used as the standard diagnostic for systemic mycoses. Although attempts to culture the microorganisms should always be pursued, culture is less effective when the fungal burden is low or depending on the clinical form or type of fungal infection. For instance, Pneumocystis jirovecii do not grow in vitro [3], and Paracoccidioides species are usually isolated in only 10 to 20% of culture examinations and can take up to a month to grow. Detection of antibodies or antigens provides valuable information about current disease and is important for the management of fungal infections. However, it is often unavailable for most mycoses. In addition, molecular approaches could be useful in detecting fungal DNA in low fungal burden cases, mainly from biological samples, but these approaches are still not well standardized (Table 1). Despite the challenges above, efforts to properly identify the pathological agent are pivotal, since early treatment, which depends on the correct diagnosis, can prevent complications and help to reduce the morbidity and mortality of the systemic fungal infections.

2. Paracoccidioidomycosis

Paracoccidioides spp. are endemic fungi restricted to Latin America ^[3][4][5][3,4,5]. The genus is composed of six human pathogenic species, Paracoccidioides brasiliensis, P. lutzii, P. americana, P. restrepiensis, P. venezuelensis [6] and the non-culturable P. loboi [7]. Brazil has a high incidence of PCM in the South, Southeast and Midwest regions, where the prevalence is related to agricultural work ^[5][8][9][5,8,9]. Rural workers are the most affected individuals, where males have a greater PCM distribution compared to females, which can be explained by female hormones [10]. The infection is triggered by the inhalation of conidia or mycelia propagules ^[5][11][5,11]. Inside the host, the inhaled fungus produces the yeast form that can disseminate to several sites, causing the acute/subacute or chronic disease forms, depending on host-parasite interaction ^[10][12][10,12].

The gold standard for definitively diagnosing PCM is the visualization of yeast cells with typical multiple budding aspects (“ship-pilot´s wheel” or “Mickey Mouse head”) in fresh examination of sputum or other clinical samples (scraped from lesion, lymph node aspiration, lesion sample, etc.). However, a low percentage of positive samples result in positive cultures. The detection of serum antibodies is diagnostically valuable and is particularly important to clinical follow-up as it allows the evaluation of the host response during specific antifungal treatment. The DID test has high specificity (100%) and sensitivity (from 65 to 100%) depending on the fungus isolate, endemic area of isolation, the culture conditions, the technique used and antigen profile ^{[11][12][13][14]}[11,12,13,14]. In a comparison between WB versus DID with a purified glycoprotein of 43,000 Daltons from P. brasiliensis (Gp43), DID showed sensitivity (80%) and specificity (>90) of the test in an endemic area (different regions of Brazil), without false positive results or cross reaction [14]. Although ELISA (88–95% of specificity) is less specific than DID, mainly due to cross-reaction with histoplasmosis [15], it is highly sensitive (up to 100%), fast and suitable for PCM high-throughput screening [16]. Overall, among available serological techniques in the detection of serum antibodies, DID is the best method in patients with suspected PCM. On the other hand, Gp43 and Gp70 were described as good markers for monitoring antigen clearance during antifungal treatment by ELISA assay ^[17][18][17,18]. The usage of the Gp43 marker has become less useful since this antigen is usually not identified in the infections caused by P. lutzii ^[16][19][16,19]. The most common specimen employed in serological diagnostics is patient sera; however, the use of cerebrospinal fluid and bronchoalveolar lavage (BAL) specimens increases the sensitivity for antigen detection in the central nervous system and in pulmonary infections, respectively [20].

Table 1.

Neglected Human Systemic Mycoses Diagnosis.

Mycosis	Diagnostic Test	Human Specimen	Time until Results	Accuracy	Advantage	Disadvantage	Infrastructural Resources	References
Paracoccidioidomycosis (PCM)	Double Immunodiffusion test	Serum	This technique requires much time.	Specificity 100% and Sensitivity 65–100%	The choice method in patients with suspected PCM without false positive results or cross reaction.	Low accuracy for determination of the patient’s cellular immunity important during the therapy.	Laborious. However, it is not expensive.	^[12]^[13]^[17][12,13,17]
	ELISA	Serum	This technique is fast.	Specificity 88–95%. Sensitivity 100%	Fast and suitable for PCM high-throughput screening.	Cross-reaction with Histoplasmmosis	Requires specific equipment and automatization.	^[15][15^[16],16]
	Nucleic Acid testing	Blood, and clinical specimen	Result is obtained in a few hours.	Specificity 100%	Genotypic studies and clinical diagnosis performed directly from samples.	Need to standardize techniques based on DNA amplification for its real implementation.	Requires a specific and high-cost equipment.	[21]
Histoplasmosis	Immunodiffusion	Serum	This technique requires much time.	Sensitivity 60–70%	Rapid turnaround time	Not be used in immunocompromised individuals, since this group may present increases in false-negative results due to the compromise of the humoral response.	Low costs and simple infrastructure	[22]
	Complement Fixation	Serum	This technique requires much time.	Sensitivity 60–70%	Rapid turnaround time		Laborious technique and requires well trained personnel	[22]
	Enzyme Immunoassay	Serum, Plasma, Urine/CSF/BAL/Other Body Fluid.	Fast. (approximately 1.5 hours)	Sensitivity ranges 95–100% in urine, over 90% in serum and BAL antigens and 78% in cerebral spinal fluid (CSF)	Particularly important in AIDS patients who have disseminated histoplasmosis and who have large fungal burden	Serologic cross-reactions to Histoplasma-like antigens with Blastomycosis, Coccidioidomycosis, PCM and Aspergillosis	Requirement of specialized laboratories, expensive equipment, and well-trained personnel	^[22]^[23][22,23]
	Nucleic Acid testing	Blood and other body fluid.	The results are obtained in a few hours.	Specificity 100% and a sensitivity 67% to 100%.	Genotypic studies and clinical diagnosis performed directly from samples	Need to standardize techniques based on DNA amplification for its real implementation.	Requires specific and high-cost equipment.	^[24]^[25][24,25]
Aspergillosis	ELISA assays galactomannan (GM) detection *	Serum, lung transplant recipients, sputum or bronchoalveolar lavage.	This technique is fast.	Sensitivity 60 to 100% and specificity 85 to 98%	GM levels are proportional to fungal burden in tissue, and present prognostic value	Both false positive and false negative results have been reported and cross reactivity.	Performed without the need for specialized equipment and reagents	^[26]^[27]^[28][26,27,28]
Aspergillosis	Nucleic Acid testing	Serum, Lung transplant recipients, sputum or bronchoalveolar lavage.	The results are obtained in a few hours.	Specificity 100%	More sensitive and quick diagnosis	The lack of sensitivity and the difficulty in distinguishing between infection and colonization.	Requires specific equipment and has a high cost.	^[29]^[30]^[31][29,30,31].
Coccidioidomycosis	Direct examination	Sputum or bronchoalveolar lavage or other biopsy material	This technique is fast.	N/A	The gold standard diagnostic method	The mold form of Coccidioides produces highly infectious arthroconidia as soon as 72 hours after initial growth.	Requires well trained personnel	^[30]^[32][30,32]
	Culture	Sputum or bronchoalveolar lavage or other biopsy material	Requires a lot of time.	N/A	The gold standard diagnostic method	This form represents a significant risk of inhalational exposure to laboratory personnel.	The potential exposure risks associated with aerosolization	^[30]^[32]^[33][30,32,33]
	Enzyme immunoassays	Serum, urinary, and cerebrospinal fluid	This technique is fast.	Sensitivity 88%. Specificity 90%	Antibody detection EIA is a sensitive and specific test, including high-risk patients’ samples, in detection of IgG and IgM antibodies	Maybe insensitive to early infection.	Performed without the need for specialized equipment and reagents	^[30]^[32]^[34][30,32,34]
Cryptococcosis	Direct examination	Biopsy material	This technique is fast.	Sensitivity 60–90%	More sensitive and quick diagnosis	Lower sensitivity in HIV-negative patients in association with a low fungal burden.	Low-resource method	^[35]^[36][35,36]
	Culture exam	cerebrospinal fluid	1 to 2 weeks for definitive results	Sensitivity 85–95%	More sensitive. A gold standard for diagnostic	Need longer incubation periods up to three weeks.	The cultures are easily performed in any microbiology laboratory.	[37]
	Nucleic Acid testing	Plasma or cerebrospinal fluid	The results are obtained in a few hours.	Specificity 100%	Allows the determination of the Cryptococcus species	Need to standardize techniques based on DNA amplification for its real implementation.	Requires a specific and high-cost equipment.	[38]
	lateral flow assay	Plasma or cerebrospinal fluid	This technique is fast.	Sensitivity 90–100%	Provides a rapid diagnosis of cryptococcosis by detecting capsular antigen of Cryptococcus spp. In serum, plasma or CSF.	Low specificity (false positive 11% to 14%)	Low-costs	^[39]^[40][39,40]

* Unusual in routine or applied in specific situations.

It is known that PCM control depends on an effective cellular immune response [41]. In this context, in order to prevent disease recurrence, it has been suggested that the suspension of treatment which is, in fact, too prolonged should occur after the patient’s cellular immune response recovery. So far, there is no specific test to assess the cellular immune response of PCM patients during treatment. Even though DID only assesses humoral immunity [17], the serological cure has often been used as an immunological cure parameter. However, this correlation is not accurate, since the DID assay has little antibody-detection power, and its low accuracy prevents the method’s validation for determining the patient’s cellular immunity. Depending on the host’s immune response and/or serological tests, patients may present high antibody titers at the end of treatment without presenting disease symptoms, while others may have low titers even in the presence of clinical symptoms [42].

Another factor that limits the use of the DID assay as a patient follow-up is the fact that about 10 to 40% of patients with PCM may not show positivity in the immunodiffusion assay [6]. This is probably due to the different antigenic profiles of the Paracoccidioides species and may affect the diagnosis of PCM. In this sense, the researchers' group recently identified a set of B-cell epitopes exclusive to the Paracoccidioides complex and a set specific to each fungal species, which were developed from an immunoproteomic approach [43]. These epitopes demonstrated promising results on serological tests (data not shown), however, they still need to be widely validated.

3. Histoplasmosis

Histoplasma capsulatum is the causative agent of American histoplasmosis in both immunocompromised and immunocompetent individuals. The mycosis is the largely distributed in North America; however, it has broken through the barriers of the endemic areas of Ohio and the Mississippi River and is found in other regions around the world ^[44][45][46][44,45,46]. The presence of microconidia or small hyphae fragments of H. capsulatum in soil contaminated by bird or bat feces is the primary scenario of host-contact, where the infection occurs via inhalation of the airborne fragments ^[47][48][47,48]. The histoplasmosis can manifest as acute, subacute, and chronic pulmonary in immunocompetent individuals, while the disseminated cases are more common in immunocompromised individuals, particularly during HIV infection ^[49][50][49,50].

Early and rapid detection of histoplasmosis is essential in preventing morbidity and mortality, but remains challenging mainly in impaired immune system patients, such as individuals suffering from AIDS. In immunocompromised individuals, histoplasmosis becomes progressive and spreads rapidly from the lungs to other organs and is known as progressive disseminated histoplasmosis (PDH) [25]. The definitive diagnosis of histoplasmosis is accomplished by isolation of H. capsulatum in culture, as well as by visualization of the yeast form in samples [22]. However, these procedures lack sensitivity and are time consuming. Thus, antibody detection methods represent the major tools currently in use for non-culture diagnosis, predominantly because of their availability and rapid turnaround time. Nevertheless, this method should not be used in immunocompromised individuals, since this group may present an increase in false-negative results due to the compromised humoral response. Furthermore, serologic cross-reactions to Histoplasma-like antigens occur in patients presenting other systemic mycoses, such as Blastomycosis, Coccidioidomycosis, PCM and Aspergillosis ^[51][52][53][51,52,53].

Due to the high specificity of H. capsulatum, the identification of anti-H and anti-M antibodies using antigenic extract (histoplasmin) from mycelial culture is notably useful in serological diagnosis of histoplasmosis. The sensitivity of antibody detection by ID or CF is between 60% and 70% [22]. On the other hand, detection of the circulating H. capsulatum polysaccharide antigen in urine and serum is particularly important in AIDS patients who have disseminated histoplasmosis. Antigenuria can be used for monitoring the host response to antifungal treatment [22], while identification of antigens in BAL is useful in pulmonary histoplasmosis [54]. Recent advances highlight MiraVista Diagnostics, a company that developed three generations of EIA assay, with sensitivity ranges of between 95–100% in urine, over 90% in serum and BAL antigens, and 78% in cerebral spinal fluid (CSF) [22]. However, the high cost of testing is still an obstacle in using the assay for the diagnosis of histoplasmosis.

4. Coccidioidomycosis

Another endemic mycosis that primarily affects the lungs is coccidioidomycosis, caused by Coccidioides spp. This genus comprises Coccidioides immitis and Coccidioides posadasii, which also cause pulmonary disease in immunocompetent individuals. The endemic region of Coccidioides spp. is that with an arid climate where the infection occurs predominantly in the dry seasons [55]. Coccidioides spp. are prevalent in Mexico and the southwestern United States (Arizona, Texas and California) where the endemic areas were determined by skin test using spherulin or coccidioidin antigen preparations ^[56][57][56,57]. In South America, Coccidioides is present in Bolivia, Paraguay, Argentina, and Brazil ^[58][59][60][58,59,60]. The infection occurs after fungi arthroconidia inhalation, which reach the host’s pulmonary system and undergo dimorphic transition to yeast or spherule infective forms ^[57][61][57,61]. The clinical manifestation of coccidioidomycosis ranges from pulmonary infection to life-threatening pneumonia, to the dissemination of the infection to the tissues of the human body [62].

Clinical information is important, but the identification of Coccidioides on pathologic examinations or the isolation of fungus in culture are the gold standards for diagnosis of coccidioidomycosis. However, the use of these techniques is less frequent compared to the large number of actual cases, lack sensitivity, are time consuming, and require a degree of expertise to recognize the fungus ^[31][33][63][31,33,63].

Serology is the most used method of diagnosis [64]. Among methods used, EIA for antibody detection is the most regularly employed. Furthermore, two methods are commercially available, the Meridian Premier Coccidioides EIA (Cincinnati, OH, USA) and the Immuno-Mycologics Inc. (IMMY) Omega Coccidioides EIA (Norman, OK, USA), which are performed in most major reference laboratories and some laboratory hospitals. An alternative test is performed by detecting immunoglobulin G (IgG) by CF or ID and immunoglobulin M (IgM) antibodies by immunodiffusion. However, EIA is simpler to perform and provides same-day results, while ID and CF are difficult to execute and require 2 to 6 days to provide results [65].

A new test was developed by MiraVista Diagnostic (MVista), which demonstrates a sensitivity for IgG and/or IgM of 88% compared to 60% for ID and 66% for CF. Furthermore, the EIA MVista maintained similarly high sensitivity in immunocompromised patients (IgG 83% and IgM 56%) for whom ID sensitivity was reduced (IgG 40% and IgM 30%). It also maintained a 90% specificity and demonstrated low-to-moderate rates of cross-reactivity with other endemic mycoses (32% histoplasmosis and 8% blastomycosis) [34].

Malo et al. (2020) evaluated three commercial enzyme immunoassay kits: the IMMY omega EIA and the Meridian Premier EIA (for IgG detection) and IgM with the new EIA test, MVista Coccidioides test, and observed that the sensitivity of the IgG antibody detection was 87.4% using the MVista test compared to 46.6% for the IMMY test and 70.9% for the Meridian test. Similarly, the specificity of IgG and IgM antibodies was higher for the MVista EIA (90% and 95.3%, respectively), indicating that the MVista Coccidioides antibody detection EIA is a sensitive and specific test, including high-risk patients’ samples, in the detection of IgG and IgM antibodies [65].

Nevertheless, serologic tests for coccidioidomycosis may be insensitive to early infection [66]. Therefore, to minimize the potential for false-negative testing, serial serological testing is recommended, necessitating an initial serology followed by a second specimen from the convalescent phase of the disease. On the other hand, an isolated positive IgM EIA test should be followed closely with clinical correlation and subsequent diagnostic testing [66]. In many instances, this will generate serial testing, repeat EIA testing for IgM and IgG, or confirmatory testing by the immunodiffusion-tube precipitin reaction [67].

Recently, a monoclonal antibody ELISA test against coccidioidal CTS1 antigen has been developed [68]. CTS1 is also known as the “CF” antigen also used in coccidioidomycosis serodiagnosis by CF and ID tests.

5. Aspergillosis

Aspergillus species are agents of pulmonary aspergillosis. Despite numbering in their hundreds, only a few species have an impact on human health. The most clinically important fungi are: Aspergillus fumigatus ^[69][70][69,70], A. flavus [71] A. nidulans [72], A. niger [73] and A. terreus [74]. Aspergillus-related pathologies are caused by inhalation of airborne conidia which are encountered in air, soil, water, outdoor plants, as well as in hospitals [75]. Aspergillus species continue to be an important cause of life-threatening infection in immunocompromised patients, particularly those under prolonged corticosteroid therapy, immunosuppressive drugs, or with hematological malignancies, or people infected with HIV or individuals suffering from AIDS [76]. Aspergillosis presents with a wide range of clinical syndromes, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and the most severe, invasive pulmonary aspergillosis (IPA), which is linked to high mortality rates ^[70][75][77][70,75,77] Chronic pulmonary aspergillosis has been recognized as an important and neglected fungal infection ^{[78][79][80][81]}[78,79,80,81]. It is estimated that approximately three million cases of CPA aspergillosis occurred annually, being 2000 to 160,000 people after tuberculosis treatment, annually, with 50% case fatality in 5 years [82].

Diagnosis of aspergilloma or invasive aspergillosis (IA) can be difficult, and currently alternatives include laboratory tests such as histopathologic/cytologic and culture examination. Serum detection of galactomannan (GM) and (1,3)-beta-D glucan (BDG) were also recommended to be used in patients with specific clinical conditions, such as hematologic malignancy as well as submitted to allogeneic hematopoietic stem cell transplant ^[83][84][83,84]. GM is a cell wall component of Aspergillus that is released by the fungus [26] and can be detected through latex agglutination or ELISA assays. It has been suggested that GM levels are proportional to the fungal burden in tissues and present prognostic value [27]. In this context, IMMY diagnostics has developed an antigen assay lateral flow device with a sensitivity of 40% and specificity of 80%. In addition, the GM assay uses sandwich EIA for the diagnosis of invasive aspergillosis, although it can also be found on the H. capsulatum and Fusarium spp. cell walls ^[85][86][85,86].

CPA is possibly the systemic mycosis where serological testing is most useful. While antigen detection tests such as galactomannan are important in IA, it is only positive in about 25% of CPA patients [87]. Anti-aspergillus tests are, however, positive in over 90% of patients. In practice, precipitation techniques were replaced by an ELISA IgG antibody detection test [88], because it was the fastest, the most sensitive [89], and easily automated, although it is lacking in specificity. In the last decade, several commercial Aspergillus-specific IgG ELISAs have been developed and are now widely used in CPA diagnosis [88].

Finally, the ABPA is a hypersensitivity reaction test to Aspergillus species (generally A. fumigatus) that occurs almost exclusively in patients with cystic fibrosis or, less commonly, with asthma. The ABPA diagnosis can be confirmed by IgE levels and A. fumigatus–specific antibody detection and by visualization of the yeast forms in clinical samples with the help of fungus-enhancing staining, such as calcofluor white, or using histopathology techniques [84].

6. Cryptococcosis

Cryptococcal meningitis, the most severe form of this systemic mycosis, it is listed in the G-Finder report, and meets the WHO and PLOS definitions of a neglected disease, affecting disproportionately populations in poverty, causing high morbidity and mortality, and being neglected by research ^[81][90][91][81,90,91].

Two complexes of the Cryptococcus species, which determine epidemiologically distinct clinical conditions, are responsible for cryptococcosis. Cryptococcus neoformans and C. gattii are associated with conditions of host cellular immunodepression. In the past, C. gattii, was considered the agent of primary cryptococcosis in apparently normal hosts, however, both have been found in immunocompromised and immunocompetent individuals ^[92][93][92,93]. Furthermore, both complexes cause meningoencephalitis, which has a severe and fatal evolution and can be accompanied by evident lung injury. Additional effects of the infection include fungemia and secondary foci for skin, bones, kidneys, and adrenal, among others ^[93][94][93,94].

Currently, the diagnosis of cryptococcosis, as well as most systemic mycoses, is performed in routine laboratories by mycological examination using histopathological and immunological tests. For better observation of the capsule, a drop of Indian ink dye should be added on a microscope slide. The sensitivity of this test assessed on CSF samples ranges from 60% to 90%, according to the analyst’s expertise and according to the fungal load. However, direct examination of blood samples has low sensitivity, and its use has not been recommended ^[35][36][95][35,36,95].

Isolation of the etiologic agent by culture allows morphological, biochemical and molecular analysis of the clinical strain. For CSF samples, the culture exam sensitivity is from 85% to 95%, according to the disease stage and fungal load [37]. The presumptive identification of the Cryptococcus genus is performed by microscopic examination of a portion of the culture that allows the presence of yeast to be checked with a capsule without the production of hyphae (some strains may form short pseudo hyphae). The positive urease test minimally complements the presumptive genus identification. The characteristic production of melanin in Guizzotia absynica agar is essential in the diagnostic laboratory, to show that it is a question of which pathogenic species is being examined [38]. For histopathological examination, the sample should be stained with Mayer’s mucicarmine, that highlights the capsule in red and is therefore particularly useful in hypocapsulated strain infections [96]. Meanwhile, the Fontana–Masson staining shows the melanin of the cell wall and, therefore, it is also specific for both Cryptococcus complexes species, because in the other Cryptococcus members the reaction is negative. Other methods, such as periodic acid from Schiff (PAS) and Groccot–Gomori can be used, however, they are non-specific. Hematoxylin–eosin staining is also useful to check the tissue reaction profile, which is quite poor in general, but eventually allows the verification of granulomatous reaction in immunocompetent patients or infections with hypocapsulated strains [97]. In addition, the MALDI-TOF MS approach allows the correct identification of species from the two complexes, provided commercial standard databases are properly enriched ^[98][99][98,99].

Immunological examination provides a rapid diagnosis of cryptococcosis by detecting the capsular antigen of Cryptococcus spp. in serum, plasma or CSF. Quantitative examination by antigen titration has a prognostic value. In cryptococcal infection, unlike other invasive mycoses, the humoral response, assessed by antibody quantification, is poor and therefore this method is not suitable for diagnosis. Otherwise, the capsular antigen (Cryptococcus Antigen-CrAg), represented by polysaccharide molecules, is soluble in several body fluids and can be detected in CSF in cases of meningitis and in the serum of patients with and without meningitis weeks to months before symptoms of the disease present [100]. All species of Cryptococcus spp. have capsular antigens and, therefore, a positive test indicates active disease, but does not allow identification of the species of the etiologic agent.

The search for capsular antigens was traditionally carried out by an agglutination test with latex particles sensitized with antibodies against Cryptococcus. A more recent qualitative and quantitative test for capsular antigens carried out on strip has been available. It was developed to be cheaper and simpler, with the point of care based on the immunochromatography reaction denominated lateral flow assay (LFA). The test stripes can be stored, for up to two years, outside refrigeration and have been designated as point-of-care tests. Other manufacturers, from France, China, and Denmark, commercialize LFA for cryptococcosis diagnosis, but few studies have been conducted for evaluating the performance of the new trend marks, in comparison to the IMMY LFA. The majority of the following information was obtained using the north American test [101]. The LFA test is more sensitive in the detection of polysaccharides from the four Cryptococcus serotypes compared to the latex test. For children, data on the accuracy of the test are limited, although it is estimated that its performance is similar to that observed in adults [102].

The LFA test has greater analytical sensitivity compared to others when it comes to antigens of both C. neoformans and C. gattii [103]. A small amount of sample is sufficient to diagnose cryptococcal meningitis, initial or advanced, with sensitivity from 97.6% to 100% (serum) and between 94.0% to 100% (CSF) ^{[40][89][104][105]}[40,89,104,105] without the need for laboratory equipment or technical expertise, which makes the test ideal for regions with limited resources [106]. The specificity of the test in serum reaches 98.1% and in CSF 98.9%. For pulmonary forms there are few studies with LFA, indicating that the test is better than latex, since the sensitivity reaches 100% with serum samples [102].

For HIV-positive patients with cryptococcal meningitis, samples of whole blood, collected on a fingertip, eluted on the LFA test strip, resulted in equal sensitivity (95%) to that obtained with serum and CSF samples. The specificity of the test, performed with sera from HIV positive patients with other diseases of the central nervous system, was 100%, but urine samples from these patients were positive in only 80% of the cases in the same study, indicating limitation in the use of this sample for antigen research [107]. In fact, the specificity of the test with urine samples is still too unreliable to recommend this procedure ^[108][109][108,109].

LFA is particularly useful in patients with HIV infection in whom early detection of cryptococcosis, followed by pre-emptive antifungal therapy with fluconazole (screen and treat strategy), reduces disease progress and mortality from meningitis ^{[109][110][111][112]}[109,110,111,112]. The WHO recommends antigen research (serum, CSF, plasma, whole blood) for screening cryptococcosis in adult and adolescent populations in regions where the prevalence of cryptococcal antigenemia is high (>3%), although it appears to be cost-effective in regions with lower antigenic prevalence, such as 0.06% ^[39][113][39,113].