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Cryptococcal lateral flow antigen assays (CLFAs) have been assessed in comparison to the latex cryptococcal antigen agglutination test but their clinical performance is unknown.Determine clinical performance of IMMY CLFA (Immuno-Mycologics Inc, Oklahoma) using patients with and without cryptococcosis as the reference standard.One-hundred ninety-seven serum samples from client-owned dogs and cats.Review of medical records of a referral population of dogs and cats that had CLFA performed between 2012 and 2020. Animals were classified as cryptococcosis positive (Cr+) or negative (Cr-) based on clinical information. Clinical diagnosis was used to calculate positive and negative percent agreement of the CLFA.Twelve specimens (4 canine, 8 feline) were obtained from Cr+ animals and had positive CLFA results. One-hundred eighty-five specimens (139 canine, 46 feline) were collected from Cr- animals. Negative CLFA results were recorded in 129 canine and 44 feline Cr- samples. Positive CLFA results were noted for 10 canine and 2 feline Cr- samples. Positive percent agreement of CLFA was 100% (confidence interval [CI], 39.8%-100% dogs; 63.1%-100% cats). Negative percent agreements were 92.8% (CI, 87.2%-96.5%) for dogs and 95.7% (CI, 85.2%-99.5%) for cats.A negative IMMY CLFA result enables reliable exclusion of cryptococcal infection in dogs and cats. By contrast, a positive result must be interpreted cautiously and further testing should be performed to verify a diagnosis of cryptococcosis.

RATIONALE Epidemiologic studies have demonstrated that exposure to molds and other fungi can play a role in a variety of allergic and pulmonary diseases in susceptible individuals. Species-specific mold antigen extracts are used in the clinical evaluation of suspected mold-related conditions, however alignment between these extracts and the species of molds identified in the indoor environment of water-damaged homes has not been rigorously evaluated. OBJECTIVES To identify the predominant genera and species of mold in the air of homes with water damage, mold growth, and/or occupants with respiratory complaints (complaint homes), and to assess their alignment with the mold antigen extracts used in clinical practice. METHODS The genera and species of molds identified in culture-type outdoor and indoor air samples collected from complaint homes throughout the U.S. and Canada from 2002-2017 were examined. Mold antigen extracts available and utilized for skin and serum testing in clinical practice were assessed and alignment between these data were evaluated. RESULTS Culture data from 24,455 indoor air samples from 7,547 complaint homes and 29,493 outdoor samples was evaluated. Mean exposure values (CFU/m3) were calculated for each genus and species, and indoor vs outdoor values compared. Penicillium was the predominant genus identified in water-damaged homes, with a mean exposure (233.3 CFU/m3) 2.9 times higher than that of the Aspergillus genus (81.4 CFU/m3). Five Penicillium (P. aurantiogriseum, P. brevicompactum, P. citrinum, P. crustosum, and P. variabile) and three Aspergillus (A. versicolor, A. sydowii, and A. niger) species were identified as the predominant indoor water-damage related fungi. However, none of these Penicillium species and only one of the Aspergillus species is currently available as an antigen extract for use in skin testing or serum testing panels. CONCLUSIONS Significant misalignment exists between the currently available mold antigen extracts and the predominant species of molds found in water-damaged homes. Improving alignment has the potential to enhance diagnosis of mold-related diseases including allergic asthma and hypersensitivity pneumonitis and to improve patient outcomes via interventions including antigen avoidance through building remediation and occupant relocation, consistent with the findings of a recent ATS Workshop Report.

Objectives Cryptococcal meningitis constitutes a significant source of mortality in the developing world. Annually, approximately 625 000 deaths occur worldwide among patients with human immunodeficiency virus (HIV) infection. This study aims to assess the cost-effectiveness of implementing cryptococcal antigen lateral flow assay (CRAG-LFA) screening in Brazil compared with the current practice. Methods An economic evaluation using a Monte Carlo microsimulation was conducted, considering the perspective of the Brazilian Public Health System, to calculate the cost-effectiveness of 4 diagnosis tests: (1) CRAG-LFA, (2) the cryptococcal antigen latex agglutination (CRAG-LA) test, (3) India ink, and (4) nontracking as a baseline. The time horizon comprised 1 year for the intervention and 5 years for the budgetary impact analysis. Two primary effectiveness outcomes were considered: years of life and quality-adjusted life-years. Results CRAG-LFA has extended dominance vis a vis CRAG-LA and India ink. CRAG-LFA would cost $418.46 more than CRAG-LA for the treatment of each symptomatic patient living with HIV, with an incremental cost effectiveness ratio of $2478.75/quality-adjusted life year. The budgetary impact analysis estimated that the incorporation of CRAG-LFA would have an additional cost of $1 959 236.50 in 5 years. Conclusions These findings suggest that, for patients living with HIV in the Brazilian Public Health System, the adoption of CRAG-LFA screening is cost-effective compared with the use of CRAG-LA and India ink. It represents an opportunity to prevent cryptococcal meningitis and its mortality in Brazil.

Establishing diagnosis of latent and active histoplasmosis is challenging. Interferon gamma-release assays (IGRAs) may provide evidence of latent and active infection. An enzyme-linked immunospot (ELISpot) assay was developed using yeast cell lysate (YCL) antigen prepared from a representative North American Histoplasma capsulatum strain. Assay parameters were optimized by measuring responses in healthy volunteers with and withoutiHistoplasma/iinfection. Assay performance as an aid for diagnosing histoplasmosis was assessed in a prospective cohort of 88 people with suspected or confirmed infection, and 44 healthy controls enrolled in two centers in North America (2013 to 2018). Antigen specificity of IFN-γ release was demonstrated using ELISpot and enzyme-linked immunosorbent assay (ELISA). Antigen-evoked, single-cell mRNA expression by memory T cells was shown using flow cytometry. The area under the receiver operating characteristic curve (AUC) was estimated at 0.89 (95% confidence interval [CI]: 78.5% to 99.9%). At optimal cutoff, sensitivity was 77.2% (95% CI: 54.6% to 92.2%) and specificity was 100% (95% CI: 89.7% to 100%). Sixteen of 44 healthy volunteers (36.4%) from a region of hyperendemicity had positive responses, suggesting detection of previously unrecognized (latent) infection. The ELISpot assay is sensitive and specific as an aid to diagnose H. capsulatum infection and disease, supporting proof of concept and further development.

Background Diagnosis of invasive candidiasis (IC) relies on insensitive cultures; the relative utility of fungal biomarkers in children is unclear. Methods This multinational observational cohort study enrolled patients aged >120 days and Results Five hundred participants were enrolled at 22 centers in 3 countries, and IC was diagnosed in 13 (2.6%). Thirteen additional blood specimens were collected and successfully spiked with Candida species, to achieve a 5.0% event rate. Valid T2Candida, Fungitell, Platelia Candida Ag Plus, and Platelia Candida Ab Plus assay results were available for 438, 467, 473, and 473 specimens, respectively. Operating characteristics for T2Candida were most optimal for detecting IC due to any Candida species, with results as follows: sensitivity, 80.0% (95% confidence interval, 59.3%–93.2%), specificity 97.1% (95.0%–98.5%), positive predictive value, 62.5% (43.7%–78.9%), and negative predictive value, 98.8% (97.2%–99.6%). Only T2Candida and Platelia Candida Ag Plus assays met the threshold for combination testing. Positive result for either yielded the following results: sensitivity, 86.4% (95% confidence interval, 65.1%– 97.1%); specificity, 94.7% (92.0%–96.7%); positive predictive value, 47.5% (31.5%–63.9%); and negative predictive value, 99.2% (97.7%–99.8%). Conclusions T2Candida alone or in combination with Platelia Candida Ag Plus may be beneficial for rapid detection of Candida species in children with concern for IC. Clinical Trials Registration NCT02220790.

Background Cryptococcal meningitis (CM) has a high morbidity and mortality due to the low detection of Cryptococcus in cerebrospinal fluid (CSF) during the early stage of the disease with traditional methods. Case presentation In addition to the traditional methods of India ink staining and cryptococcal antigen (CrAg), we used nanopore sequencing and next-generation sequencing (NGS) to detect pathogenic DNA in CSF samples of three patients with CM. The CSF samples of all three patients were positive by India ink staining and CrAg. NGS also detected Cryptococcus in all three CSF samples. Nanopore sequencing detected Cryptococcus in two CSF samples. Conclusion Nanopore sequencing may be useful in assisting with the clinical diagnosis of CM. Further research is needed to determine the sensitivity and specificity of nanopore sequencing of CSF.

Therapies for plantar warts remain subjective and unclear, which has led to continual pursuit of an optimal treatment. As a consequence, many intralesional therapies have emerged over the last decade. This warrants a systematic review from a clinical lens which provides updates on intralesional treatment options for plantar warts from the last decade.A PubMed/MEDLINE literature search was performed, in accordance with PRISMA reporting guidelines for systematic reviews. Original peer-reviewed articles on safety/efficacy of intralesional plantar wart treatments, published from January 2012 to January 2021, were considered for inclusion.Twenty-6 studies were included and the following intralesional modalities were identified (median cure rates): vitamin D3 (80%), bleomycin (74%), 5-fluorouracil (59%), Candida antigen (66%), zinc sulfate (70%), and purified protein derivative (67%).Intralesional vitamin D3, in particular, demonstrated promising results as a potential second- or even first-line agent although not accessible in the United States. Candida antigen and bleomycin are less effective than intralesional vitamin D3, but given their greater accessibility and superiority to cryotherapy, should continue to be considered for treating recalcitrant plantar warts. Moreover, the quadrivalent human papillomavirus (HPV) vaccine, showing success in case reports, warrants further attention for both the treatment and prevention of plantar warts. J Drugs Dermatol. 2022;21(12):1322-1329. doi:10.36849/JDD.6735.

Pulmonary cryptococcosis (PC) is a rare fungal lung infection that usually occurs in immunocompromised hosts. We report a rare case of PC with asymptomatic and mixed lesions of infiltrative and nodular shadows. A woman in her 50s with a 7-year history of methotrexate treatment for rheumatoid arthritis (RA) presented with abnormal chest shadows on annual examination. Chest CT revealed two tiny nodules and two infiltrative shadows in the right lower lobe of the lung. We suspected lung cancer, cryptogenic organising pneumonia or RA-interstitial lung disease. However, transbronchial lung biopsy and positivity for serum cryptococcal antigen confirmed the diagnosis of PC. We initiated treatment with fluconazole, which drastically reduced the chest shadows without any adverse events. Since PC can present with several pulmonary shadows, it should be considered as a differential diagnosis of any pulmonary lesion.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on β1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of “probable” invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4—deviating from the 0.5 threshold recommended by the manufacturer—sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

The detection of antibodies against Histoplasma capsulatum remains a frequently relied-on approach to diagnose histoplasmosis. We retrospectively assessed the performances of complement fixation (CF) and immunodiffusion (ID) assays for anti-iHistoplasma/iantibody detection in patients with culture-confirmed histoplasmosis at Mayo Clinic (Rochester, MN) over a 10-year period (2011 to 2020). Among 67 culture-confirmed patients who also had H. capsulatum CF/ID testing ordered, 51 (67.1%) were immunocompromised, 34 (50.7%) had localized disease, and 51 (76.1%) presented withlt;3 months of symptoms before testing. H. capsulatum CF and/or ID testing was positive in 47 (70.1%) patients, with both assays being positive in 39 cases. CF was positive in 44 (65.7%) patients, with reactivity against both H. capsulatum mycelial and yeast antigens in 30 (68.2%) cases, whereas 11 (25%) and 3 (6.8%) individuals had antibodies to the CF yeast or mycelial antigen only, respectively. H. capsulatum ID was positive in 42 (62.7%) patients, with the presence of the M-band only or the H- and M-bands in 27 (64.3%) and 15 (35.7%) cases, respectively. Among 18 serially tested patients, 12 remained ID and/or CF positive at the final time point (median, 154 days; range, 20 to 480 days). Serial CF testing showed that antibodies to the mycelial antigen serorevert to negative more frequently (6/11) than antibodies to the yeast antigen (2/13). There was no statistically significant difference in antibody positivity relative to patient immune status, degree of disease dissemination, or symptom duration. Serologic testing remains a valuable asset to support the diagnosis of histoplasmosis, particularly when direct detection methods fail to identify an infection.

Background: People living with advanced HIV disease are at high risk of morbidity and mortality. We assessed the prevalence of cryptococcal antigenemia (CrAg) and clinical outcomes among patients newly presenting with CD4 ≤100 cells/μL in Vietnam. Setting: Twenty-two public HIV clinics in Vietnam. Methods: During August 2015–March 2017, antiretroviral therapy (ART)-naïve adults presenting for care with CD4 ≤100 cells/μL were screened for CrAg. Those who consented to study enrollment were followed up for up to 12 months and assessed for clinical outcomes. Results: Of 3504 patients with CD4 results, 1354 (38.6%) had CD4 ≤100 cells/μL, of whom 1177 (86.9%) enrolled in the study. The median age was 35 years (interquartile range 30–40); 872 (74.1%) of them were men, and 892 (75.8%) had CD4

Background Increasing incidence of invasive infections caused by rare fungi was observed over the recent years. Case Here, we describe the first reported case of an infection caused by the thermophilic mold Talaromyces thermophilus. Cultivation and, hence, identification of this fastidious organism is challenging since standard incubation conditions are not sufficient. Retrospective analysis of patient samples and in vitro experiments demonstrated that testing for fungal antigens, i.e., the cell wall components galactomannan and β-1,3-d-glucan, is a promising tool.

Objectives Histoplasmosis and cryptococcosis are important public health problems in people living with HIV (PLHIV) in Central America. Conventional laboratory assays, based on microscopy and culture, are not optimal for the diagnosis of either disease. However, antigen (Ag) assays are rapid and highly accurate for the diagnosis of these infections. Methods Laboratory surveillance of PLHIV was carried out in four hospitals in Panama, Honduras and Nicaragua, between 2015 and 2019. Detection of Histoplasma antigens in urine was performed by enzyme immunoassay (EIA), and Cryptococcus antigen detection in sera and cerebrospinal fluid specimens was performed by lateral flow assay (LFA). Results A total of 4,453 PLHIV with clinical suspicion of histoplasmosis (n = 1,343) or cryptococcosis (n = 3,110; 2,721 sera and 389 CSF) were tested. Of 1,343 patients suspected of having histoplasmosis, 269 (20%) were Histoplasma Ag positive. Of 3,110 patients tested using the Cryptococcus Ag assay, 329 (11%) were positive. Honduras reported the highest positivity rates (32% for Histoplasma Ag, and 16% for Cryptococcus Ag); Panama reported the largest number of patients testing positive using the Histoplasma Ag assay (n = 201); and Nicaragua reported the largest number of patients testing positive using the Cryptococcus Ag assay (n = 170). Conclusion Here, we show how the implementation of rapid diagnostics assays impacted case detection and was useful for the care of people with advanced HIV. Rapid and accurate diagnosis could reduce mortality associated with histoplasmosis and cryptococcosis in PLHIV.

Paracoccidioidomycosis (PCM) is caused by the fungi Paracoccidioides spp. The main antigens recognized by IgE are known for P. brasiliensis species complex, but not for P. lutzii. Current research investigated the major P. lutzii (LDR2) antigens recognized by IgE, in comparison to P. restrepiensis and P. americana (former P. brasiliensis species complex), besides IgG recognition. Cell-free antigens (CFA) from P. lutzii (LDR2), P. restrepiensis (B339) and P. americana (LDR3) were analyzed by ELISA and immunoblotting (IB) by detecting specific IgG and IgE from sera from patients with PCM presumable by P. brasiliensis species complex (n = 24). Additionally, somatic antigen (SA) was analyzed by IB. P. lutzii (LDR2) antigens showed significantly lower reactivity than P. restrepiensis (B339) and P. americana (LDR3) in ELISA for both IgE and IgG (p

Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic tools are restricted. We carried-out a 1-year prospective cohort study at a referral hospital in São Paulo, Brazil. Participants had > or =18 years old, were hospitalized due to any indication and had CD4+ < 200 cells/µl. A urine commercial monoclonal Histoplasma galactomannan enzyme-linked immunosorbent assay (IMMY, Norman, OK, USA) and ‘in house’ Histoplasma blood nested PCR were performed in all cases. Probable/proven DH cases were defined according to international guidelines. Conventional mycological methods were available in routine conditions to investigate suspected DH cases. Treatment of participants followed the institutional routine. One-hundred six participants were included. Median age (interquartile range [IQR]) was 39.5 years (30.0–47.3) and 80 individuals (75.5%) were males. Median (IQR) CD4 cell count was 26.5 (9.4–89.3) cells/mm3. DH was diagnosed in 8/106 patients (7.5%). Antigen assay and/or PCR were positive in 4.7% (5/106) of patients. The antigen assay and/or PCR identified 37.5% (3/8) of DH cases, which had not been diagnosed with conventional mycological methods, but had clinical manifestations compatible with HD. In conclusion, the use of Histoplasma urine antigen and Histoplasma blood PCR guided by CD4 status contributed to the diagnosis of DH in hospitalized individuals. These assays were complementary to conventional mycologic methods and are urgently needed in our setting. Lay Summary In this prospective cohort study carried-out in a referral center in São Paulo, Brazil, we found a high frequency of AIDS-related disseminated histoplasmosis (8/106, 7.5%). We used urine antigen test and blood PCR assay to improve the diagnosis of this opportunistic disease.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients’ sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β- and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

There is scarce information about HIV-related cryptococcosis in the Brazilian Amazon basin where laboratory infrastructure is limited. The serum cryptococcal antigen (CrAg) lateral flow assay (LFA) has simplified diagnosis of cryptococcosis and is recommended for screening in advanced HIV disease. We evaluated the prevalence of cryptococcal antigenemia using finger-prick CrAg LFA in the Brazilian Amazon basin. We enrolled a prospective cohort of outpatients and hospitalized individuals with advanced HIV disease at two centers in Santarém Municipality, Northern Brazil. All individuals were 18 years old with advanced HIV disease, regardless of antiretroviral therapy (ART) status and with no prior or current history of confirmed cryptococcal meningitis. We tested CrAg LFA on finger-prick whole blood using an exact volume transfer pipette. From August 2018 to October 2019, 104 individuals were enrolled (outpatients 62 [60%] and hospitalized 42 [40%]). Median age was 38 years (interquartile range [IQR] 30-46), and 84 (81%) were male. Sixty-five (63%) individuals were ART-naïve. Prevalence of finger-prick CrAg LFA-positive was 10.6%; 95% CI, 5.4 to 18.1%. Prevalence of finger-prick CrAg LFA-positive among individuals without neurological symptoms was 6.0%; 95% CI, 1.7-14.6%. The number needed to test to detect one CrAg-positive individual was 9.4 persons (95% CI, 5.5-18.5). Prevalence of cryptococcal antigenemia using finger-prick whole blood CrAg LFA was high. Point-of-care approach was important for the diagnosis and screening of cryptococcosis in resource-limited settings. Screening and preemptive therapy strategy should be urgently implemented in individuals with advanced HIV disease in the Brazilian Amazon basin.This prospective cohort study was carried-out in the Brazilian Amazon basin. We used a cryptococcal rapid test in patients with AIDS. We included 104 participants, and 11 (10.6%) of them had positive results showing a high prevalence of cryptococcal antigenemia.

Cryptococcal antigen (CrAg) detection could direct the timely initiation of antifungal therapy. We searched MEDLINE and Embase for studies where CrAg detection in serum/cerebrospinal fluid (CSF) and CSF fungal culture were done on adults living with human immunodeficiency virus (HIV) who had suspected cryptococcal meningitis (CM). With Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2), we evaluated the risk of bias in 11 included studies with 3600 participants, and used a random-effects meta-analysis to obtain summary sensitivity and specificity of serum and CSF CrAg, as well as agreement between CSF CrAg and CSF culture. Summary sensitivity and specificity of serum CrAg were 99.7% (97.4–100) and 94.1% (88.3–98.1), respectively, and summary sensitivity and specificity of CSF CrAg were 98.8% (96.2–99.6) and 99.3% (96.7–99.9), respectively. Agreement between CSF CrAg and CSF culture was 98% (97–99). In adults living with HIV who have CM symptoms, serum CrAg negativity may rule out CM, while positivity should prompt induction antifungal therapy if lumbar puncture is not feasible. In a first episode of CM, CSF CrAg positivity is diagnostic.

Meningitis due to the fungal pathogen Cryptococcus neoformans is estimated to cause nearly 200,000 deaths annually, mostly in resource-limited regions. We previously identified cryptococcal protein antigens which, when delivered in glucan particles, afford vaccine-mediated protection against an otherwise lethal infection. Many of these proteins exhibit significant homology to other similar cryptococcal proteins leading us to hypothesize that protection may be augmented by immunologic cross-reactivity to multiple members of a protein family. To examine the significance of protein cross-reactivity in vaccination, we utilized strains of Cryptococcus that are genetically deficient in select antigens, yet are still lethal in mice. Vaccination with a protein without homologs (e.g., Mep1 and Lhc1) protected against challenge with wild-type Cryptococcus, but not against a deletion strain lacking that protein. Contrastingly, vaccination with a single chitin deacetylase (Cda) protein protected against the corresponding deletion strain, presumably due to host recognition of one or more other family members still expressed in this strain. Vaccination with a single Cda protein induced cross-reactive antibody and interferon-gamma (IFNγ) immune responses to other Cda protein family members. Paradoxically, we saw no evidence of cross-protection within the carboxypeptidase family of proteins. Factors such as in vivo protein expression and the degree of homology across the family could inform the extent to which vaccine-mediated immunity is amplified. Together, these data suggest a role for prioritizing protein families in fungal vaccine design: increasing the number of immune targets generated by a single antigen may improve efficacy while diminishing the risk of vaccine-resistant strains arising from gene mutations.

Cryptococcal antigen (CrAg) is a capsule polysaccharide antigen that can be detected in the fluids of patients with cryptococcal infections. Cryptococcal Antigen Latex Agglutination System (CALAS), enzyme-linked immunosorbent assays (EIA), and lateral flow assay (LFA) are the main methods available. Two main commercial LFA kits are available: CryptoPS (Biosynex, Illkirch Graffenstaden, France) and CrAg LFA (IMMY, Inc. USA). In our lab, we prospectively used CryptoPS as a screening tool in serum for confirmed positive results with CALAS. We investigated the rigor of the CryptoPS test in serum in a multicentric evaluation over 3 years. To improve the specificity of CryptoPS in serum, we additionally implemented and evaluated a pretreatment protocol before CryptoPS testing. A total of 43 serum samples collected from 43 patients were investigated. We found that the CryptoPS assay is hampered by a high rate of false-positive results in serum with a high rate of CryptoPS-positive but CrAg LFA-negative and CALAS-negative sera in patients with no proof of Cryptococcus infection (n = 29). Using a simple pretreatment procedure (5 min incubation at 100°C and centrifugation) we were able to reverse false-positive results, suggesting that there could be interferent material present in the serum. Pretreatment also impacted the CryptoPS results (negative result) in two patients with the cryptococcal disease, one with isolated antigenemia and one with cryptococcal meningitis. Comparing the titers obtained with CALAS and CrAg LFA, we noticed that the titer obtained with CrAg LFA was almost 10-fold higher than those with CALAS. This study showed that Biosynex CryptoPS in serum could give false-positive results even in the absence of cryptococcal disease. These could be reduced by applying an easy pretreatment procedure to the serum before testing, with little but existing impact on the sensitivity.Lateral flow assays are useful to detect the cryptococcal antigen in human fluids. We investigated CryptoPS-positive results and observed that true false-positive results occurred. The false-positive results can be reduced by applying an easy pretreatment procedure.

Background: Exposure to fungal allergens poses a serious threat to human health, especially to mould-allergic individuals. The prevalence of fungal allergic disease is increasing globally but is poorly studied in Africa. Here, we aimed to identify and characterize fungal proteins that were immunoreactive against serum samples from fungal-sensitized Zimbabweans from Shamva district to inform the development of diagnostics and therapeutics. Methods: Crude protein extracts of the Ascomycota Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Penicillium chrysogenum, and Saccharomyces cerevisiae as well as mucoromycota Rhizopus nigricans were individually separated by one-dimensional gel electrophoresis for protein staining and immunoblotting. A pool of eight sera from fungi-sensitive Zimbabwean children aged 3–5 years was used to screen the crude extracts to determine their immunoreactivity. Protein bands recognized by the sera were subjected to mass spectrometry to identify the individual proteins reactive with the sera. Results: The pooled serum sample reacted with 20 bands, which resolved to 34 distinct proteins, most of which were novel immunogens. The pool was most reactive to A. alternata. The proteins identified included peptidases (8/34), hydrolases (6/34), oxidoreductases (5/34), and glucosidases (4/34), while 11/34 were unknown. Eight of the proteins were predicted to be allergens using the Structural Database of Allergenic Proteins (SDAP). Conclusions: We identified novel immunogens from fungi expanding the number of known fungal allergens. These form a potential basis for diagnostics specific for the Zimbabwean population. Validation assays will now need to be carried out to further evaluate the cross-reactivity of the identified allergen candidates as well as investigate their potential recognition in a larger cohort of patients. Furthermore, there is now a need to conduct studies relating sensitization to these immunogens and clinical diseases in the population.

Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause cryptococcosis, a life-threatening systemic mycosis of global distribution affecting mainly immunocompromised adults. Although a humoral response occurs during cryptococcosis, the role of antibody production against this mycosis is not fully understood. We aimed to determine total and specific antibodies against cryptococcal protein antigens in sera from people with and without a diagnosis of cryptococcosis from Colombia. Using ELISA, total and specific levels of immunoglobulin (Ig)G, IgA and IgM were determined in sera from children and adults with (n = 109) and without (n = 119) cryptococcosis. Specific antibodies were those binding Cn- and Cg-protein antigens. In general, the mean of the total IgG production was higher in cryptococcosis patients than in controls (13 942.32 vs. 6459.91 µg/ml), while levels of IgA (488.13 vs. 1564.53 µg/ml) and IgM (775.69 vs. 1014.72 µg/ml) were higher in controls than in cryptococcosis patients (P ≤ .05). In patients with cryptococcosis, total IgG, IgA and IgM levels were higher in HIV + compared with HIV- (P ≤ .05). Specific antibodies tended to be higher in cryptococcosis patients than in controls and in adults than in children, with a positive correlation between antibody reactivity and age. All immunoglobulins were more reactive against Cn-proteins than Cg-proteins. Overall, a positive weak correlation between total and specific antibodies was found, although not always statistically significant. In patients with cryptococcosis from Colombia, the levels of immunoglobulins, total and specific, differ with respect to people without cryptococcosis. Variations in antibody production among adults and children with cryptococcosis and between Cn- and Cg-protein antigens were as well established. Our findings encourage further studies to determine the role of humoral immunity for host defense against cryptococcosis.Differential IgG, IgA, and IgM production and their reactivity with cryptococcal proteins, both among children and adults with and without a diagnosis of cryptococcosis from Colombia, lead to reappraise the study of the potential role of antibody production as host defense against this fungal infection.

Early diagnosis of cryptococcal meningitis among people living with HIV (PLHIV) is crucial for its therapeutic success. The objective of this study was to diagnose cryptococcal meningitis in PLHIV cases using the available laboratory techniques for its confirmation in resource limited setting. This cross-sectional prospective study was conducted among 72 PLHIV with clinical suspicion of meningitis. Each cerebrospinal fluid (CSF) sample received at the National Public Health Laboratory, Kathmandu was processed for India ink staining, cryptococcal antigen lateral flow assay, and fungal culture following standard protocols. The laboratory-confirmed cryptococcal meningitis cases were between 24 and 69 years of age (median age 39 years) with 87.5% (12/14) of cases being male. Cryptococcus was detected in 22.22% (16/72) by any of the three tests, 19.44% (14/72) by cryptococcal antigen lateral flow assay, 16.66% (12/72) by India ink staining, and 8.33% (6/72) by culture. High percentage of cryptococcal meningitis among PLHIV warrants early microbiological diagnosis for better case management. Cryptococcal antigen detection immunoassay should be the priority test for laboratory diagnosis of cryptococcal meningitis in PLHIV. Alternatively, very simple and economic India ink staining of CSF specimens could be used in resource limited settings.

Cryptococcosis is a neglected tropical infection and a major cause of morbidity and mortality, especially in HIV–positive persons in Africa. Efforts to manage HIV infection have not had any significant impact on the fatalities due to cryptococcosis. An integrated healthcare approach that includes universal care coverage for Africans, expanded national care guidelines to include CrAg screening for vulnerable groups in all African countries, collaborative research, infection surveillance, and data sharing within Africa will mark a turnaround point.

Cryptococcal disease (CD) is a leading cause of mortality among individuals with advanced HIV disease (AHD). Screening with serum cryptococcal antigen (sCrAg) lateral flow assay (LFA) enables early detection of subclinical disease but requires venipuncture and laboratory processing. Clinic-based point of care (POC) CrAg screening tests using urine or fingerprick whole blood could facilitate early diagnosis of CD. We evaluated the diagnostic performance of POC clinic-based fingerprick whole blood and urine CrAg compared to the gold standard laboratory sCrAg LFA in screening for CD among asymptomatic individuals with CD4 counts of 200 cells/μL in Harare, Zimbabwe. sCrAg positive participants who consented to a lumbar puncture also had cerebrospinal fluid (CSF) CrAg testing and titers for CSF-positive specimens. A total of 1,333 individuals were screened, and over half (56.6%) were males. The median (interquartile range) CD4 count was 27.5 (11-46) cells/μL. We found a sensitivity of 63.8% (95% CI: 54.8-72.1) and specificity of 84.0% (95% CI: 81.7-86.0) for urine CrAg, and a sensitivity of 48.0% (95% CI: 39.1-57.1) and specificity of 99.5% (95% CI: 98.9-99.8) was found for fingerprick whole blood. The sensitivity of both POC CrAg tests increased in individuals with sCrAg titers of ≥1:160, CD4 count of50 cells/μL and disseminated central nervous system (CNS) disease. Clinic-based POC urine and fingerprick whole blood CrAg testing performed better in screening for CD among AHD patients with CNS disease. More sensitive assays to identify AHD patients with asymptomatic CD are needed.

The Genital warts are common sexually transmitted diseases caused by definite types of human papillomavirus. There are many strategies for the treatment of genital wart and intralesional immunotherapy is considered to be a safe and effective treatment modality. However, there are lack of studies that comparing the clinical effectiveness of intralesional purified protein derivative (PPD) and Candida antigen (CA) in genital wart treatment. To investigate the effectiveness and safety of PPD and CA in the treatment of genital warts. Eighty patients were enrolled in this study and were randomly divided into 2 groups with 40 patients in each. Each antigen was injected intralesionally at a dose of 0.1 ml into the largest wart every 2 weeks until complete improvement or for a maximum of four sessions. Complete clinical response was demonstrated in 65%, 62.5% in PPD and CA groups, respectively. There was no statistically difference between both groups. After the 3-month follow-up period, 72.5%, 85% of patients showed complete clearance in PPD and CA groups respectively. Side effects were mild and insignificant in both groups. Recurrence was observed in only one patient in each group. Immunotherapy by intralesional PPD and CA injection is considered to be effective and well-tolerated modalities in treatment of genital wart with minimal side effects and recurrence rate compared to other modalities.

ObjectiveTo compare the diagnostic accuracy of metagenomic next-generation sequencing (mNGS) for cryptococcosis in patients with different immune statuses with that of conventional detection.MethodsA total of 1442 specimens including 71 specimens from patients with cryptococcosis were analyzed in the study. The chi square test was used to screen the sensitivity and specificity of different detection methods for different specimen types. One-way ANOVA was used to compare the mNGS results with age, CD4, lymphocytes, IFN, IL-6, IL-2 and serum antigen assay.ResultsThe sensitivity of mNGS was 44.29% in Cryptococcus infection cases. The positive rate of mNGS results for bronchoalveolar lavage fluid (BALF, 87.50%) from immunocompromised patients was higher than that of BALF from immunocompetent patients (40.00%, p=0.04). The sensitivity of the serum Cryptococcus capsular antigen assay was 80.00% in immunocompetent patients and 96.42% in immunocompromised patients (p = 0.049). A positive rate of detection of Cryptococcus from mNGS was higher when cryptococcal antigen ≥1:160 (p=0.022) in immunocompromised patients. A positive rate of detection of Cryptococcus from mNGS was higher when lymphocyte counts were lower in both immunocompetent patients(p=0.017) and in immunocompromised patients(p=0.029).ConclusionsThe sensitivity of mNGS is lower than that of serum cryptococcal antigen assay and histopathology in immunocompetent patients. However, BALF detection is recommend for immunocompromised patients compared with tissue and CSF. The positive mNGS result was correlated with lower lymphocyte counts, higher IL-2 and higher serum antigen assay in immunocompromised patients.