Detection Of Cryptococcal Antigen Research Articles

Establishment and methodological evaluation of a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes based on CRISPR-Cas12a technology

PurposeThe opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method. MethodsRecombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis. ResultsThis study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/μL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %. ConclusionIn this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.

Diagnostic Performances of an in-House Immunochromatography Test Based on the Monoclonal Antibody 18B7 to Glucuronoxylomannan for Clinical Suspected Cryptococcosis: a Large-Scale Prototype Evaluation in Northern Thailand.

Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7. We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485. The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis. The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030.

Prevalence of Cryptococcal Antigenemia and Lateral Flow Assay Accuracy in Severely Immunosuppressed AIDS Patients.

This study aimed to estimate the prevalence of cryptococcal antigenemia detected by lateral flow assay (LFA) in AIDS patients and its accuracy in the diagnosis of cryptococcosis. Conducted at a university hospital in Brazil from March 2015 to July 2017, it included AIDS patients over 18 years old with a CD4+ count ≤ 200 cells/mm3. Cryptococcal antigen (CrAg) detection using LFA and latex agglutination (LA), along with blood and urine cultures, were performed. The reference standard was the identification of Cryptococcus spp. in clinical specimens through microbiological or histopathological examination. Among 230 patients, the prevalence of CrAg detected by LFA (CrAg LFA) was 13.0%. Factors associated with cryptococcal antigenemia included fever, vomiting, seizures, and a lack of antiretroviral therapy. The sensitivity and specificity of CrAg LFA were 83.9% and 98.0%, respectively. The positive predictive value (PPV) was 86.7%, the negative predictive value (NPV) was 97.5%, and overall accuracy was 96.1%. Cross-reactions were observed in patients with histoplasmosis and paracoccidioidmycosis, but not with aspergillosis or positive rheumatoid factor. The study concludes that the LFA is a useful tool for detecting cryptococcal antigenemia in severely immunocompromised AIDS patients due to its high NPV, specificity, and PPV.

Rapid duplex flap probe-based isothermal assay to identify the Cryptococcus neoformans and Cryptococcus gattii.

Cryptococcosis is a life-threatening invasive fungal infection with significantly increasing mortality worldwide, which is mainly caused by Cryptococcus neoformans and Cryptococcus gattii. These two species complexes have different epidemiological and clinical characteristics, indicating the importance of accurate differential diagnosis. However, the clinically used culture method and cryptococcal capsular antigen detection couldn't achieve the above goals. Herein, we established a novel duplex flap probe-based isothermal assay to identify the Cryptococcus neoformans and Cryptococcus gattii within 1 hour. This assay combined the highly sensitive nucleic acid isothermal amplification and highly specific fluorescence probe method, which could effectively distinguish the sequence differences of the two species complexes using two different fluorescence flap probes in a single reaction system. This novel method showed excellent detection performance with sensitivity (10 copies/μL each) and specificity (100%) compared to traditional culture and sequencing methods. Furthermore, we applied this method to spiked clinical samples, 30 cerebrospinal fluids and 30 bronchoalveolar lavage fluids, which kept good detection performance. This novel rapid duplex flap probe-based isothermal assay is a promising and robust tool for applications in differential diagnosis of the Cryptococcus neoformans and Cryptococcus gattii in clinical settings, especially when clinical suspicion for cryptococcal disease is high and epidemiological studies.

Cryptococcosis in kidney transplant recipients: Current understanding and practices.

Cryptococcosis is the third most commonly occurring invasive fungal disease in solid organ transplant recipients (SOT). It is caused by encapsulated yeast, Cryptococcus species, predominantly Cryptococcus neoformans and Cryptococcus gattii. Though kidney transplant recipients are at the lowest risk of cryptococcosis when compared to other solid organ transplant recipients such as lung, liver or heart, still this opportunistic infection causes significant morbidity and mortality in this subset of patients. Mortality rates with cryptococcosis range from 10%-25%, while it can be as high as 50% in SOT recipients with central nervous system involvement. The main aim of diagnosis is to find out if there is any involvement of the central nervous system in disseminated disease or whether there is only localized pulmonary involvement as it has implications for both prognostication and treatment. Detection of cryptococcal antigen (CrAg) in cerebrospinal fluid or plasma is a highly recommended test as it is more sensitive and specific than India ink and fungal cultures. The CrAg lateral flow assay is the single point of care test that can rapidly detect cryptococcal polysaccharide capsule. Treatment of cryptococcosis is challenging in kidney transplant recipients. Apart from the reduction or optimization of immunosuppression, lipid formulations of amphotericin B are preferred as induction antifungal agents. Consolidation and maintenance are done with fluconazole; carefully monitoring its interactions with calcineurin inhibitors. This review further discusses in depth the evolving developments in the epidemiology, pathogenesis, diagnostic assays, and management approach of cryptococcosis in kidney transplant recipients.

Assessment of an Antigen Lateral Flow Assay (LFA) For Point of Care Diagnosis of Cryptococcosis in Senegal

Background: Cryptococcal meningitis is a medical emergency. Therefore, simple, rapid and efficient diagnostic techniques are needed. New rapid diagnostic tests based on the detection of cryptococcal antigen have been developed. The aim of our study was to evaluate the performance of the Dynamiker Cryptococcal Antigen Lateral Flow Assay (LFA) in our routine diagnosis activities. Materials and Methods: We performed a prospective study from November 2017 to August 2018 at the ParasitologyMycology laboratory of the CHNU of Fann in Dakar. Biological samples collected from HIV-infected patients hospitalized at the infectious diseases department of CHUN de Fann were used. Serum, CSF and urine samples received in the laboratory were examined using mycological techniques and the cryptococcal latex agglutination test (CLAT). The performance of the Dynamiker test was evaluated considering CLAT as the reference method. Results: A total of 39 samples were examined: 14 CSF, 24 sera and 1 urine sample. The average age was 44.5 years with a predominance of female patients (64.1%). The mean CD4 count was 73 cells/mm3 . Among the 39 samples tested, 23.1% were CLAT positive while 30.8% were LFA positive. The sensitivity and specificity rates were 100% and 75% respectively. The percentage of correlation between the two methods was 92.3%. The intensity of the bands on the LFA was high en 66.7% of cases. Conclusion: The excellent sensitivity of the rapid Cryptococcal Antigen Lateral Flow Assay and the rapidity of obtaining results (less than 15 minutes) make it suitable for routine diagnosis of cryptococcal meningitis.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic.

Infectious meningitis and encephalitis (ME) are life-threatening conditions are caused by various pathogens. Conventional laboratory tests with low sensitivity and specificity cannot help with early diagnosis. A prospective study using the novel multiplex PCR detection for 18 pathogens of ME (MME-18) was conducted to investigate the clinical utilization and the epidemiology characteristics of ME in southwestern China. Patients with suspected intracranial infection were recruited between May and October 2019 at West China Hospital of Sichuan University. The MME-18 was used to detect cerebrospinal fluid, and conventional experiments including cryptococcal capsular antigen detection, GeneXpert, real-time PCR, and clinical feedback were used to verify the result of MME-18. Among 581 tested patients, 139 eligible individuals were enrolled in the study. Among them, Mycobacterium tuberculosis was the most common pathogen in mono-infection. Viruses and Cryptococcus neoformans were also frequently detected. Of 139 infected patients, 12 cases were diagnosed by MME-18 only, 57 patients by conventional testing only, and 70 cases by both comparator tests and MME-18. There were 96.3% (79/82) diagnoses made by MME-18 had a favorable outcome, and two of twelve diagnoses, made solely by MME-18, had a likely unclear clinical significance. The MME-18 showed satisfactory consistency with expert clinical consensus for patients presenting with ME. Combined with conventional testing and clinical suspicion, MME-18 may help clinicians with the early identification of pathogens.

RECURRENT CRYPTOCOCCAL MENINGITIS IN PATIENT WITH ANTI-GM-CSF AUTOANTIBODIES

<h3>Introduction</h3> Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor and pro-inflammatory cytokine. The involvement of anti-GM-CSF autoantibodies in the development of protein alveolar proteinosis (PAP) has been previously elucidated, however its role in increasing susceptibility to opportunistic infections remains less clear. <h3>Case Description</h3> A 33 year-old previously healthy man developed recurrent disseminated cryptococcal infections. He was hospitalized several times due to severe headaches and detection of cryptococcal antigen in his serum and CSF. He underwent multiple courses of antifungal induction therapy followed by antifungal prophylaxis and dexamethasone taper, yet remained symptomatic with persistent cryptococcal antigen in his CSF. Blood work was notable for the presence of anti-GM-CSF autoantibodies, low IgG (463), low IgM (26), normal IgA (69), 3/23 protective pneumococcal titers, and normal tetanus/diphtheria antibodies. Flow cytometry is currently pending. <h3>Discussion</h3> Anti-GM-CSF autoantibodies neutralize cell signaling resulting in impaired phagocytosis and host immune responses. Autoantibodies against GM-CSF increase susceptibility to opportunistic infections, particularly cryptococcal meningitis, in otherwise immunocompetent patients via the inhibition of GM-CSF STAT5 phosphorylation and GM-CSF-induced MIP-1alpha protein production. This is a rare case of a patient who developed recurrent cryptococcal meningitis in the setting of anti-GM-CSF autoantibodies and hypogammaglobulinemia. Further workup is underway to explore the possibility of concurrent CVID versus secondary hypogammaglobulinemia due to prolonged dexamethasone tapers. Patients with recurrent cryptococcal infections should be screened for anti-GM-CSF autoantibodies, which can help tailor treatment including GM-CSF therapy and anti-CD20 therapy. Patients would also benefit from a basic immunologic workup and routine screening for PAP.

Pulmonary Cryptococcosis.

Pulmonary cryptococcosis describes an invasive lung mycosis caused by Cryptococcus neoformans or Cryptococcus gattii complex. It is often a high-consequence disease in both immunocompromised and immunocompetent populations, and may be misdiagnosed as pulmonary malignancy, leading to a delay in therapy. Epidemiology follows that of cryptococcal meningoencephalitis, with C. gattii infection more common in certain geographic regions. Diagnostic tools include histopathology, microscopy and culture, and the detection of cryptococcal polysaccharide antigen or Cryptococcus-derived nucleic acids. All patients with lung cryptococcosis should have a lumbar puncture and cerebral imaging to exclude central nervous system disease. Radiology is key, both as an adjunct to laboratory testing and as the initial means of detection in asymptomatic patients or those with non-specific symptoms. Pulmonary cryptococcomas (single or multiple) may also be associated with disseminated disease and/or cryptococcal meningitis, requiring prolonged treatment regimens. Optimal management for severe disease requires extended induction (amphotericin B and flucytosine) and consolidation therapy (fluconazole) with close clinical monitoring. Susceptibility testing is of value for epidemiology and in regions where relatively high minimum inhibitory concentrations to azoles (particularly fluconazole) have been noted. Novel diagnostic tools and therapeutic agents promise to improve the detection and treatment of cryptococcosis, particularly in low-income settings where the disease burden is high.

Cryptococcal antigen prevalence in HIV patients from a tertiary care centre in South India.

Cryptococcosis is an opportunistic mycosis, caused by Cryptococcus neoformans. Cryptococcal meningitis is one of the most fatal opportunistic infections associated with human immunodeficiency virus (HIV) infection. The aim of this study was to find the prevalence of cryptococcal antigenemia in people living with HIV (PLHA) and also to find the prevalence of opportunistic infections among these patients. A total of 204 non duplicate samples were collected from people with HIV aged above 18 years. Samples with CD4 count less than 300 were included in the study. Cryptococcal antigen detection was done by CrAg Lateral flow assay. None of the patients in our study were positive for cryptococcal antigen. Opportunistic infections were observed in 82 (40.2%) HIV positive patients. Candidiasis, tuberculosis and Pneumocystis jiroveci pneumonia were the most common opportunistic infections. This is the first study from the southern state of Kerala on the prevalence of Cryptococcal antigenemia among HIV positive individuals. The study showed that routine screening for cryptococcal antigen will not be cost effective in our population. Similar to other studies, eventhough candidiasis, tuberculosis and PCP were more commonly seen among people with CD4 count < 200 cells/mm3, there was no statistically significant association.

PULMONARY CRYPTOCOCCUS: THE GREAT IMPERSONATOR

PULMONARY CRYPTOCOCCUS: THE GREAT IMPERSONATOR

P259 Rare presentations of Cryptococcosis: a case series

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Cryptococcus spp. is usually opportunistic pathogens affecting immunocompromised individuals causing meningitis primarily. Non-CNS presentations are a rare entity and we hereby present a series of 3 cases in the past 1 year (2021-2022).MethodsCase records of the three patients were studied. Detailed history, demographic details, investigations, treatment were noted.ResultsPatient-1 was a 14-year-old girl who came with complaints of fever, pain, swelling, and restricted movements of the right wrist, elbow, and ankle joints with multiple subcutaneous swellings initially on the thigh followed by elbows, arms, and forearms. The swellings became hemorrhagic bullae bursting to form ulcers. She had a history of being treated 4 times for tuberculous lymphadenopathy. KOH-Calcofluor white mount of biopsy and pus aspirate samples showed circular yeast cells which were confirmed by cryptococcal antigen detection. All the samples had grown Cryptococcus neoformans on culture except blood, BAL, and CSF. She responded to Liposomal amphotericin-B drastically. Retesting of pus swabs from the ulcers after a week of antifungal therapy were negative for C. neoformans. Subcutaneous nodules and joint swellings decreased but she developed reactions to amphotericin B and was changed to fluconazole. She is on regular follow-up with no recurrence.Patient-2 was a 22-year-old male, a known case of Hodgkin Lymphoma stage 4 who underwent Autologous stem cell transplantation (ASCT) and was on immunosuppressants. He presented with fever, dyspnea, and cough which got worsened along with multiple cervical, hilar and abdominal lymphadenopathy. KOH-Calcofluor white mount of biopsy samples demonstrated circular yeast cells which were confirmed by cryptococcal antigen detection test of biopsy and BAL samples. Cryptococcus neoformans was grown on culture from all the samples. He succumbed to ARDS and cardiorespiratory arrest before any treatment could be initiated.Patient-3 was a 38-year-old female, known case of SLE with lupus nephritis, presented with intermittent fever, dyspnea, chest pain, decreased urinary output, and gradual swelling of the body starting from the face and progressing to the whole body. She further developed synpneumonic effusion, multiple erythematous tender papules over the right thigh, and cellulitis of the right lower limb. She was started on voriconazole in view of HRCT findings suggestive of fungal pneumonia. As galactomannan antigen test was negative, voriconazole was stopped. Pleural tap fluid flagged positive in Bactec and C. neoformans grown on subculture. Her condition worsened with septic shock and succumbed to the disease before any treatment could be initiated.ConclusionSubcutaneous, joint, and pulmonary involvement is rare, without a primary focus on the central nervous system. Culture and antigen detection can aid in early detection and hence early initiation of therapy.

P406 Rise in HIV negative Cryptococcal infection in liver disease patient: epidemiology, risk factor, antifungal susceptibility profile from tertiary care hepatobiliary center

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjective Cryptococcus neoformans is an encapsulated yeast found ubiquitously in the environment, considered important in immunocompromised individuals with HIV. Non-HIV susceptible groups include malignancies, long-term use of corticosteroids, solid organ transplantation, sarcoidosis, immunosuppressive therapy, and cirrhosis. Cryptococcal infections are rising in HIV-negative cirrhotic patients, accounting for 6-21% of the systemic infections. Chronic liver disease (CLD) is a neglected risk factor for fungal infections. To analyze epidemiology, risk factors, and antifungal susceptibility profile of HIV-negative liver disease patients.Material MethodsThe retrospective study was conducted in tertiary care hepatobiliary center in New Delhi, India from January 2017 to March 2022 after ethical approval. Demographic data, laboratory data, and clinical history of the patients admitted with liver disease and suspected cryptococcal infections were obtained from Hospital records for epidemiology and risk factor analysis. Blood, CSF, serum, urine, and body fluid (Ascitic fluid, peritoneal fluid, pleural fluid, MiniBAL) samples were processed for fungal culture on SDA and SDA with cyclohexamide. Blood and body fluid were also inoculated in an automated blood culture system (BacT/ALERT 3D bioMérieux). Samples were processed for gram stain, India ink, and Cryptococcal antigen. Identification was done by an automated identification system (VITEK 2 compact bioMérieux). The micro-broth dilution method (Sensititre YeastOne colorimetric plate, Thermo Fisher Scientific, MA, United States) was used to determine the susceptibility of all Cryptococcus strains to the six antifungal drugs, fluconazole, 5-fluorocytosine, amphotericin B, itraconazole, posaconazole, and voriconazole. The results were reported as wild-type (WT) or non-wild-type (non-WT) in accordance with the epidemiological cutoff value (ECV) set for Cryptococcus spp. (Espinel-Ingroff et al., 2012a, b; CLSI, 2018).ResultsWe analyzed 30 patients of suspected cryptococcal infection and obtained 40 C. neoformans isolates from different samples from these patients. Out of 40 samples, C neoformans was isolated from blood (25,62.5%), urine (6,15%), ascitic fluid (4,10%), mini-BAL (2,5%), bone marrow (1,2.5%), CSF (1,2.5%), pleural fluid (1,2.5%). All samples were positive for Cryptococcal antigen. India ink positivity was observed in 56%. Alcoholic liver disease was the most common risk predictive factor seen in 30% of patients. Hepatitis B and C-associated with CLD was seen in 20%. Other risk predictive factors were AKI (70%), diabetes mellitus (20%), TB (10%), autoimmune hepatitis (6.6%), autoimmune disease (autoimmune hemolytic anemia, Sjogren syndrome) (6.6%), Sarcoidosis (3.3%), Hepatocellular carcinoma (3.3%), HIV (3.3%). Hepatic encephalopathy was seen in 70% of patients which mimics the symptoms of C. meningitis.7.5% (3/40), 5% (2/40), 2.5% (1/40), 7.5% (3/40), and 2.5% (1/40) of C. neoformans strains were non-WT to fluconazole, 5-fluorocytosine, amphotericin B, posaconazole, and itraconazole, respectively, but all strains were WT to voriconazole. Overall mortality was 66.6%.ConclusionAbove study shows that liver diseases are an important risk factor for cryptococcal infection owing to immunosuppressed state, use of steroids, and associated comorbidities like DM, TB, AKI, autoimmune disease, Sarcoidosis, hepatocellular carcinoma. Therefore, it is necessary to investigate and be vigilant about the isolation of Cryptococcus. The use of appropriate antifungals can improve clinical outcomes. As there is increasing resistance to amphotericin B, 5-fluorocytosine, azoles; antifungal susceptibility testing should be done. Cryptococcal antigen detection can be useful in non-CSF samples as well.

P485 Hiv-positive, solid organ transplant (SOT), and non-Hiv-positive/non-transplant (NHNT) associated with cryptococcosis in Brazil: First national multicenter cohort study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesTo describe the characteristics, mortality, and therapeutic response among hospitalized patients with cryptococcosis in Brazil.MethodsThis is a multicenter retrospective cohort study of seven Brazilian public tertiary hospitals (Figure 1). Medical records of patients admitted from January 2014 to December 2019 were evaluated. Confirmed cases of the first episode of cryptococcosis were included. Hosts were classified as HIV-positive, solid organ transplant (SOT), and non-HIV-positive/non-transplant (NHNT). Mortality was defined as the time of patient admission to in-hospital death from any cause. Statistical analysis was performed using the software R and JAMOVI.ResultsA total of 384 patients were included; the median (25th-75th) age was 39 (31-48) years and 283 (73.7%) were men. Hosts were 304 (79.2%) HIV-positive, 16 (4.2%) SOT, and 64 (16.7%) NHNT. More frequent diagnosis tools were culture, direct microscopic examination of infected body fluids using India ink, histological examination of tissue samples, and detection of cryptococcal polysaccharide antigen in body fluids (CrAg) using latex. Central nervous system (CNS) cryptococcosis had a significantly higher counting level across disease categories, with 313 cases or 81.5%. NHNT were more likely to have CNS cryptococcosis than people HIV-positive (84.4% vs. 81.9%, respectively). SOT patients had more pulmonary form infections (31.2%) as compared with HIV-positive (3.3%) and NHNT (1.6%). Other extrapulmonary sites category had HIV-positive and NHNT basically the same percentage of disease involvement (14%) compared with SOT (6.2%) (P < .001, Figure 2). Among cases with identification of specie, 56% were Cryptococcus neoformans and 4.4% were C. gattii. A total of 271 (70.6%) patients were discharged home with total or partial improvement and 113 (29.4%) patients died during hospitalization. In-hospital mortality among HIV-positive, SOT, and NHNT patients was 30.3% (92/304), 12.5% (2/16), and 29.7% (19/64), respectively. Induction therapy with AMB had the conventional deoxycholate mainly in combination with fluconazole (234, 84.2%). Only 80 (22.3%) patients received an AMB lipid formulation (liposomal AMB, n = 35 and AMB lipid complex, n = 45). The median (25th-75th) length of AMB therapy was 20 (14–32) days. Death patients had more age when compared with discharged-to-home cases (43 vs. 38 years, P < .002). Patients with CNS cryptococcosis had lower mortality (83/313, 26.5%) when compared with the other categories [pulmonary, 5/16 (31.2%) and other extrapulmonary sites, 25/55 (45.4%)] (P = .017). Survival benefits were seen for patients who received monotherapy or combination therapy. However, D-AMB alone showed a higher mortality rate, although not statistically significant (P = .537).ConclusionHIV infection is the most important condition among patients with cryptococcosis in Brazil and CNS involvement is the commonest manifestation in all hosts, mainly HIV-positive and NHNT. The proportion of pulmonary cryptococcosis is relevant in SOT patients. Mortality was high in all categories of hosts. Understanding the epidemiology and characteristic of patients admitted to our hospitals will help to understand the burden and causes of mortality and identify strategies to improve this scenario. Optimized diagnosis (i.e., lateral flow assay) and treatment (i.e., AMB lipid formulation plus flucytosine) are urgently necessary for our setting.

Clinical utility of cryptococcal antigen detection in transthoracic needle aspirate by lateral flow assay for diagnosing non-HIV pulmonary cryptococcosis: A multicenter retrospective study.

Lateral flow immunoassay (LFA) detection of cryptococcal capsular polysaccharide antigen (CrAg) is reported to be the most rapid and convenient laboratory method for diagnosing cryptococcosis. Its clinical diagnostic use, however, is not well studied. We retrospectively analyzed the data from 97 patients with suspected pulmonary cryptococcosis (PC) at 2 tertiary care centers. CrAg in both serum and lung aspirate specimens were examined by LFA. We divided the patients who were diagnosed with PC into group I, patients positive for CrAg in both the serum and lung aspirate, and group II, patients positive for CrAg in the lung aspirate but not in the serum. We analyzed the differences in imaging distribution, morphological characteristics, and concomitant signs between the 2 groups. Of all 97 patients, 47 were diagnosed with PC. Lung aspirates were positive for CrAg in 46/47 patients with PC (sensitivity 97.9%, specificity 100%, positive predictive value = 100%, negative predictive value = 98%). There were no false positive results in the noncryptococcosis patients, revealing a diagnostic accuracy of 99%. Serum CrAg tests were positive in 36/47 patients with PC (sensitivity 76.6%, specificity 100%, accuracy 88.7%, positive predictive value = 100%, negative predictive value = 82%). Chest imaging data showed a statistically significant greater number of single lesions in group II than in group I (P < .05). More lesions accompanied by halo signs were showed in group I (P < .01), whereas more accompanied by pleural stretch signs were found in group II (P < .01). The LFA-positive rate of CrAg in lung aspirate samples was higher than that of the serum samples, especially in patients with single pulmonary lesion or in those accompanied by pleural stretch. The direct measurement of CrAg in lung aspirate is a rapid, useful alternative diagnostic method for PC confirmation.

Cryptococcal meningitis in people living with human immunodeficiency virus in Nepal: Perspectives from resource limited setting.

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.

Pleural involvement in cryptococcal infection.

Pleural involvement of cryptococcal infection is uncommon and is more commonly observed in immunocompromised hosts than in immunocompetent ones. Pleural involvement in cryptococcal infections can manifest with or without pleural effusion. The presence of Cryptococcus spp. in the effusion or pleura is required for the diagnosis of cryptococcal pleural infection, which is commonly determined by pleural biopsy, fluid culture, and/or detection of cryptococcal antigen in the pleura or pleural fluid.

Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes.

Cryptococcus neoformans and Cryptococcus gattii are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target Cryptococcus do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The cyt b-directed assay accurately detected and identified all eight C. neoformans/gattii genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The Cryptococcus-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.

Evaluation of Lateral Flow Assay for the Diagnosis of Cryptococcal Meningitis and its Comparison with the Gold Standard and Other Laboratory Tests

Introduction: Cryptococcosis is a potentially fatal fungal disease affecting both immunocompromised and immunocompetent individuals. Hence, a Point Of Care Test (POCT) is required with higher sensitivity and specificity (100%), for the rapid detection of cryptococcosis which will be life saving for the patient. Aim: To evaluate the usefulness of Lateral Flow Assay (LFA) for rapid detection of Cryptococcal Antigen (CrAg) from Cerebrospinal Fluid (CSF) sample. Materials and Methods: This diagnostic study was conducted in Mycology Laboratory, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India, over a period of six months (June 2019-November 2019). The CSF samples (n=37) from patients with suspected meningitis were considered for the study. Direct microscopy with gram stain, negative stain and histopathological stains was performed for all the CSF samples. Culture was done on basal medium, enriched media and special media. Cryptococcus genus specific Polymerase Chain Reaction (PCR) was also performed to confirm the isolates grown on the culture. Serological tests like Latex Agglutination Test (LAT) and LFA were also performed on all the samples. The LFA results were compared with microscopy, culture and LAT. Results: Among the 37 suspected meningitis patients, three grew Cryptococcus in culture which was considered as the gold standard in the diagnosis of Cryptococcal Meningitis (CM). Direct microscopic techniques had 100% specificity however their sensitivity was less i.e., 67%. LAT had a sensitivity of 100% and specificity of 94%. However, LFA in present study had a very good sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) (100%). Conclusion: LFA for the detection of CM is considered to be an effective method when compared to the other conventional and serological methods with higher sensitivity and specificity. Hence, in present study authors evaluated the use of LFA in diagnosis of CM and found that this assay when used as a POCT, can give the results in short duration. Considering this as a pilot study, further studies including higher number of samples are essential to confirm the effectiveness of the findings.

Evaluation of a cryptococcal antigen lateral flow assay test for rapid detection of cryptococcal infection in HIV-negative patients in Ibadan, Nigeria

Background: A number of studies have been conducted in Nigeria on the prevalence of cryptococcal infections mostly on HIV-infected patients using culture, India ink and/or latex agglutination tests. These tests are either laborious, time-consuming and expensive or have low sensitivity, thus limiting their use. Cryptococcal antigen lateral flow assays (LFA) were introduced in the last decade as rapid user-friendly tests for diagnosis. In this study, we sought to determine the diagnostic accuracy of an LFA kit for the detection of cryptococcal antigen in the serum of HIV-negative patients with or without cerebrovascular accident (CVA) or stroke in University College Hospital, Ibadan, Nigeria.Methodology: The diagnostic accuracy of Dynamiker CrAg LFA was tested against BiosynexR CryptoPS on serum samples of 100 HIV-negative patients with and without stroke. Samples were tested and results interpreted in accordance with the manufacturer’s instructions. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios of the Dynamiker CrAg LFA were calculated by comparing with the BiosynexR CryptoPS as ‘gold standard’.Results: Overall, a total of 98 valid patient sample results were analysed; 17 samples (17.3%) were positive with Dynamiker CrAg LFA cryptococcal antigen and 16 samples (16.3%) were positive with BiosynexR CryptoPS. The sensitivity, specificity, PPV and NPV of Dynamiker CrAg LFA compared to the BiosynexR CryptoPS were 100%, 98.8%, 94.1% and 100% respectively, while the positive and negative likelihood ratios were 82 and 0 respectively.Conclusion: In comparison to the BiosynexR CryptoPS, the Dynamiker CrAg LFA is a highly sensitive and specific test for the detection of cryptococcal antigen in serum. The test kit should be considered as a screening device for cryptococcal infection both in outreach and clinical settings, especially in antiretroviral therapy (ART) centres.&#x0D; Keywords: Cryptococcus; evaluation; lateral flow assay; HIV-negative; stroke