CrAg Lateral Flow Assay Research Articles

Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay for the Diagnosis of HIV-Associated Cryptococcosis.

Cryptococcal meningitis is a leading cause of meningitis in sub-Saharan Africa. Given the need for rapid point-of-care testing, we evaluated the diagnostic performance of the Dynamiker cryptococcal antigen (CrAg) lateral flow assay (LFA). We assessed the diagnostic performance of the Dynamiker CrAg LFA compared to the IMMY CrAg LFA as the reference standard. We tested 150 serum, 115 plasma, and 100 cerebrospinal fluid (CSF) samples from HIV patients with symptomatic meningitis and 113 serum samples from patients with suspected asymptomatic cryptococcal antigenemia. Compared to the IMMY CrAg LFA, sensitivity of Dynamiker CrAg LFA was 98% in serum, 100% in plasma, 100% in CSF from symptomatic patients and 96% in serum from asymptomatic patients. Specificity was 66% in serum, 61% in plasma, and 91% in CSF from symptomatic patients, and 86% in serum from asymptomatic patients. The positive predictive value was 85% in serum, 82% in plasma, and 96% in CSF from symptomatic patients, and 69% in serum from asymptomatic patients. The negative predictive value was 94% in serum, 100% in plasma, and 100% in CSF from symptomatic patients, and 99% in serum from asymptomatic patients. The interassay reproducibility was 100% across the four sample types with no observed discordant results when Dynamiker CrAg LFA was tested in duplicate. However, a high number of false positives were observed on serum of symptomatic patients (11%), serum of asymptomatic patients (11%) and plasma of symptomatic patients (14%). The Dynamiker CrAg LFA had excellent sensitivity but poor specificity, particularly when tested on serum and plasma.

Epidemiology of Cryptococcal antigenemia among HIV infected patients in South-western Nigeria

Background: Cryptococcosis is a life-threatening fungal infection that presents diversely with no specific pathognomonic features. Cryptococcal disease is one of the most important opportunistic infections, and a major contributor to early mortality in HIV infected subjects. Cryptococcal antigenemia occurs in Nigeria, but the magnitude of this disease remains unclear. This study was carried out to determine the prevalence of CrAg among HIV seropositive and seronegative individuals attending Adeoyo Maternity Teaching Hospital (AMTH), Yemetu, Ibadan. Methods and materials: This was a case-control study in which 114 cases and 228 controls were recruited. Cases were HIV seropositive individuals while controls were HIV seronegative individuals receiving care at AMTH. Adult male and female (age range: 18–65 years) who agree to participate in the study were recruited. Simple random sampling was used to select subjects. Semi-structured interviewer administered questionnaire was used to collect data from subjects and retrospective review of CD4 count records in HIV infected subjects. Five millilitres venous blood was collected from each participant. Serum Cryptococcal antigen testing was done using CrAg Lateral Flow Assay. Data was analyzed using descriptive statistics and bivariate analysis at 5% level of significance. Results: Mean age of cases was 41.2 ± 10.0 years and 85 (74.6%) were females while mean age of control subjects was 38.9 ± 13.7 years 156 (68.4%) were females. The cumulative prevalence of CrAg was 8.5%. However, the prevalence of CrAg among cases was 11.4% and 7.0% among controls. Age groups 41-50 years and 21-30 years were the most affected among cases and controls respectively. HIV positive subjects were about two times more likely to test positive for CrAg. Individuals with CD4 counts of ≤100cells/μl were 20 times more likely to have positive serum cryptococcal antigen than individuals with CD4 counts >100 cells/μl (OR: 20.3, 95%CI: 5.23-78.9). Conclusion: Cryptococcal antigen was present among both cases and controls; however, the prevalence was higher among cases. Since asymptomatic cryptococcal antigenemia predicts impending cryptococcal infection (pneumonia and meningitis) with probable mortality, screening for CrAg should be part of the routine tests amongst all confirmed HIV seropositive cases

Evaluation of a Novel Semiquantitative Cryptococcal Antigen Lateral Flow Assay in Patients with Advanced HIV Disease.

Higher cryptococcal antigen (CrAg) titers are strongly associated with mortality risk in individuals with HIV-associated cryptococcal disease. Rapid tests to quantify CrAg levels may provide important prognostic information and enable treatment stratification. We performed a laboratory-based validation of the IMMY semiquantitative cryptococcal antigen (CrAgSQ) lateral flow assay (LFA) against the current gold standard CrAg tests. We assessed the diagnostic accuracy of the CrAgSQ in HIV-positive individuals undergoing CrAg screening, determined the relationship between CrAgSQ scores and dilutional CrAg titers, assessed interrater reliability, and determined the clinical correlates of CrAgSQ scores. A total of 872 plasma samples were tested using both the CrAgSQ LFA and the conventional IMMY CrAg LFA, of which 692 were sequential samples from HIV-positive individuals undergoing CrAg screening and an additional 180 were known CrAg-positive plasma samples archived from prior studies. Interrater agreement in CrAgSQ reading was excellent (98.17% agreement, Cohen's kappa 0.962, P < 0.001). Using the IMMY CrAg LFA as a reference standard, CrAgSQ was 93.0% sensitive (95% confidence interval [CI] 80.9% to 98.5%) and 93.8% specific (95% CI, 91.7% to 95.6%). After reclassification of discordant results using CrAg enzyme immunoassay testing, the sensitivity was 98.1% (95% CI, 90.1% to 100%) and specificity 95.8% (95% CI, 93.9% to 97.2%). The median CrAg titers for semiquantitative score categories (1+ to 4+) were 1:10 (interquartile range [IQR], 1:5 to 1:20) in the CrAgSQ 1+ category, 1:40 (IQR, 1:20 to 1:80) in the CrAgSQ 2+ category, 1:640 (IQR, 1:160 to 1:2,560) in the CrAgSQ 3+ category, and 1:5,120 (IQR, 1:2,560 to 1:30,720) in the CrAgSQ 4+ category. Increasing CrAgSQ scores were strongly associated with 10-week mortality. The IMMY CrAgSQ test had high sensitivity and specificity compared to the results for the IMMY CrAg LFA and provided CrAg scores that were associated with both conventional CrAg titers and clinical outcomes.

Prevalence of cryptococcal antigen (CrAg) among HIV-positive patients in Eswatini, 2014-2015.

BackgroundCryptococcal meningitis is a leading cause of death amongst people living with HIV. However, routine cryptococcal antigen (CrAg) screening was not in the national guidelines in Eswatini.ObjectivesA cross-sectional study was conducted between August 2014 and March 2015 to examine CrAg prevalence at Mbabane Government Hospital in Eswatini.MethodsWe collected urine and whole blood from antiretroviral-therapy-naïve patients with HIV and a cluster of differentiation 4 (CD4) counts < 200 cells/mm3 for plasma and urine CrAg lateral flow assay (LFA) screening at the national HIV reference laboratory. Two CD4 cut-off points were used to estimate CrAg prevalence: CD4 < 100 and < 200 cells/mm3. Sensitivity and specificity of urine CrAg LFA was compared to plasma CrAg LFA.ResultsPlasma CrAg prevalence was 4% (8/182, 95% confidence interval [CI]: 2–8) amongst patients with CD4 counts of < 200 cells/mm3, and 8% (8/102, 95% CI: 3–15) amongst patients with CD4 counts of < 100 cells/mm3. Urine CrAg LFA had a sensitivity of 100% (95% CI: 59–100) and a specificity of 80% (95% CI: 72–86) compared with plasma CrAg LFA tests for patients with CD4 < 200 cells/mm3. Forty-three per cent of 99 patients with CD4 < 100 were at World Health Organization clinical stages I or II.ConclusionThe prevalence of CrAg in Eswatini was higher than the current global estimate of 6% amongst HIV-positive people with CD4 < 100 cell/mm3, indicating the importance of initiating a national screening programme. Mechanisms for CrAg testing, training, reporting, and drug and commodity supply issues are important considerations before national implementation.

Underlying Cryptococcal Diseases and the Correlation With Serum Cryptococcal Antigen Titers in Hospitalized HIV-Infected Patients Screened Positive for Cryptococcal Antigenemia.

Background: The prevalence of different underlying cryptococcal diseases in human immunodeficiency virus (HIV)-infected patients screened positive for cryptococcal antigenemia and the association between cryptococcal diseases and serum cryptococcal antigen (CrAg) titers were understudied.Methods: HIV-infected patients with CD4 < 200 cells/ul, admitted to the second hospital of Nanjing, Nanjing, China, from January 2016 to September 2019, were retrospectively analyzed. Integrated into routine HIV care, all these patients were screened for cryptococcal antigenemia with CrAg lateral flow assay. Positive patients received extensive laboratory and radiological studies to evaluate underlying cryptococcal diseases.Results: A total of 872 HIV inpatients were screened for serum CrAg. The prevalence of cryptococcal antigenemia in the study population was 10.3% (95% CI, 8.3–12.3%), 87.6% of which with cryptococcal antigenemia had clinically cryptococcal diseases. The prevalence of cryptococcal meningitis (CM), cryptococcemia and pulmonary cryptococcosis (PC) in patients with cryptococcal antigenemia were 58.4% (95% CI, 48.0–68.9%), 50.7% (95% CI, 39.1–62.2%), and 68.5% (95% CI, 58.7–78.4%), respectively. The median (range) serum CrAg titers in severe cryptococcal diseases (CM or cryptococcemia), localized PC (without co-existing CM or cryptococcemia) and isolated cryptococcal antigenemia were 1:2560 (1:10–1:2560), 1:20 (1:2–1:320), and 1:5 (1:2–1:320), respectively. Serum CrAg titers ≥1:320 were independently associated with CM (adjusted OR 26.88; 95%CI, 8.36–86.42). Severe cryptococcal diseases were found in all patients with serum CrAg titers ≥1:640. None of the patients with serum CrAg titers ≤ 1:5 had CM.Conclusion: The prevalence of cryptococcal antigenemia was high in HIV inpatients, supporting routine CrAg screening. Clinical cryptococcal diseases, most commonly the PC, existed in the majority of the patients with cryptococcal antigenemia. Since serum CrAg titer is correlated with cryptococcal disease severity, it may possibly guide anti-fungal treatment.

Evaluation of low cryptococcal antigen titer as determined by the lateral flow assay in serum and cerebrospinal fluid among HIV-negative patients: a retrospective diagnostic accuracy study

Cryptococcosis is one of the most common opportunistic infections in both immunocompetent and immunocompromised hosts. Although the cryptococcal antigen (CrAg) lateral flow assay (LFA) has been widely used in clinical settings due to its high sensitivity and specificity, the diagnostic value of a low CrAg LFA titers remains unclear. In this study, we performed a retrospective analysis of 149 HIV-negative patients with low CrAg LFA titers (≤1:10) in a Chinese tertiary hospital from January 2013 to December 2017, to evaluate the diagnostic value of low CrAg LFA titers in serum and cerebrospinal fluid (CSF) at different thresholds. Sensitivity and specificity of low CrAg LFA titers in patients with definitive diagnoses of cryptococcosis were 39.6% (95% CI, 29.7–50.1%) and 100% (95% CI, 69.2–100%), respectively, at a threshold of 1:10 in serum. A sensitivity of 72.9% (95% CI, 62.9–81.5%) and a decreased specificity of 70.0% (95% CI, 34.8–93.3%) were observed at a threshold of 1:5 in serum. No false-positive cases were identified in patients with low CrAg titers in CSF and all positive predictive values (PPVs) were 100%. Among the cases with low serum CrAg titers, lumbar puncture was performed in 97 patients and positive CSF CrAg titers were reported in 6 patients. In conclusion, the results of this study imply that low CrAg LFA titer, either in serum or CSF, is crucial for early diagnosis of cryptococcosis in HIV-negative patients, and lumbar puncture is recommended to be performed routinely for CSF testing when a positive low serum titer is reported. Cryptococcal meningitis should be considered seriously when the CSF CrAg titer is positive.

Prevalence and mortality of cryptococcal disease in adults with advanced HIV in an urban tertiary hospital in Sierra Leone: a prospective study

BackgroundThe global annual estimate for cryptococcal disease-related deaths exceeds 180,000, with three fourth occurring in sub-Saharan Africa. The World Health Organization (WHO) recommends cryptococcal antigen (CrAg) screening in all HIV patients with CD4 count < 100/μl. As there is no previous published study on the burden and impact of cryptococcal disease in Sierra Leone, research is needed to inform public health policies. We aimed to establish the seroprevalence and mortality of cryptococcal disease in adults with advanced HIV attending an urban tertiary hospital in Sierra Leone.MethodsA prospective cohort study design was used to screen consecutive adult (18 years or older) HIV patients at Connaught Hospital in Freetown, Sierra Leone with CD4 count below 100 cells/mm3 from January to April 2018. Participants received a blood CrAg lateral flow assay (IMMY, Oklahoma, USA). All participants with a positive serum CrAg had lumbar puncture and cerebrospinal fluid (CSF) CrAg assay, and those with cryptococcal diseases had fluconazole monotherapy with 8 weeks followed up. Data were entered into Excel and analysed in Stata version 13.0. Proportions, median and interquartile ranges were used to summarise the data. Fisher’s exact test was used to compare categorical variables.ResultsA total of 170 patients, with median age of 36 (IQR 30–43) and median CD4 count of 45 cells/mm3 (IQR 23–63) were screened. At the time of enrolment, 54% were inpatients, 51% were newly diagnosed with HIV, and 56% were either ART-naïve or newly initiated (≤ 30 days). Eight participants had a positive blood CrAg, giving a prevalence of 4.7% (95% CI: 2.4–9.2%). Of those with a positive CrAg, CSF CrAg was positive in five (62.5%). Five (62.5%) CrAg-positive participants died within the first month, while the remaining three were alive and established on ART at 8 weeks.ConclusionA substantial prevalence of cryptococcal antigenaemia and poor outcome of cryptococcal disease were demonstrated in our study. The high mortality suggests a need for the HIV programme to formulate and implement policies on screening and pre-emptive fluconazole therapy for all adults with advanced HIV in Sierra Leone, and advocate for affordable access to effective antifungal therapies.

253. Evaluation of a Cryptococcal Antigen Lateral Flow Assay and the Burden of Cryptococcal Disease: A Cohort Study at Grady Memorial Hospital in Atlanta, Georgia

BackgroundWhile Cryptococcus neoformans is a major cause of morbidity and mortality among HIV-infected persons worldwide, there is scarce recent data on disease prevalence in the United States, including in Southeastern states, where HIV rates are high. We sought to determine the prevalence of cryptococcal disease and compare the performance of a cryptococcal antigen (CrAg) lateral flow assay (LFA) vs. latex agglutination (LA) test.MethodsAll patients from Grady Memorial Hospital in Atlanta, Georgia who had a serum or cerebrospinal fluid (CSF) sample sent for CrAg LA testing as part of routine management from November 2017 to July 2018 were included. The LFA was performed on all samples by research staff; results were not available to clinicians. Rates of disease and agreement between the LA test and LFA were calculated.ResultsAmong 467 patients, 570 LA tests were performed; 417 on serum and 153 on CSF (87 patients with multiple tests performed). Mean age was 44 years, and most were male (n = 322, 69%). Most patients had HIV (n = 371, 79%); median CD4 count was 73 cells/mm3 and 77% were not receiving ART. Among HIV-infected individuals, testing was performed equally in the inpatient and outpatient setting. Cryptococcal testing was done in 53 persons without apparent risk factors. Thirty-three (7%) patients had a positive serum or CSF test. Five (1%) patients had both a positive serum and CSF LA test and LFA. While the overall agreement between the LA test and LFA was substantial to high for CSF (κ = 0.71) and serum (κ = 0.93), respectively, there were important discrepancies. Four patients with a negative serum LA test had a positive serum LFA. Five patients had false-positive CSF LA tests, determined by negative CSF LFA testing, India ink, and CSF and fungal cultures. All were treated with amphotericin and flucytosine with one patient experiencing a severe anaphylactic reaction to amphotericin.ConclusionWe found a moderately high rate of cryptococcal disease and important discrepancies between the LA test and LFA. The LFA appeared to be more sensitive for cryptococcemia and more specific for meningitis. Clinical implications of these findings include earlier detection and treatment of cryptococcemia, and averting unnecessary treatment of meningitis with costly medications associated with high rates of adverse events. Disclosures All authors: No reported disclosures.

Epidemiology of Cryptococcal antigenemia among HIV infected patients in southwestern Nigeria

Cryptococcosis is a life-threatening fungal infection that presents diversely with no specific pathognomonic features. Cryptococcal disease is one of the most important opportunistic infections, and a significant contributor to early mortality in HIV infected subjects. Cryptococcal antigenemia occurs in Nigeria, but the magnitude of this disease remains unclear. This study was carried out to determine the prevalence of CrAg among HIV infected and HIV seronegative subjects and to assess the relationship between CD4 count and CrAg in HIV-positive subjects attending Adeoyo Maternity Teaching Hospital, Yemetu, Ibadan. In an hospital-based case-control study using simple random sampling, 114 HIV-seropositive individuals (cases) and 228 HIV-seropositive individuals (controls) were recruited. A semi-structured interviewer-administered questionnaire was used to collect data from subjects and retrospective review of CD4 count records in HIV infected subjects. Five millilitres of venous blood were collected from each participant. Serum Cryptococcal antigen testing was done using CrAg Lateral Flow Assay. Data was analyzed using descriptive statistics and bivariate analysis at 5% level of significance. Mean age of cases was 41.2 ± 10.0 years and 85 (74.6%) were females while mean age of controls was 38.9 ± 13.7 years and 156 (68.4%) were females. The prevalence of CrAg among cases was 11.4% and 7.0% among controls. Cases were about two times more likely to test positive for CrAg. However, the association was not statistically significant (OR: 1.71, 95%CI: 0.79 - 3.68). Individuals with CD4 counts of ≤100 cells/µl were 20 times more likely to have positive serum cryptococcal antigen than individuals with CD4 counts >100 cells/µl (OR: 20.3, 95%CI: 5.23-78.9). This study has demonstrated significant prevalence of Cryptococcal antigenemia among the study population; however, prevalence was higher among cases. Screening for CrAg should therefore be part of routine tests amongst all confirmed HIV seropositive cases, since asymptomatic cryptococcal antigenemia predicts impending cryptococcal infection with probable mortality.

Point-of-Care Cryptococcal Antigen Screening Predicts Cryptococcal Meningitis and Mortality Among People Living with HIV in a South African Township

Background: Cryptococcal meningitis remains a leading cause of HIV-related mortality. Point-of-care tests for cryptococcal antigen (CrAg) may identify HIV-associated cryptococcal infections for administering anti-fungal preemptive treatment to prevent cryptococcal meningitis and death. Methods: We conducted a prospective study of adults presenting for HIV testing in Umlazi Township, South Africa. Among people living with HIV, we performed laboratory-based serum CrAg enzyme immunoassay (EIA) testing, and clinic-based testing of fingerprick capillary blood, venous whole blood, and urine with a rapid CrAg lateral flow assay (LFA) (Immy Diagnostics, Norman, USA). We followed participants for 12 months to assess cryptococcal meningitis and all-cause mortality using medical charts, hospital records, phone calls, and the national death registry. We used Cox proportional hazards to measure associations between baseline CrAg testing and progression to cryptococcal meningitis and/or death, controlling for age, sex, baseline CD4 count, and initiation of antiretroviral therapy. Findings: Among 2,379 HIV-infected adults, 1,370 (58%) were female; median CD4 count was 317 (IQR 173-491) cells/mm3. Overall, 50 (3.2%) participants were CrAg LFA positive by fingerprick capillary blood, 32 (2.0%) by venous whole blood, and 132 (8.4%) by urine. Among all participants after twelve months, 22 (0.9%) participants developed cryptococcal meningitis, 87 (3.7%) participants died, and 104 (4.4%) either developed cryptococcal meningitis or died. In unadjusted analyses, positive fingerprick and venous blood, but not urine, CrAg LFA results were associated with cryptococcal meningitis. In adjusted analyses, positive CrAg LFA results from fingerprick or venous blood had a 3.8 (95% CI 1.7-8.4) and 4.3 (95% CI 2.0-9.2) greater hazard of cryptococcal meningitis or death, with most events occurring within six months. Positive lab-based serum EIA had a 6.3 (95% CI 2.9-14.0) greater hazard of cryptococcal meningitis or death. Interpretation: Clinic-based CrAg LFA screening results strongly predict HIV-associated cryptococcal meningitis or death, and could provide a strategy for rapid anti-fungal prophylaxis or treatment initiation to improve HIV-associated outcomes. Funding: This work was supported by the Infectious Disease Society of America Education & Research Foundation and National Foundation for Infectious Diseases (PKD); Massachusetts General Hospital Executive Committee on Research (PKD); the Harvard University Center for AIDS Research [P30 AI060354] (PKD); and the National Institute of Allergy and Infectious Diseases [K23 AI108293] (PKD) and [R21AI110264] (IVB). Declaration of Interest: We declare that we have no competing interests. Ethical Approval: The study was approved by the University of Washington’s Institutional Review Board (IRB #49563) and the University of KwaZulu-Natal’s Medical Research Ethics Committee (Protocol #BF052/13). All study participants provided written informed consent.

Prevalence and factors associated with cryptococcosis among human immunodeficiency virus-infected patients of a tertiary hospital in Northwestern Nigeria

Background: Cryptococcosis is an important opportunistic infection and major contributor to mortality in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients. There have been increasing reports of varied prevalence for cryptococcosis among HIV patients in Nigeria. Aims: The aim of this study was to determine the prevalence and factors associated with risk of cryptococcosis among HIV/AIDS patients from Usmanu Danfodiyo University Teaching Hospital (UDUTH), Sokoto. Materials and Methods: A descriptive cross-sectional study at HIV clinic of UDUTH, Sokoto, Nigeria, was conducted. The participants were HIV positive enrolled by systematic random sampling. Information on sociodemographics and clinical features of the participants was recorded. Blood was collected to determine the CD4 count/presence of cryptococcosis among the participants. Their CD4 counts were determined, and the presence of cryptococcal antigen was detected in the blood sample using IMMY CrAg LFA (cryptococcal antigen lateral flow assay test kit). Statistical analysis using Epi Info version 7.2/SPSS version 20.0 was done. Univariate, bivariate, and multivariate analyses were done. Results: Of 326 HIV-positive patients recruited, 36 of them were positive to cryptococcal antigen (prevalence of 11%). The highest proportion of patients positive to cryptococcosis (13 [36.1%) had CD4 values <50 cells/μL. Weight loss was the most common presenting symptom (11 [30.6%]) among those who were Cryptococcus positive. Conclusions: This study demonstrates that cryptococcosis is a burden among HIV patient in Sokoto, and its occurrence is associated with decreasing levels of CD4.

Antibody Responses in HIV-Infected Patients With Advanced Immunosuppression and Asymptomatic Cryptococcal Antigenemia.

BackgroundThere are no host biomarkers of risk for HIV-associated cryptococcal meningitis (CM) except CD4+ T-cell deficiency. At present, serum cryptococcal antigen (CrAg) screening of those with CD4 <100 cells/µL is used to identify persons at risk for HIV-associated CM. We determined if plasma antibody profiles could discriminate CrAg+ from CrAg- patients.MethodsWe performed serological analyses of 237 HIV-infected asymptomatic Zimbabwean patients with CD4 <100 cells/µL; 125 CrAg- and CrAg+ but cerebrospinal fluid CrAg- by CrAg lateral flow assay. We measured plasma immunoglobulin M (IgM), immunoglobulin G (IgG) 1, and IgG2 concentrations by Luminex, and titers of Cryptococcus neoformans (Cn) glucuronoxylomannan (GXM) polysaccharide and naturally occurring Laminarin (natural Lam, a β-(1–3)-glucan linked polysaccharide)-binding IgM and IgG by enzyme-linked immunosorbent assay.ResultsGXM-IgG, -IgM, and -IgG2 levels were significantly higher in CrAg+ patients, whereas natural Lam-IgM and Lam-IgG were higher in CrAg- patients before and after adjustment for age, sex, and CD4 T-cell count, despite overlap of values. To address this variability and better discriminate the groups, we used Akaike Information Criteria to select variables that independently predicted CrAg+ status and included them in a receiver operating characteristic curve to predict CrAg status. By inclusion of CD4, GXM-IgG, GXM-IgM, and Lam-IgG, -IgG2, and -IgM, this model had an 80.4% probability (95% confidence interval, 0.75–0.86) of predicting CrAg+ status.ConclusionsStatistical models that include multiple serological variables may improve the identification of patients at risk for CM and inform new directions in research on the complex role that antibodies may play in resistance and susceptibility to CM.

The performance of serum cryptococcal capsular polysaccharide antigen test, histopathology and culture of the lung tissue for diagnosis of pulmonary cryptococcosis in patients without HIV infection.

BackgroundClinicians may fail to make an early diagnosis of pulmonary cryptococcosis (PC) without HIV infection. Serum cryptococcal capsular polysaccharide antigen (CrAg) test, histopathology and culture of lung tissue play different roles in diagnosis of PC.ObjectiveTo investigate the performance of serum CrAg test, histopathology and culture of the lung tissue in diagnosis of PC without HIV infection.Patients/methodsFrom January 2011 to September 2017, patients with proven PC were recruited from a teaching hospital in southern China. Those patients with HIV infection, PC confirmed by surgery or PC with probable or possible diagnosis were excluded from the study. Latex agglutination test and CrAg lateral flow assay were used for detection of serum CrAg. Lung biopsy and needle aspiration were performed under computed tomography guidance.ResultsEighty-nine patients with proven PC including 41 male (46.1%) and 48 female (53.9%) were enrolled. Fifty-one (57.3%) patients had underlying disease. Positive CrAg test was found in 83 (93.3%) cases. Among six cases with negative CrAg test, PC was confirmed by histology in two cases and positive culture in four cases. The histopathological results of 77 (86.5%) cases revealed cryptococcal granuloma and 12 cases showed chronic inflammation, which was confirmed by positive culture. Among 65 cases, the diseased tissue of 46 (70.8%) cases presented Cryptococcus neoformans in the culture and one case was diagnosed with lung cancer coexisting with PC.ConclusionOur findings showed that serum CrAg test is rapid and sensitive in diagnosing PC, histology is important for confirming PC and culture plays a complementary role. Biopsied lung tissue should be submitted for cultures whenever feasible.

Brief Report: Point of Care Cryptococcal Antigen Screening: Pipetting Finger-Prick Blood Improves Performance of Immunomycologics Lateral Flow Assay.

Cryptococcal antigen (CrAg) screening at the point of care could improve cryptococcal meningitis prevention where laboratory resources are limited. We evaluated the accuracy of Immunomycologics (IMMY, Norman, OK) CrAg lateral flow assay (LFA) using different techniques at point of care. Two tertiary-level hospitals in Johannesburg and a community health clinic in Soweto, South Africa. A case-control diagnostic validation study and a prospective clinic-based implementation study using the IMMY CrAg LFA on finger-prick blood. Accuracy, using direct application of LFA to sample, or pipette to transfer sample to diluent, and reading after 10 and 20 minutes, was compared with laboratory-based plasma testing. The validation study tested 64 CrAg-positive and 152 CrAg-negative patients with no symptoms or signs of meningitis, identified by routine laboratory screening, recruited by convenience sampling. Consecutively diagnosed HIV-infected adults (n = 654) were included in the implementation study. Sensitivity was 82% and 20% when the LFA was read 10 minutes after direct application to finger-prick blood in the validation and implementation studies, respectively. Using a pipette to transfer blood and reading after 20 minutes improved sensitivity to 100%, while retaining 100% specificity, in both studies. Although the IMMY CrAg LFA performs well when applied directly to finger-prick blood for diagnosing cryptococcal meningitis, this technique may not provide adequate volume to detect low concentrations of CrAg when screening asymptomatic patients. Using a pipette to transfer larger volumes of blood to diluent before CrAg LFA testing and reading results after 20 minutes is a more reliable point-of-care method.

A qualitative evaluation of an implementation study for cryptococcal antigen screening and treatment in Uganda.

Cryptococcal meningiti s causes 15% of AIDS-related deaths globally. Screening and preemptive treatment for cryptococcal antigen (CrAg) in the blood of persons with advanced HIV/AIDS reduces mortality. National and international HIV guidelines recommend CrAg screening; however, implementation studies and evaluations of how to integrate CrAg screening programs into existing HIV care infrastructure are lacking.During a CrAg screening program in Kampala, Uganda, we interviewed 15 health care workers (2 coordinating research nurses and 13 clinic personnel) from 6 HIV clinics between March and April 2017, to identify barriers to implementation as well as facilitating factors for program success. The interviews were coded and themes compiled.We found key factors for successful implementation of a CrAg screening program were: adequate supplies of fluconazole and CrAg lateral flow assay (LFA) point-of-care tests, timely patient follow-up, and quick turnaround time of laboratory results. Although both CrAg LFA kits and fluconazole are on the national formulary, stockouts are common, affecting patient care. The CrAg screening recommendation by national HIV guidelines remains integral to the success of the program, as overburdened clinics are otherwise reluctant to adopt additional screening. Collaboration with Ministries of Health for support with enforcing national guidelines, and procuring supplies is paramount to a successful CrAg screening program.Development of a CrAg screening and treatment program within the HIV clinic infrastructure has a number of barriers. Education and training of clinic staff, along with partnership with the Ministry of Health to ensure adequate supplies, facilitated the program.

Performance of cryptococcal antigen lateral flow assay in serum, cerebrospinal fluid, whole blood, and urine in HIV-infected patients with culture-proven cryptococcal meningitis admitted at a Brazilian referral center.

ABSTRACTCryptococcal meningitis is the most common cause of opportunistic meningitis in HIV-infected patients in Brazil and causes unacceptable high mortality rates. In this study, HIV-infected patients with a first episode of culture-proven cryptococcal meningitis in cerebrospinal fluid (CSF) were prospectively included in order to evaluate sensitivity of cryptococcal antigen (CrAg) lateral flow assay (LFA) in serum, CSF, whole blood (fingerstick), and fresh urine. In addition, HIV-infected patients with other neurological confirmed diseases were included in order to evaluate the specificity of CrAg LFA in serum. Twenty patients with cryptococcal meningitis were included and in 19 of them, CrAg LFA in CSF, serum, and whole blood were positive (95% sensitivity). In 18 patients, India ink test was positive in CSF (90% sensitivity), and in 16 cases, CrAg LFA was positive in urine (80% sensitivity). Thirty-six HIV-infected patients with other neurological diseases had negative results of CrAg LFA in serum (100% specificity). In conclusion, CrAg LFA in serum, CSF, and whole blood showed high sensitivity and specificity. Whole blood CrAg LFA seems to be a good and reliable strategy to improve AIDS-related cryptococcal meningitis diagnosis in Brazil.

Evaluation of a point-of-care immunoassay test kit 'StrongStep' for cryptococcal antigen detection.

BackgroundHIV-associated cryptococcal meningitis is the leading cause of adult meningitis in Sub-Saharan Africa, accounting for 15%–20% of AIDS-attributable mortality. The development of point-of-care assays has greatly improved the screening and diagnosis of cryptococcal disease. We evaluated a point-of-care immunoassay, StrongStep (Liming Bio, Nanjing, Jiangsu, China) lateral flow assay (LFA), for cryptococcal antigen (CrAg) detection in cerebrospinal fluid (CSF) and plasma.MethodsWe retrospectively tested 143 CSF and 77 plasma samples collected from HIV-seropositive individuals with suspected meningitis from 2012–2016 in Uganda. We prospectively tested 90 plasma samples collected from HIV-seropositive individuals with CD4 cell count <100 cells/μL from 2016–2017 as part of a cryptococcal antigenemia screening program. The StrongStep CrAg was tested against a composite reference standard of positive Immy CrAg LFA (Immy, Norman, OK, USA) or CSF culture with statistical comparison by McNemar’s test.ResultsStrongStep CrAg had a 98% (54/55) sensitivity and 90% (101/112) specificity in plasma (P = 0.009, versus reference standard). In CSF, the StrongStep CrAg had 100% (101/101) sensitivity and 98% (41/42) specificity (P = 0.99). Adjusting for the cryptococcal antigenemia prevalence of 9% in Uganda and average cryptococcal meningitis prevalence of 37% in Sub-Saharan Africa, the positive predictive value of the StrongStep CrAg was 50% in plasma and 96% in CSF.ConclusionsWe found the StrongStep CrAg LFA to be a sensitive assay, which unfortunately lacked specificity in plasma. In lower prevalence settings, a majority of positive results from blood would be expected to be false positives.

Cryptococcal antigen screening by lay cadres using a rapid test at the point of care: A feasibility study in rural Lesotho.

IntroductionCryptococcal meningitis is one of the leading causes of death among people with HIV in Africa, primarily due to delayed presentation, poor availability and high cost of treatment. Routine cryptococcal antigen (CrAg) screening of patients with a CD4 count less than 100 cells/mm3, followed by pre-emptive therapy if positive, might reduce mortality in high prevalence settings. Using the cryptococcal antigen (CrAg) lateral flow assay (LFA), screening is possible at the point of care (POC). However, critical shortages of health staff may limit adoption. This study investigates the feasibility of lay counsellors conducting CrAg LFA screening in rural primary care clinics in Lesotho.MethodsFrom May 2014 to June 2015, individuals who tested positive for HIV were tested for CD4 count and those with CD4 <100 cells/mm3 were screened with CrAg LFA. All tests were performed by lay counsellors. CrAg-positive asymptomatic patients received fluconazole, while symptomatic patients were referred to hospital. Lay counsellors were trained and supervised by a laboratory technician and counsellor activity supervisor. Additionally, nurses and doctors were trained on CrAg screening and appropriate treatment.ResultsDuring the study period, 1,388 people were newly diagnosed with HIV, of whom 129 (9%) presented with a CD4 count <100 cells/mm3. Of these, 128 (99%) were screened with CrAg LFA and 14/128 (11%) tested positive. Twelve of the 14 (86%) were asymptomatic, and received outpatient fluconazole. All commenced ART with a median time to initiation of 15.5 days [IQR: 14–22]. Of the asymptomatic patients, nine (75%) remained asymptomatic after a median time of 5 months [IQR; 3–6] of follow up. One (8%) became co-infected with tuberculosis and died and two were transferred out. The two patients with symptomatic cryptococcal meningitis (CM) were referred to hospital, where they later died.ConclusionsCrAg LFA screening by lay counsellors followed by pre-emptive fluconazole treatment for asymptomatic cases, or referral to hospital for symptomatic cases, proved feasible. However, regular follow-up to ensure proper management of cryptococcal disease was needed. These early results support the wider use of CrAg LFA screening in remote primary care settings where upper cadres of healthcare staff may be in short supply.

Low Cryptococcus Antigen Titers as Determined by Lateral Flow Assay Should Be Interpreted Cautiously in Patients without Prior Diagnosis of Cryptococcal Infection.

Detection of Cryptococcus antigen (CrAg) is invaluable for establishing cryptococcal disease. Multiple different methods for CrAg detection are available, including a lateral flow assay (LFA). Despite excellent performance of the CrAg LFA, we have observed multiple cases of low-titer (≤1:5) positive CrAg LFA results in patients for whom cryptococcosis was ultimately excluded. To investigate the accuracy of low-titer positive CrAg LFA results, we performed chart reviews for all patients with positive CrAg LFA results between June 2014 and December 2016. During this period, serum and/or cerebrospinal fluid (CSF) samples from 3,969 patients were tested with the CrAg LFA, and 55 patients (1.5%) tested positive. Thirty-eight of those patients lacked a history of cryptococcal disease and were the focus of this study. Fungal culture or histopathology confirmed Cryptococcus infection for 20 patients (52.6%), and CrAg LFA titers in serum and CSF samples ranged from 1:5 to ≥1:2,560. For the 18 patients (47.4%) without culture or histopathological confirmation, the CrAg LFA results were considered true-positive results for 5 patients (titer range, 1:10 to ≥1:2,560), due to clinical improvement with targeted therapy and decreasing CrAg LFA titers. The remaining 13 patients had CrAg LFA titers of 1:2 (n = 11) or 1:5 (n = 2) and were ultimately diagnosed with an alternative condition (n = 11) or began therapy for possible cryptococcosis without improvement (n = 2), leading to an overall CrAg LFA false-positive rate of 34%. We recommend careful clinical correlation prior to establishing a diagnosis of cryptococcal infection for patients with first-time positive CrAg LFA titers of 1:2.

Cryptococcal Antigenemia in Nigerian Patients With Advanced Human Immunodeficiency Virus: Influence of Antiretroviral Therapy Adherence.

Background. Cryptococcal meningitis has a high mortality in human immunodeficiency virus (HIV)-infected persons in Africa. This is preventable with early screening and preemptive therapy. We evaluated the prevalence of cryptococcal disease by antigen testing, possible associated factors, and outcomes in HIV-infected patients being managed in a tertiary hospital in Lagos, Nigeria.Methods. Sera were collected from 214 consenting HIV-infected participants with CD4+ counts <250 cells/mm3, irrespective of their antiretroviral therapy (ART) status, between November 2014 and May 2015. A cryptococcal antigen (CrAg) lateral flow assay was used for testing. Pertinent clinical data were obtained from patients and their case notes.Results. Of the 214 participants, females (124; 57.9%) outnumbered males. Mean age was 41.3 ± 9.4 (standard deviation) years. The majority (204; 95.3%) were ART experienced. The median CD4+ cell count was 160 cells/mm3 (interquartile range, 90–210). The overall seroprevalence of cryptococcal antigenemia was 8.9% (19 of 214); 6 of 61 (9.8%) in those with CD4+ cell counts <100 cells/mm3, 4 of 80 (5.0%) in the 100–200 group, and 9 of 73 (12.3%) in 200–250 cells/mm3 group. Among ART-naive patients, 1 of 10 (10%) was CrAg positive. Twenty-seven of 214 (12.6%) had associated oral thrush. Potential baseline meningitis symptoms (3 of 214 [1.4%] experienced neck pain or stiffness and 21 of 214 [9.8%] experienced headache) were common in the study group, but the result was not statistically significant in relation to CrAg positivity. Two of 19 (10.5%) CrAg-positive patients died, 10 of 19 (52.6%) were lost to follow up, and 7 of 19 (36.8%) were alive. Empirical fluconazole was routinely given to those with low CD4 counts <100 cells/mm3, which was unrelated to CrAg positivity (P = .018).Conclusions. We report a prevalence of 8.9% cryptococcal antigenemia in a setting where first-line antifungals are not readily available. We recommend CrAg screening for HIV-infected patients, even for patients on ART.