P-206. Change in Cryptococcal Antigen Titer Over Time in Uganda

ABSTRACTCryptococcal 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 of <50 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.IMPORTANCE Cryptococcal disease (CD) remains a leading cause of morbidity and mortality among individuals with advanced HIV disease (AHD). Identifying point of care (POC) approaches to screening for CD in asymptomatic individuals is important to guide therapeutic management. We evaluated the use of POC fingerprick whole blood and urine testing for cryptococcal disease in patients with AHD as compared with laboratory-based serum antigen testing. POC fingerprick whole blood and urine testing had low sensitivity and specificity in asymptomatic individuals with AHD. Most analysis has focused on evaluating test performance in symptomatic individuals. Here we show that POC testing with whole blood and urine samples should not be used to screen for asymptomatic CD in AHD.

Characteristics and prognostic risk factors of patients with sequence type 5 lineage-associated cryptococcosis in China

There is a paucity of data on clinical epidemiology of osteonecrosis in HIV-infected patients. We aimed to describe patients' characteristics and natural history of this poorly known condition. All cases of symptomatic HIV-related osteonecrosis diagnosed from 1990 through 2003 in 19 Spanish clinics were reviewed. Functional status at the last visit was assessed with the validated Western Ontario and McMaster Universities Index questionnaire. Of 54 patients analyzed, 29 (53.7%) had a single bone necrosis, and 25 (46.3%) had 2 or more sites involved. Progression of symptoms happened more often in patients with hip involvement (17/39 vs 0/8 patients; P = 0.019). Twenty patients (37%) required surgical intervention. Male sex and higher CD4 cell count were associated with surgery on multivariable analysis. Overall, at the end of the follow-up period, half of the patients had moderate to severe disability (Western Ontario and McMaster Universities Index score > or =60). During a follow-up period of 137 person-years, only 2 new episodes of osteonecrosis were observed (rate of recurrences, 1.5/100 person-years; 95% confidence interval, 0.4-5.1). HIV-related osteonecrosis is associated with significant disability over time. Location of bone necrosis, sex, and CD4 cell count may influence the outcome. The risk for recurrences for patients who have experienced 1 episode is low.

To evaluate the association between a positive serum cryptococcal antigen (CRAG) test at baseline and mortality during the first 12 weeks on antiretroviral therapy (ART). Cryptococcal meningitis is a leading cause of HIV-related mortality in Africa, but current guidelines do not advocate CRAG testing as a screening tool. Between May 2003 and December 2004, we enrolled HIV-1 infected individuals into a study of ART monitoring in rural Uganda. CRAG testing was conducted retrospectively on stored pre-ART serum samples of participants whose baseline CD4 cell count was <100 cells/mul and who were without symptoms suggestive of disseminated cryptococcal disease at enrolment. Of 377 participants, 5.8% had serum CRAG titre >/=1:2. Of these, 23% died during follow-up. Controlling for CD4 cell count, HIV-1 viral load, anaemia, active tuberculosis and body mass index, relative risk of death during follow-up among those with asymptomatic cryptococcal antigenemia at baseline was 6.6 [95% confidence interval (CI) 1.86-23.61, P = 0.0036]. The population attributable risk for mortality associated with a positive CRAG at baseline was 18% (CI 2-33%), similar to that associated with active tuberculosis (19%, CI 1-36%). Asymptomatic cryptococcal antigenemia independently predicts death during the first 12 weeks of ART among individuals with advanced HIV disease in rural Uganda. Routine screening and provision of azole antifungal therapy prior to or simultaneous with the start of ART should be evaluated for the potential to prevent mortality in this population.

Evidence to inform cryptococcal antigen (CrAg)-screening guidelines among ART-experienced populations is lacking. We performed a study evaluating the utility of reflex CrAg screening in Gaborone, Botswana. CD4 count data were collected from the HIV reference laboratory from 2014-2016. CrAg screening was performed on samples with CD4 ≤100 cells/µL beginning January 2015. The proportion of CD4 counts ≤100 cells/µL was determined and the frequency of repeat CrAg testing described. Analyses ascertained the impact of ART status on CrAg prevalence and outcomes, and whether CrAg titers could be used for risk stratification. Overall, 5.6% (3335/59 300) of individuals tested had CD4 ≤100 cells/μL; 2108 samples with CD4 ≤100 cells/μL from 1645 unique patients were CrAg tested. Over half of samples were from ART-experienced individuals: 40.9% (863) on ART and 12.1% (255) defaulters; 22% (463) of CrAg tests were on repeat samples. CrAg prevalence was 4.8% (72/1494; 95% CI, 3.8-6.0%) among outpatients and 21.9% (32/151; 95% CI, 15.3-28.5%) among inpatients. CrAg prevalence rates did not differ by ART status, but 6-month mortality was significantly lower in CrAg-positive individuals on ART at screening. Ten CrAg positives were identified through repeat testing. A CrAg titer cutoff ≥1:80 provided the best discrimination for 6-month survival. CrAg-positivity rates in an ART-experienced population were comparable to those seen in ART-naive populations. Repeat screening identified individuals who seroconverted to CrAg positivity and were at risk of cryptococcal disease. CrAg titers ≥1:80 can help identify the individuals at highest risk of death for more intensive management.

Retrospective data suggest that cryptococcal antigen (CrAg) screening in patients with late-stage human immunodeficiency virus (HIV) initiating antiretroviral therapy (ART) may reduce cryptococcal disease and deaths. Prospective data are limited. CrAg was measured using lateral flow assays (LFA) and latex agglutination (LA) tests in 645 HIV-positive, ART-naive patients with CD4 counts ≤100 cells/µL in Cape Town, South Africa. CrAg-positive patients were offered lumbar puncture (LP) and treated with antifungals. Patients were started on ART between 2 and 4 weeks and followed up for 1 year. A total of 4.3% (28/645) of patients were CrAg positive in serum and plasma with LFA. These included 16 also positive by urine LFA (2.5% of total screened) and 7 by serum LA (1.1% of total). In 4 of 10 LFA-positive cases agreeing to LP, the cerebrospinal fluid (CSF) CrAg LFA was positive. A positive CSF CrAg was associated with higher screening plasma/serum LFA titers.Among the 28 CrAg-positive patients, mortality was 14.3% at 10 weeks and 25% at 12 months. Only 1 CrAg-positive patient, who defaulted from care, died from cryptococcal meningitis (CM). Mortality in CrAg-negative patients was 11.5% at 1 year. Only 2 possible CM cases were identified in CrAg-negative patients. CrAg screening of individuals initiating ART and preemptive fluconazole treatment of CrAg-positive patients resulted in markedly fewer cases of CM compared with historic unscreened cohorts. Studies are needed to refine management of CrAg-positive patients who have high mortality that does not appear to be wholly attributable to cryptococcal disease.

Our study's primary objective was to compare 1-year survival rates between serum cryptococcal antigen (sCrAg)-positive and sCrAg-negative HIV-positive individuals with CD4+ cell counts less than 100 cells/μl without symptoms of meningitis in Zimbabwe. This was a prospective cohort study. Participants were enrolled as either sCrAg-positive or sCrAg-negative and followed up for 52 weeks or less, with death as the outcome. Lumbar punctures were recommended to all sCrAg-positives and inpatient management with intravenous amphotericin B and high-dose fluconazole was recommended to those with disseminated Cryptococcus. Antiretroviral therapy was initiated immediately in sCrAg-negatives and after at least 4 weeks following initiation of antifungals in sCrAg-positives. Multivariable logistic regression models were used to determine risk factors for mortality. We enrolled 1320 participants and 130 (9.8%) were sCrAg positive, with a median sCrAg titre of 1 : 20. Sixty-six (50.8%) sCrAg-positives had lumbar punctures and 16.7% (11/66) had central nervous system (CNS) dissemination. Cryptococcal blood cultures were performed in 129 sCrAg-positives, with 10 (7.8%) being positive. One-year (48-52 weeks) survival rates were 83.9 and 76.1% in sCrAg-negatives and sCrAg-positives, respectively, P = 0.011. Factors associated with increased mortality were a positive sCrAg, CD4+ cell count less than 50 cells/μl and having presumptive tuberculosis (TB) symptoms. Our study reports a high prevalence of subclinical cryptococcal antigenemia and reiterates the importance of TB and a positive sCrAg as risk factors for mortality in advanced HIV disease (AHD). Therefore, TB and sCrAg screening remains a crucial component of AHD package, hence it should always be part of the comprehensive clinical evaluation in AHD patients.

In 2017, the World Health Organization (WHO) published guidelines for the management of advanced human immunodeficiency virus (HIV) disease within a public health approach. Recent data suggest that more than a third of people starting antiretroviral therapy (ART) do so with advanced HIV disease, and an increasing number of patients re-present to care at an advanced stage of HIV disease following a period of disengagement from care. These guidelines recommend a standardized package of care for adults, adolescents, and children, based on the leading causes of morbidity and mortality: tuberculosis, severe bacterial infections, cryptococcal meningitis, toxoplasmosis, and Pneumocystis jirovecii pneumonia. A package of targeted interventions to reduce mortality and morbidity was recommended, based on results of 2 recent randomized trials that both showed a mortality reduction associated with delivery of a simplified intervention package. Taking these results and existing recommendations into consideration, WHO recommends that a package of care be offered to those presenting with advanced HIV disease; depending on age and CD4 cell count, the package may include opportunistic infection screening and prophylaxis, including fluconazole preemptive therapy for those who are cryptococcal antigen positive and without evidence of meningitis. Rapid ART initiation and intensified adherence interventions should also be proposed to everyone presenting with advanced HIV disease.

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

Cryptococcal meningitis (CM) remains a common AIDS-defining illness in Africa and Asia. Subclinical cryptococcal antigenemia is frequently unmasked with antiretroviral therapy (ART). We sought to define the cost-effectiveness of serum cryptococcal antigen (CRAG) screening to identify persons with subclinical cryptococcosis and the efficacy of preemptive fluconazole therapy. There were 609 ART-naive adults with AIDS who started ART in Kampala, Uganda, and who had a serum CRAG prospectively measured during 2004-2006. The number needed to test and treat with a positive CRAG was assessed for > or = 30-month outcomes. In the overall cohort, 50 persons (8.2%) were serum CRAG positive when starting ART. Of 295 people with a CD4(+) cell count < or = 100 cells/microL and without prior CM, 26 (8.8%; 95% confidence interval [CI], 5.8%-12.6%) were CRAG positive, of whom 21 were promptly treated with fluconazole (200-400 mg) for 2-4 weeks. Clinical CM developed in 3 fluconazole-treated persons, and 30-month survival was 71% (95% CI, 48%-89%). In the 5 CRAG-positive persons with a CD4(+) cell count < or = 100 cells/microL treated with ART but not fluconazole, all died within 2 months of ART initiation. The number needed to test and treat with CRAG screening and fluconazole to prevent 1 CM case is 11.3 (95% CI, 7.9-17.1) at costs of $190 (95% CI, $132-$287). The number needed to test and treat to save 1 life is 15.9 (95% CI, 11.1-24.0) at costs of $266 (95% CI, $185-$402). The cost per disability-adjusted life year saved is $21 (95% CI, $15-$32). Integrating CRAG screening into HIV care, specifically targeting people with severe immunosuppression (CD4(+) cell count < or = 100 cells/microL) should be implemented in treatment programs in resource-limited settings. ART alone is insufficient treatment for CRAG-positive persons.

IntroductionThere is limited data in children on whether persistence of HIV‐1 infected cells is affected by age at initiating antiretroviral therapy (ART), its duration or any subsequent ART interruption. We therefore investigated the effects of both age of ART initiation and duration of ART interruption on HIV‐1 DNA decay in children.MethodsWe investigated HIV‐1 DNA decay in three groups of children on ART: Group‐1 (n = 7) started uninterrupted ART within eight days of life; Group‐2 (n = 8) started uninterrupted ART at a median of five months of age; and Group‐3 (n = 23) started ART at a median age of 1.8 months for either 40 or 96 weeks, then interrupted ART (median of seven months), and restarted ART based on CD4 count and clinical criteria. Total HIV‐1 DNA was assayed using a sensitive HIV‐1 subtype C‐adapted quantitative PCR for integrase. The duration of ART was square root transformed to fit the observed slowing of HIV‐1 DNA decay rate. For each group, point estimates for decay rates were determined after six months of continuous suppressive ART in groups 1 and 2 or six months after restarting ART in Group‐3. Groups‐2 and 3 were combined using a mixed effect regression model to investigate covariates of HIV‐1 DNA decay rate.Results and DiscussionAt six months of continuous suppressive ART, the HIV‐1 DNA t½ (95% CI) was shorter in Group‐1 (n = 7): 2.7 months (2.1 to 3.8), than 9.2 months (7.4 to 12.1) in Group‐2 (n = 8); and 9.6 months (7.6 to 12.6) in Group‐3 (n = 23) (p < 0.01). In multivariable analyses, HIV‐1 DNA before treatment (p < 0.001) and the change in HIV‐1 DNA during interruption (p < 0.01) were independent predictors of slower HIV‐1 DNA decay.ConclusionsThese data suggest that ART initiation within the first week of life can reduce the persistence of long‐lived infected cells. Delaying ART is associated with slower decay of infected cells.

To examine the interaction between CD4 cell count, viral load suppression and duration of antiretroviral therapy (ART) on mortality. Cohort analysis of HIV-infected patients initiating ART between April 2004 and June 2011 at a large public sector clinic in Johannesburg, South Africa. A log-linear model with Poisson distribution was used to estimate risk of death as a function of the interaction between current CD4 count, current viral load suppression and duration on ART in 12-month intervals. We calculated predicted mortality using estimated coefficients within combinations of predictors. Amongst 14932 ART patients, 1985 (13.3%) died. Current CD4 was the strongest predictor of death (<50 vs. ≥550cells/mm(3) - RR: 46.3; 95% CI: 26.8-80), while unsuppressed current viral load vs. suppressed (RR: 1.8; 95% CI: 1.5-2.1) and short duration of ART (0-11.9 vs. 66-71.9months RR: 1.7; 95% CI: 1.2-2.3) also predicted death. Our interaction model showed that mortality was highest in the first 12months on treatment across all CD4 and viral load strata. As current CD4 and duration on ART increased and viral load suppression occurred, mortality dropped. CD4 count was the strongest predictor of death. The relative effect of current CD4 count varied strongly by viral load and duration of ART (from 1.3 to 55). Lack of suppression increased the risk of mortality upwards of six-fold depending on time on ART and current CD4. Our findings show that while CD4 count is the strongest predictor of death, the effect is modified by viral load and the duration of ART. Assessment of risk should take into account all three factors.

BackgroundReports on the sexual behavior of people on antiretroviral therapy (ART) are inconsistent. We selected 14 articles that compared the sexual behavior of people with and without ART for this analysis.MethodsWe included both cross-sectional studies that compared different ART-naïve and ART-experienced participants and longitudinal studies examining the behavior of the same individuals pre- and post-ART start. Meta-analyses were performed both stratified by type of study and combined. Outcome variables assessed for association with ART experience were any sexual activity, unprotected sex and having multiple sexual partners. Random-effect models were applied to determine the overall odds ratios. Sub-group analyses and meta-regression analyses were performed to examine sources of heterogeneity among the studies. Sensitivity analysis was also conducted to evaluate the stability of the overall odds ratio in the presence of outliers.ResultsThe meta-analysis failed to show a statistically significant association of any sexual activity with ART experience. It did, however, show an overall statistically significant reduction of any unprotected sex, having multiple sexual partners and unprotected sex with HIV negative or unknown HIV status with ART experience. Meta-regression showed no interaction between duration of ART use or recall period of sexual behavior with the sexual activity variables. However, there was an association between the percentage of married or cohabiting participants included in a study and reductions in the practice of unprotected sex with ART.ConclusionIn general, this meta-analysis demonstrated a significant reduction in risky sexual behavior among people on ART in sub-Saharan Africa. Future studies should investigate the reproducibility and continuity of the observed positive behavioural changes as the duration of ART lasts a decade or more.

BackgroundMeningitis remains an important cause of morbi-mortality in adults in sub-Saharan Africa. Data on the etiological investigation of meningitis in adults in Mozambique is limited and most studies were conducted in southern Mozambique. Identification of the etiology of meningitis in adults are crucial to guide prevention and treatments strategies. In this study, we determine the burden of fungal and bacterial meningitis among adults at the three largest hospitals in Mozambique.MethodWe performed analysis of data from the routine sentinel surveillance system for meningitis in Mozambique from January 2016 to December 2017. Cerebrospinal fluid (CSF) samples were collected from eligible adults (≥18 years old) who met World Health Organization (WHO) case definition criteria for Meningitis. All samples were tested by cryptococcal antigen (CrAg) lateral flow assay (LFA), culture and triplex real-time polymerase chain reaction (qPCR) assay and all patients were tested for human immunodeficiency virus (HIV) using the national algorithm for HIV testing.ResultsRetrospective analysis of 1501 CSF samples from adults clinically suspected of meningitis revealed that 10.5% (158/1501) were positive for bacterial and fungal meningitis. Of these 158 confirmed cases, the proportion of Cryptococcal meningitis and pneumococcal meningitis was38.6% (95% CI: 31.0% to 46.7%) and 36.7% (95% CI: 29.2% to 44.7%), respectively. The other bacterial agents of meningitis identified include Neisseria meningitidis (8.9%; 14/158), Escherichia coli (6.3%; 10/158), Haemophilus influenzae (5.1%; 8/158) and S. aureus (4.4%; 7/158), which represent (24.7%; 39/158) of the total confirmed cases.ConclusionAltogether, our findings show a high burden of Cryptococcal meningitis among adults in Mozambique, especially in people living with HIV, followed by pneumococcal meningitis. Our findings suggest that rollout of CrAg Lateral Flow Assay in the health system in Mozambique for early detection of cryptococcus neoformans is necessary to improve overall patient care.

Even in antiretroviral therapy (ART)-suppressed human immunodeficiency virus (HIV)-infected individuals, there are heterogeneous populations of HIV-expressing cells exhibiting variable degrees of progression through blocks to HIV transcriptional initiation, elongation, completion, and splicing. These HIV-transcribing cells likely contribute to HIV-associated immune activation and inflammation as well as the viral rebound that occurs after stopping ART. However, it is unclear whether the blocks to HIV transcription are present before ART and how the timing and duration of ART may affect the clearance of cells expressing HIV transcripts that differ in their processivity and/or presence of mutations. To investigate these questions, we quantified different types of HIV transcripts and the corresponding HIV DNA regions/proviruses in longitudinal blood samples obtained before ART initiation (T1) and after 6 months (T2) and 1 year (T3) of ART in 16 individuals who initiated ART during acute HIV infection. Before ART, the pattern of HIV transcripts suggested blocks to elongation and splicing, and only ~10% of intact proviruses were transcribing intact HIV RNA. During the first 6 months of ART, we detected progressively greater reductions in initiated, 5'-elongated, mid-transcribed, completed, and multiply spliced HIV transcripts. Completed HIV RNA decayed faster than initiated or 5'-elongated HIV RNA, and intact HIV RNA tended to decay faster than defective HIV RNA. HIV DNA and RNA levels at T1-T3 correlated inversely with baseline CD4+ T-cell counts. Our findings suggest the existence of immune responses that act selectively to reduce HIV transcriptional completion and/or preferentially kill cells making completed or intact HIV RNA.IMPORTANCEEven in virologically suppressed HIV-infected individuals, expression of viral products from both intact and defective proviruses may contribute to HIV-associated immune activation and inflammation, which are thought to underlie the organ damage that persists despite suppressive ART. We investigated how the timing of ART initiation and the duration of ART affect the heterogeneous populations of HIV-transcribing cells, including a detailed characterization of the different HIV transcripts produced before ART and the rate at which they decay after ART initiation during acute HIV infection. Even during untreated infection, most cells (~90%) have blocks at some stage of transcription. Furthermore, different HIV transcripts decline at different rates on ART, with the fastest decay of cells making completed and intact HIV RNA. Our results suggest that intrinsic or extrinsic immune responses act selectively to either reduce particular stages of HIV transcription or cause selective killing of cells making particular HIV transcripts.