Achieving UNAIDS Viral Suppression Goal: Experience from a Tertiary Care Public Hospital, Western India.

INTRODUCTION: Targeted HIV 1 viral load testing has been recommended in 2010 only for suspected cases of antiretroviral therapy failure. India is committed to achieve UNAIDS '90-90-90' target by 2020. The third 90 target was to ensure all people receiving antiretroviral therapy (ART) are virologically suppressed. Implementation of routine viral load testing in national programme helps us in assessing early treatment failure and the need to switch to second line therapy; thus eventually reducing drug resistance and improving patient outcomes. AIMS: Study was aimed to determine the proportion of patients responding to antiretroviral therapy, correlates of viral suppression & the discordance between virological and immunological failure. DESIGN: Retrospective analysis. MATERIAL & METHODS: As per the NACO policy, all patients diagnosed as HIV positive are started on antiretroviral therapy and are monitored regularly. The patient's adherence details are noted down during regular follow up visit and patient is referred for routine HIV 1 VL and/or CD4 testing as per National guidelines. Analysis of data was carried out retrospectively for all patients referred for HIV 1 viral load and/or CD4 testing during the study period from July 2019 to June 2020. Confidentiality of the patient was maintained at all times as per routine protocol. RESULTS: A total of 7601 PLHIV on antiretroviral therapy, 3813 samples were tested for both HIV 1 VL and CD4 counts and these results were further analyzed. 3616 (94.8%) showed virological suppression and 197(5.2%) showed virological failure. Among virologically failed group, 46.2% (91/197) underwent retesting after adherence counseling and among these 48.4%(44/91) showed viral suppression. Virological failure was significantly high in younger PLHIV receiving second or third line ART for less than 5 years duration who were non adherent. Immunological discordance was seen in 28.3 % of PLHIV. CONCLUSION: In the present study, 95.99% patients showed virological suppression indicating that the third "90"target is being exceeded.

Seroprevalence of Toxoplasma gondii in newly diagnosed HIV seropositive patients.

BACKGROUND & OBJECTIVES: Immunocompromised individuals mainly HIV infected patients are at a great risk for developing toxoplasmosis. The presence of toxoplasmosis among HIV-infected patients directly correlates with the prevalence of anti- Toxoplasma gondii antibodies and the degree of immunosuppression (measured by CD4 counts). The data regarding the seroprevalence of toxoplasmosis in HIV-infected patients are scarce in India. Therefore, this study was initiated to find out the seroprevalence of toxoplasmosis in treatment-naïve HIV seropositive patients and to determine its association with CD4 counts, if any. METHODS: Four hundred newly diagnosed antiretroviral therapy (ART) naïve adult HIV positive patients coming for CD4 count estimation were tested for the presence of anti- Toxoplasma IgG antibodies. Risk factors for acquisition of toxoplasmosis as well as the age, gender and CD4 counts of the patient were noted down. RESULTS: Toxoplasma IgG was positive in 292 (73%) patients, and the positivity was not related to their CD4 counts. The proportion of anti- Toxoplasma IgG positivity showed no significant association with age, gender and risk factors of the patients. INTERPRETATION & CONCLUSIONS: In the absence of any specific vaccine or prophylaxis for toxoplasmosis, it is pertinent to screen all HIV-positive patients for Toxoplasma IgG at diagnosis, irrespective of their CD4 counts, and sensitize them about the means to prevent either acquisition or activation of infection to avert the development of toxoplasmic encephalitis.

Performance characteristics and operational feasibility assessment of a CRISPR based tata MD CHECK diagnostic test for SARS-CoV-2 (COVID-19).

BACKGROUND: Tata MD CHECK SARS-CoV-2 kit 1.0, a CRISPR based reverse transcription PCR (TMC-CRISPR) test was approved by Indian Council of Medical Research (ICMR) for COVID-19 diagnosis in India. To determine the potential for rapid roll-out of this test, we conducted performance characteristic and an operational feasibility assessment (OFA) at a tertiary care setting. INTERVENTION: The study was conducted at an ICMR approved COVID-19 RT-PCR laboratory of King Edward Memorial (KEM) hospital, Mumbai, India. The TMC-CRISPR test was evaluated against the gold-standard RT-PCR test using the same RNA sample extracted from fresh and frozen clinical specimens collected from COVID-19 suspects for routine diagnosis. TMC-CRISPR results were determined manually and using the Tata MD CHECK application. An independent agency conducted interviews of relevant laboratory staff and supervisors for OFA. RESULTS: Overall, 2,332 (fresh: 2,121, frozen: 211) clinical specimens were analysed of which, 140 (6%) were detected positive for COVID-19 by TMC-CRISPR compared to 261 (11%) by RT-PCR. Overall sensitivity and specificity of CRISPR was 44% (95% CI: 38.1%-50.1%) and 99% (95% CI: 98.2%-99.1%) respectively when compared to RT-PCR. Discordance between TMC-CRISPR and RT-PCR results increased with increasing Ct values and corresponding decreasing viral load (range: <20% to >85%). In the OFA, all participants indicated no additional requirements of training to set up RT PCR. However, extra post-PCR steps such as setting up the CRISPR reaction and handling of detection strips were time consuming and required special training. No significant difference was observed between manual and mobile app-based readings. However, issues such as erroneous results, difficulty in interpretation of faint bands, internet connectivity, data safety and security were highlighted as challenges with the app-based readings. CONCLUSION: The evaluated version-Tata MD CHECK SARS-CoV-2 kit 1.0 of TMC-CRISPR test cannot be considered as an alternative to the RT-PCR. There is a definite scope for improvement in this assay.

First detection of Varicella Zoster Virus clade 9 cases in India during mpox surveillance.

BACKGROUND: The multi-country mpox outbreak across the globe has led to the systematic surveillance of mpox cases in India. During the surveillance of mpox, we encountered cases of Varicella Zoster Virus (VZV) in suspected mpox cases amongst children & adults. This study focused on the genomic characterization of VZV in India. METHODS: A total of 331 mpox suspected cases were tested for VZV through real-time PCR, and the positive samples were subjected to next-generation sequencing to retrieve the whole genome of VZV using CLC genomics software. Phylogenetic analysis has been done in MEGA 11.0 software to identify circulating clades. RESULT: Of the 331 suspected cases, 28 cases with vesicular rashes were found to be positive for VZV. The maximum genome could be retrieved from the clinical specimens of 16 cases with coverage greater than 98% when mapped with reference strain Dumas (NC 001348). The phylogenetic analyses of these sequences determined the circulation of clades 1, 5, and 9 in India. Further, the sequence analysis demonstrated non-synonymous single nucleotide polymorphism (SNPs) among specific ORF of VZV including ORF 14, ORF 22, ORF 36, ORF 37 and ORF 51. Although clade 1 and 5 has been reported earlier, the circulation of clade 9 of VZV has been determined for the first time in India. CONCLUSION: Although the circulation of different clades of VZV was reported from India, the presence of clade 9 was detected for the first time during the mpox surveillance.

Clinical Course and Outcomes of COVID-19 in Kidney Transplant Recipients.

Introduction: Kidney transplant recipients (KTR) are at increased risk of morbidity and mortality due to coronavirus disease 2019 (COVID-19). This study aimed to explore the clinical characteristics and outcomes of COVID-19 in KTR. Methods: We reviewed the clinical profile, outcomes, and immunological responses of recipients admitted with COVID-19. We determined the risk factors for mortality and severe COVID-19. Results: Out of 452 recipients on follow-up, 60 were admitted with COVID-19. Prevalent comorbidities were hypertension (71%), diabetes (40%), lung disease (17%). About 27% had tuberculosis. The median Sequential Organ Failure Assessment score at presentation was 3 (interquartile range [IQR] 1-5). There was a high incidence of diarrhea (52%) and anemia (82%). Treatment strategies included antimetabolite withdrawal (85%), calcineurin inhibitor decrease or withdrawal (64%), increased steroids (53%), hydroxychloroquine (21%), remdesivir (28.3%), and tocilizumab (3.3%). Severe COVID-19 occurred in 34 (56.4%) patients. During a median follow-up of 42.5 days (IQR 21-81 days), 83% developed acute kidney injury (AKI) and eight (13%) died. Mortality was associated with the baseline graft dysfunction, hypoxia at admission, lower hemoglobin and platelets, higher transaminases, higher C reactive protein, diffuse radiological lung involvement, hypotension requiring inotropes, and Kidney Diseases Improving Global Outcomes (KDIGO) stage 3 AKI (univariate analysis). Around 57% of patients remained RT-PCR positive at the time of discharge. By the last follow-up, 66.6% of patients developed IgM (immunoglobulin M) antibodies and 82.3% of patients developed IgG antibodies. Conclusion: COVID-19 in kidney transplant recipients is associated with a high risk of AKI and significant mortality.

Comparison of CBNAAT and conventional real time RT PCR for HIV 1 viral load testing.

PURPOSE: HIV viral load testing is now recommended for monitoring of anti-retroviral treatment failure in PLHIV. Xpert® HIV-1 Viral Load is a fully automated CB-NAAT. A reduced turnaround time leads to prompt clinical management. Hence the current study was undertaken to compare Xpert® HIV-1 Viral Load with the routinely used conventional real time RT PCR. METHODS: The study was conducted in the Department of Microbiology of a tertiary care medical college after ethics committee approval. 100 HIV positive samples were tested by both CB-NAAT and conventional real time RT PCR for HIV 1 viral load. Results were analyzed using Spearman's correlation co-efficient and Bland Altman plot for agreement. The number of samples with inter assay differences in viral loads exceeding 0.5 log copies/ml was also recorded. The sensitivity, specificity, PPV and NPV as well as the possible misclassification were calculated at the clinically significant value of 1000 copies/ml. RESULTS: 25 samples in each of the four groups with log 10 value of <3, 3 to <4, 4 to <5 and ≥5 respectively were included. The log difference between the groups varied from 0 to 1.54. CB-NAAT has shown a statistically significant correlation with conventional real time RT PCR by Spearman's rank correlation (R = 0.972) (P < 0.01) and acceptable level of agreement with Bland Altman plot. The sensitivity, specificity, PPV, NPV and diagnostic accuracy was 80%, 100%, 100%, 93.75% and 95% respectively. The overall concordance was 95% with an upward misclassification of 6.25% and downward misclassification of 0%. CONCLUSIONS: Point of care technology with sample in/answer out approach makes it an excellent choice especially in resource constrained and remote settings.

Routine viral load monitoring and enhanced adherence counselling at a public ART centre in Mumbai, India.

BACKGROUND: Routine viral-load (VL) measurements along with enhanced adherence counselling (EAC) are recommended to achieve virological suppression among people living with HIV/AIDS (PLHA) on anti-retroviral therapy (ART). The Mumbai Districts AIDS Control Society along with Médecins Sans Frontières has provided routine VL measurements and EAC to PLHA on ART at King Edward Memorial (KEM) hospital, Mumbai since October-2016. This study aims to describe the initial VL results and impact of EAC on viral suppression and factors associated with initial viral non-suppression among patients with an initial detectable VL, in a cohort of patients tested between October-2016 and September-2018. METHODS: This is a descriptive study of PLHA on ART who received VL testing and EAC during October-2016 to September-2018. Log-binomial regression was used to identify factors associated with a high VL. RESULTS: Among 3849 PLHA who underwent VL testing, 1603(42%) were female and median age was 42 years (IQR:35-48). Majority were referred for routine testing (3432(89%)) and clinical/immunological failure (233(6%)). Overall, 3402(88%) PLHA had suppressed VL at initial testing. Among 3432 tested for routine monitoring, 3141(92%) had VL suppressed. Of 291 with VL>1000c/ml, 253(87%) received EAC and after repeat VL, 70(28%) had VL<1000c/ml. Among 233 referred for clinical/immunological failure, 122(52%) had VL>1000c/ml and 109 have been switched to second-line ART. CD4 count<500 (aOR-5.0[95%CI 3.8-6.5]), on ART for<5 years (aOR-1.5[1.1-2.0]) and age<15 years (aOR-5.2[3.0-8.9]) were associated with an initial VL>1000c/ml. Factors associated with follow-up VL suppression included EAC (p<0.05) and being on second-line ART (p<0.05). CONCLUSION: Results from a routine VL program in public sector in India were encouraging and in line with UNAIDS 90-90-90 targets. Routine VL monitoring along with EAC resulted in early switch to alternative optimised regimens while also preventing unnecessary switches. Along with the vital scale up of routine VL monitoring, implementation of enhanced adherence strategies for patients with detectable viral load should be ensured.

Profiles of HIV-infected anti-retroviral therapy naïve children from Mumbai, India.

BACKGROUND: This study aimed to investigate the demographic profiles of human immunodifficiency virus (HIV) infected anti-retroviral therapy (ART) naïve children in our hospital and their relations to the clinical, immunological and nutritional status. METHODS: A cross-sectional study was conducted in an Integrated Counselling and Testing Center (ICTC) at a tertiary care hospital in Mumbai. ART naïve HIV positive children were enrolled in the study. The demographic profiles, clinical features, immunological (CD4%/CD4 count) and nutritional status of these children were recorded. The agreement between clinical, immunological and nutritional staging was determined using Cohen's kappa test. RESULTS: In 192 HIV-infected ART naive children enrolled with a median age of 9 years (range 3 months-14 years), 97.4% acquired infection through vertical transmission. The most common clinical presentation was fever (39.6 %), followed by generalized lymphadenopathy (32.3%), cough (22.4%) and diarrhoea (9.9%). Tuberculosis was seen in 22.9% of the children. The agreement was fair between clinical and immunological staging, and slight between nutritional, immunological and clinical staging. CONCLUSION: Perinatal transmission is the most common mode of acquiring HIV infection in children. The Prevention of Parent to Child Transmission (PPTCT) program should be strengthened for lowering the transmission rate by providing extended ART to mothers during pregnancy and breast-feeding. Tuberculosis remains a major concern in HIV-infected children. The poor correlation between WHO clinical and immunological staging emphasizes the importance of making CD4 facilities available in HIV prevalent areas. Malnutrition cannot be used as a surrogate marker for predicting stage or severity as it is common at all stages of HIV disease.

Monitoring HIV Epidemic in Pregnant Women: Are the Current Measures Enough?

Introduction. Burden of HIV in pregnant women follows overall epidemic in India. Hence, it is imperative that prevalence calculations in this group be accurate. The present study was carried out to determine prevalence of HIV in pregnant women attending our hospital, to determine trend of HIV infection and to compare our results with reported prevalence. Methods. All pregnant women are routinely counselled for HIV testing using opt-out strategy. Year-wise positivity and trend were determined in these patients over a period of five years. The positivity in different age groups was determined. Results. 31,609 women were tested of which 279 (0.88%) were positive. Positivity showed a declining trend over study period and significant quadratic trend (biphasic, P < 0.05) was observed. The positivity in older age group ≥35 years (1.64%) was significantly more than younger age groups (0.76% in 15-24-year and 0.94% in 25-34-year age group) (P = 0.0052). Conclusion. A significant decline in HIV positivity was seen over the study period. Taking into account heterogeneous nature of HIV epidemic even within the same district, analysis at local levels especially using the prevention of parent to child transmission of HIV program data is critical for HIV programming and resource allocation.

CD4 counts in laboratory monitoring of HIV disease--experience from western India.

BACKGROUND: CD4 counts vary in different populations. The present study was conducted to determine CD4 counts in different World Health Organization (WHO) clinical stages in antiretroviral therapy naive individuals and to find out optimum CD4 cutoffs. METHOD: Data of adult HIV seropositive patients who underwent CD4 count and total lymphocyte count (TLC) testing were included for analysis. The severity of immunosuppression was graded based on WHO criteria. To establish optimum CD4 cutoff values, receiver-operator characteristics (ROC) curves were generated. RESULTS: Of 754 patients, 52.2% had CD4 counts <200 cells/mm3, but only 2.3% belonged to WHO stage IV. Newer CD4 cutoffs generated were 280, 120-280, <120 cells/mm3. Spearman rank correlation between CD4 counts and TLC was found to be weak (r = .32). CONCLUSION: The cutoff values of CD4 counts for HIV disease may need to be revised for India. Regular CD4 count estimation is a must for monitoring disease progression in people living with HIV/AIDS.

CD4 counts in laboratory monitoring of HIV disease--experience from western India.

BACKGROUND: CD4 counts vary in different populations. The present study was conducted to determine CD4 counts in different World Health Organization (WHO) clinical stages in antiretroviral therapy naive individuals and to find out optimum CD4 cutoffs. METHOD: Data of adult HIV seropositive patients who underwent CD4 count and total lymphocyte count (TLC) testing were included for analysis. The severity of immunosuppression was graded based on WHO criteria. To establish optimum CD4 cutoff values, receiver-operator characteristics (ROC) curves were generated. RESULTS: Of 754 patients, 52.2% had CD4 counts <200 cells/mm3, but only 2.3% belonged to WHO stage IV. Newer CD4 cutoffs generated were 280, 120-280, <120 cells/mm3. Spearman rank correlation between CD4 counts and TLC was found to be weak (r = .32). CONCLUSION: The cutoff values of CD4 counts for HIV disease may need to be revised for India. Regular CD4 count estimation is a must for monitoring disease progression in people living with HIV/AIDS.

Hepatitis B Virus (HBV) Infection in Liver Disease Patients in Mumbai, India with Special Reference to Hepatitis B Surface Antigen (HBsAg) Mutant Detection.

OBJECTIVES: To determine the prevalence of Hepatitis B Surface antigen (HBsAg) in patients attending the Hepatology Out Patient Department (OPD) of a tertiary care hospital and to compare the routinely used HBsAg detection kit with the mutant detection kit to find out the presence of mutants in a given setting. MATERIALS AND METHODS: A cross-sectional study was carried out in adult patients with liver disease attending the Hepatology OPD, of a tertiary care hospital in Mumbai, India. Age, gender and clinical history of the patient were recorded. Blood specimen was tested for HBsAg (Microscreen(TM) ELISA, Span diagnostics, India) and HBsAg mutants (Hepanostika HBsAg Ultra(TM) ELISA, Biomerieux, France). The samples with discordant results between these two ELISAs were confirmed by Hepatitis B Virus (HBV) Deoxyribonucleic Acid (DNA) Polymerase Chain Reaction (PCR) (Cobas Taqman(TM), Roche Molecular Systems, USA). RESULTS: Seven hundred and eighteen patients were enrolled in the study. The mean age of patients in the study group was 41 years (range 17 to 69 years). Four hundred and ninety seven (69.22%) were males and remaining were females. The prevalence of HBsAg was found to be 17.4%. The positivity amongst the male population was 18.1% which was higher than the female population (15.8%). Of the 718 samples tested, 120 were positive for HBsAg by Microscreen(TM) ELISA and 132 were positive by Hepanostika HBsAg ultra(TM). Of the 12 discordant samples, HBV DNA was detected in five samples indicating 0.7% prevalence of mutants. CONCLUSION: Hepatitis B is prevalent in liver disease patients. The mutant detecting assay is recommended in set-ups where missing HBsAg in patients would have tremendous impact on the outcome such as in blood donors, organ or tissue donors and antenatal screening of mothers. It is also helpful in chronic liver disease patients where the routine HBsAg detection test is negative and the other causes of chronic liver disease have been ruled out. However, it is not recommended for use in routine diagnostic set-ups where high false positivity would lead to over-diagnosis of the condition.

Performance of immunological response in predicting virological failure.

In HIV-infected individuals on antiretroviral therapy (ART), the decision on when to switch from first-line to second-line therapy is dictated by treatment failure, and this can be measured in three ways: clinically, immunologically, and virologically. While viral load (VL) decreases and CD4 cell increases typically occur together after starting ART, discordant responses may be seen. Hence the current study was designed to determine the immunological and virological response to ART and to evaluate the utility of immunological response to predict virological failure. All treatment-naive HIV-positive individuals aged >18 years who were eligible for ART were enrolled and assessed at baseline, 6 months, and 12 months clinically and by CD4 cell count and viral load estimations. The patients were categorized as showing concordant favorable (CF), immunological only (IO), virological only (VO), and concordant unfavorable responses (CU). The efficiency of immunological failure to predict virological failure was analyzed across various levels of virological failure (VL>50, >500, and >5,000 copies/ml). At 6 months, 87(79.81%), 7(5.5%), 13 (11.92%), and 2 (1.83%) patients and at 12 months 61(69.3%), 9(10.2%), 16 (18.2%), and 2 (2.3%) patients had CF, IO, VO, and CU responses, respectively. Immunological failure criteria had a very low sensitivity (11.1-40%) and positive predictive value (8.3-25%) to predict virological failure. Immunological criteria do not accurately predict virological failure resulting in significant misclassification of therapeutic responses. There is an urgent need for inclusion of viral load testing in the initiation and monitoring of ART.

Laboratory tests for malaria: a diagnostic conundrum?

OBJECTIVES: To detect malarial parasites using the peripheral blood smear (PBS) and to compare the PBS with the immunochromatographic antigen test (i.e. OptiMAL and polymerase chain reaction (PCR)). METHODS: Six ml of blood was collected from each of 170 patients clinically suspected of having malaria. These samples were used to perform PBS examination, the OptiMAL test and PCR by standard protocol. RESULTS: PBS examination found malarial parasites in 86 (50.6%) samples. In comparison, 71 (41.8%) samples were positive by OptiMAL test whereas PCR could detect malarial parasites in only 44 (25.9%) samples. All 84 (49.4%) samples which were negative by PBS were negative by both OptiMAL and PCR. The sensitivity and specificity were respectively 85.54% and 100% for OptiMAL and 51.12% and 100% for PCR. CONCLUSION; Depending on the tests' operational feasibility, and the availability of adequate trained personnel, equipment and laboratory management systems, and considering its sensitivity and cost-effectiveness, peripheral blood smear remains the test of choice for malaria, especially in endemic areas.

HIV-2 Infection: Where Are We Today?

CONTEXT: The choice of antiretroviral therapy for HIV-2 differs from that for HIV-1, underscoring the importance of differentiating between the two. AIMS: The current study was planned to find out the prevalence of HIV-2 infection at our center and to find out the utility of the current diagnostic algorithm in identifying the type of HIV infection. SETTING AND DESIGN: Retrospective analysis in a tertiary care teaching institute over a period of three years. MATERIALS AND METHODS: All patients diagnosed as HIV infected using NACO/WHO HIV testing strategy III were included in the study. They were classified as HIV-1 infected, HIV-2 infected and HIV-1 and HIV-2 co-infected based on their test results. For discordant samples, immunoblotting result from National Reference Laboratory was considered as final. STATISTICAL ANALYSIS USED: Comparison between HIV-1, HIV-2 and HIV-1+2 positive groups for age, gender, route of transmission was made using chi squared test. P value < 0.05 was considered as significant. RESULTS: Of the total of 66,708 patients tested, 5,238 (7.9%) were positive for HIV antibodies. 7.62%, 0.14%, 0.08% and 0.004% were HIV-1, HIV-2, HIV-1 and HIV-2 co-infected and HIV type indeterminate (HIV-1 Indeterminate, 2+) respectively. The current algorithm could not differentiate between the types of HIV infection (as HIV-1 or HIV-2) in 63 (1.2%) cases. CONCLUSION: In areas like the Indian subcontinent, where epidemic of both HIV-1 and HIV-2 infections are ongoing, it is important to modify the current diagnostic algorithms to diagnose and confirm HIV-2 infections.

Transmitted HIV drug resistance among HIV-infected voluntary counseling and testing centers (VCTC) clients in Mumbai, India.

A survey for transmitted HIV drug resistance (HIVDR) was conducted according to WHO guidelines among clients newly diagnosed with HIV-1 infection at two voluntary counseling and testing centers (VCTC) in Mumbai. HIVDR testing was performed using the ViroSeq RT-PCR method (Abbott). Out of 50 successfully amplified and sequenced specimens, analysis of the first 34 consecutively collected specimens revealed no nucleoside reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor, or protease inhibitor mutations from the 2007 WHO list of mutations for surveillance of transmitted HIVDR, indicating that the prevalence of transmitted HIVDR to all three drug classes was <5% among recently infected VCTC clients in Mumbai. The phylogenetic analysis revealed that all samples belonged to HIV-1 subtype C. Continued ART program monitoring and further evaluation of transmitted HIV drug resistance in coming years are essential in Mumbai as well as in other regions of the country in which ART is being scaled up rapidly.