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.

A single-center observational study on clinical features and outcomes of 21 SARS-CoV-2-infected neonates from India.

Coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing pandemic with significant morbidity and mortality. Neonates represent a vulnerable population, in which we have limited knowledge of its natural history, optimal management, and outcomes. In this retrospective observational study from a low-middle-income setting, clinical characteristics and outcomes of neonatal SARS-CoV-2 infection were evaluated. We report an incidence of 10.6% of SARS-CoV-2 infection (21 neonates), among a group of 198 neonates with suspected infection. Most of the SARS-CoV-2-infected neonates were term (80.9%) and none required any resuscitation. The infection was detected by a positive nasopharyngeal swab reverse transcriptase-polymerase chain reaction (RT-PCR) for SARS-CoV-2. Neonatal COVID-19 manifestations developed in one-third (33.3%) of the infected neonates. Most of them demonstrated the involvement of respiratory (33.3%) and gastrointestinal systems (4.8%). Laboratory parameters suggested multi-systemic involvement, with elevated creatine kinase (CK) (76.2%), creatine kinase-myocardial band (CK-MB) (76.2%), and lactate dehydrogenase (LDH) (71.4%) levels. Supportive treatment was given to infected neonates with intensive care required in six neonates (28.6%). This included four preterm and two term neonates, of which two received non-invasive and one received invasive ventilation with intra-tracheal surfactant instillation. IgM antibodies against COVID-19 were detected in one neonate. All neonates with COVID-19 improved and were successfully discharged.Conclusion: SARS-CoV-2 in neonates has a wide clinical spectrum. Further studies are needed which are adequately powered to completely understand the course of this infection in neonates, its implications not only in the neonatal period but also on long-term follow-up. What is Known: • SARS-CoV-2 infection has a predilection for all age groups but with limited literature on clinical profile, outcomes, and long-term follow-up in neonates. What is New: • SARS-CoV-2 infection in neonates has a wide clinical spectrum and displays a significant overlap with common neonatal conditions. • Most neonates with COVID-19 improved with supportive care, though a subset required intensive care, emphasizing the need for cautious monitoring and management.

Effect of different methods of pasteurization on bactericidal action of human milk: A prospective observational study.

Background & objectives: Pasteurization involves not only inactivation of pathogens, but also loss of immunological functions and bactericidal action of human milk. Hence, this study was aimed to explore the stability of such bactericidal action after subjecting human milk samples to thermal pasteurization under different condition of time and temperature. Methods: In this observational study 48 human milk samples were analyzed over a period of three months. The effect of holder and flash methods of pasteurization on bactericidal action against Escherichia coli was evaluated compared to the control sample before and after 72 h of storage at -18°C. Results: Both holder and flash methods of pasteurization showed significant reduction in the E. coli growth to 46.4 and 25.5 per cent, respectively, after 24 h of incubation (P <0.001). The bactericidal activity was significantly more in samples subjected to holder method compared to flash method before and after 72 h of storage (46.41±15.38 vs. 25.50±30.74, P <0.001 and 42.27±20.38 vs. 18.33±28.55, P <0.001). Interpretation & conclusions: Our results showed that the bactericidal activity of human milk was better preserved by the holder method of pasteurization. Further well-powered and well-designed randomized trials are needed to confirm the findings.

Single-dose and Steady-state Pharmacokinetics of Vancomycin in Critically Ill Patients Admitted to Medical Intensive Care Unit of India.

RATIONALE: Vancomycin remains the standard of care for gram-positive bacterial infections, though there are significant developments in newer antibacterial agents. Efficacy can be improved by linking pharmacokinetic with pharmacodynamic principles, thus leading to optimum antibiotic exposure. There is scarcity of pharmacokinetic data in Indian intensive care unit (ICU) population. MATERIALS AND METHODS: Fifteen subjects with suspected or proven gram-positive bacterial infection of either gender between 18 years and 65 years of age were enrolled. Vancomycin at the dose of 1 g every 12 hours was administered over 1-hour period and pharmacokinetic assessments performed on blood samples collected on days 1 and 3. Vancomycin concentrations were measured on validated liquid chromatography mass spectrometry method. Pharmacokinetic parameters were calculated using Winnonlin (Version 6.3; Pharsight, St. Louis, MO). RESULTS: The mean C max, elimination half-life, AUC0-12hours, volume of distribution, and clearance of single dose were 36.46 µg/mL (±14.87), 3.98 hours (±1.31), 113.51 µg/mL (±49.51), 52.01 L (±31.31), and 8.90 mL/minute (±3.29), respectively, and at steady state were 40.87 µg/mL (±19.29), 6.27 hours (±3.39), 147.94 µg/mL (±72.89), 56.39 L (±42.13), and 6.98 mL/minute (±4.48), respectively. The elimination half-life increased almost two-fold at steady state. The steady state mean AUC0-24 was 295.89 µg/mL (±153.82). Out of 45 trough levels, 32 (71.11%) concentrations were below recommended range. CONCLUSION: Recommended AUC0-24hours and trough concentrations were not achieved in majority of patients with current dosing, suggesting reevaluation of current vancomycin dosing. Individualized treatment based on close monitoring of vancomycin serum concentrations in critically ill patients is imperative. HOW TO CITE THIS ARTICLE: Mali NB, Deshpande SP, Wandalkar PP, Gupta VA, Karnik ND, Gogtay NJ, et al. Single-dose and Steady-state Pharmacokinetics of Vancomycin in Critically Ill Patients Admitted to Medical Intensive Care Unit of India. IJCCM 2019;23(11):513-517.

Acetic acid dressings: Finding the Holy Grail for infected wound management.

BACKGROUND: Wounds have since long, contributed majorly to the health-care burden. Infected long-standing non-healing wounds place many demands on the treating surgeon and are devastating for the patients physically, nutritionally, vocationally, financially, psychologically and socially. Acetic acid has long been included among agents used in the treatment of infected wounds. In this study, we have evaluated the use of acetic acid for topical application in the treatment of infected wounds. MATERIALS AND METHODS: A total of 100 patients with infected wounds were treated with topical application of 1% acetic acid as dressing material after appropriate cleaning. A specimen of wound swab was collected before first application and further on days 3, 7, 10 and 14. Daily dressings of wounds were done similarly. Minimum inhibitory concentration (MIC) of acetic acid against various organisms isolated was determined. RESULTS: The patients treated ranged between 9 and 60 years, with the mean age 33 years. Nearly 70% of patients were male. Aetiologies of wounds: infective 35, diabetic 25, trauma 20, burns 10, venous ulcers 5 and infected graft donor site 5. Various microorganisms isolated include Pseudomonas aeruginosa (40%), Staphylococcus aureus (2%), Acinetobacter (12%), Escherichia Coli (5%), Proteus mirabilis (3%), Klebsiella (18%), methicillin-resistant S. aureus (10%), Streptococcus (2%) and Enterococcus (1%), Citrobacter (1%). Few wounds (6%) also isolated fungi. About 28%, 64% and 8% of patients isolated no growth on culture after 7, 14 and 21 days, respectively. MIC of all isolated organisms was ≤0.5%. CONCLUSION: pH of the wound environment plays a pivotal role in wound healing. Acetic acid with concentration of 1% has shown to be efficacious against wide range of bacteria as well as fungi, simultaneously accelerating wound healing. Acetic acid is non-toxic, inexpensive, easily available and efficient topical agent for effective elimination of wound infections caused due to multi-drug resistant, large variety of bacteria and fungus.

The Ever Increasing Reservoirs of Infection in the Health Care Environment - Time for a Sixth Moment of Hygiene.

OBJECTIVES: The study was carried out to determine the extent and type of contamination of the hands and accessories of staff from different settings and also to determine the phenotypic similarity between the isolates recovered from the same staff. DESIGN: Prospective cross-sectional study. SETTING: Tertiary care center. PARTICIPANTS: Health care workers (HCWs') and administrative staff. METHODS: Samples were collected and processed for bacteriology from the dominant hand, mobiles, aprons, stethoscopes and computer keyboards of 280 staff working in different settings after consent. Isolates were identified and antimicrobial susceptibility testing was carried out. A comparison of data sets was performed using chi square test. p value < 0.05 was considered significant. RESULTS: 817 samples were screened, 616 yielded growth. Contamination rate was found to be 75%. Of the 1254 isolates recovered, Gram positive and gram negative accounted for 80% and 18% respectively. Hands and accessories of HCWs from clinical settings showed significant contamination with potential pathogens as compared to the non-clinical settings. (p<0.0001) All computer keyboards and stethoscopes used by ICU doctors were contaminated. MRSA was recovered only from clinical settings. Two salmonella isolates were isolated from apron of laboratory health care workers. In 102 staff, similar isolates were recovered from hand and any accessory. CONCLUSIONS: The risk of contamination of any accessory with potential pathogens is high in the clinical setting. The five moments of hand hygiene should be strengthened. A policy for decontamination of all accessories should be formulated.

Prevalence of HIV, syphilis, hepatitis B, and hepatitis C in patients attending STI/RTI clinic of a tertiary care teaching hospital.

BACKGROUND: Patients attending Sexually transmitted infection/ Reproductive tract infection (STI/RTI) clinics are investigated for HIV and syphilis under the National AIDS Control Program (NACP). Although sexual contact is one of the modes of transmission of hepatitis B and C, they are not investigated under NACP. This study was planned to find the prevalence of HIV, syphilis, hepatitis B, and C in patients attending STI/RTI clinics and to identify the predictive risk factors. METHODS: A prospective cross-sectional study was carried out over 5 years on 500 consenting adults. 10 ml blood was collected and tests were performed as per standard protocol for HIV, syphilis, hepatitis B, and C. Risk factors for the sexually transmitted diseases were queried. RESULTS: 500 samples were tested, 117(23.4%) men and 383 (76.6%) women. 26(22.2%), 20(17.1%), 11(9.4%) and 01(0.9%) men and 8(2.1%), 36(9.4%), 01(0.3%) and 0(0%) women were positive for HIV, RPR, hepatitis B and C respectively. Dual infection for HIV and syphilis was detected in four (0.8%) men and HIV and hepatitis B in three (0.6%) men. CONCLUSION: To investigate all patients attending STI/RTI clinics for Hepatitis B and to integrate Hepatitis B testing into the National AIDS Control Program.

Antibody response to non-mRNA SARS-CoV-2 vaccine in kidney transplant recipients.

BACKGROUND: Kidney transplant recipients (KTRs) show poor antibody response to the SARS-CoV-2 vaccine. There is limited data on immune response to non-mRNA vaccines in KTRs. We studied the antibody response to the SARS-CoV-2 non-mRNA vaccine in a cohort of kidney transplant recipients. METHODS: We included KTRs following up in the tertiary care transplant outpatient clinic from February to April 2022. SARS-CoV-2 spike protein IgG antibody titers were measured using chemiluminescence immunoassay. Data on demographic, clinical, and laboratory characteristics were collected, and patients were characterized by the history of Coronavirus disease 2019 (COVID-19) infection in the past and the number of vaccine doses received. Predictors of antibody response were obtained using multivariate regression analysis. RESULTS: S1/S2 IgG anti-SARS-CoV-2 antibodies were detected in 197 (87.94%) of 224 KTRs with a median [IQR] titers of 307.5 AU/ml [91 AU/ml - 400 AU/ml]. Neutralizing range antibody titers were found in 170/224 (75.9%) KTRs. Diabetes at the time of vaccination was associated with poorer antibody response (aOR 0.31, 95% confidence interval [CI] - 0.10, 0.90; p = 0.032) and vaccination with Covishield™ (ChAdOx1 nCoV- 19 Recombinant CoronaVirus Vaccine) showed higher antibody response as compared to Covaxin™ (BBV152) (aOR 5.04, 95% CI - 1.56, 16.22; p = 0.007). Graft dysfunction at baseline was associated with poorer antibody response. CONCLUSIONS: KTRs showed good antibody response after SARS-CoV-2 vaccination with non-mRNA vaccines. Diabetes and graft dysfunction were associated with poor seroconversion rates.

Development and validation of a pentaplex PCR assay for rapid detection of blaCTX-M, blaOXA-1, blaCMY, blaNDM and the PBP3 insert in Enterobacterales.

BACKGROUND: There is a high diversity of beta-lactamases in gram negative pathogens, making them difficult to treat. In the presence of OXA-1 and ampC, PTZ is no longer clinically relevant when treating Enterobacterales expressing ESBLs. Further, MBL infections are often treated with the combination of ceftazidime/avibactam with aztreonam. . It has recently been reported that NDM-expressing E. coli isolates co-harboring PBP3 insert develops resistance to this triple combination. METHODS: A pentaplex PCR is developed and validated to simultaneously detect blaCTX-M, blaOXA-1, blaCMY, blaNDM, and the PBP3 insert in whole genome sequenced E. coli and K. pneumoniae isolates. In addition, the isolates chosen for pentaplex PCR evaluation were tested for their minimum inhibitory concentrations (MICs) against piperacillin/tazobactam, cefoperazone/sulbactam (C/S), ertapenem, imipenem, meropenem, ceftazidime/avibactam, aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. RESULTS: The developed pentaplex PCR showed 100 % reproducibility with the antimicrobial resistance profile generated from whole genome sequenced data. PTZ and C/S are not effective against ESBL and/or OXA-1 expressing E. coli and K. pneumoniae isolates and do not offer any activity against CMY co-producers. Further, the combined effect of CMY, NDM and PBP3 inserts impacts aztreonam/avibactam activity and reduces the susceptibility to 40 % in E. coli isolates. While, aztreonam/avibactam showed potent activity against NDM-expressing K. pneumoniae isolates. Importantly, cefepime/taniborbactam and cefiderocol showed limited activity against NDM-expressing E. coli and K. pneumoniae isolates. CONCLUSION: The pentaplex PCR was effective in detecting four beta-lactamases (blaCTX-M, blaOXA-1, blaCMY, blaNDM) as well as PBP3 inserts. It is expected that using pentaplex PCR as a diagnostic test for resistance detection in clinical practice will improve patient outcomes by providing prompt and targeted treatment.

Utility of laboratory diagnostic tests in women suspected of genital tuberculosis attending a tertiary care teaching hospital.

Background: Genital Tuberculosis is a form of extrapulmonary tuberculosis, which if not diagnosed early can lead to complications. The objective of this study was to determine the sensitivity and specificity of Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay in genital tuberculosis (TB) in comparison with culture as a gold standard. Methods: The results of the Xpert MTB/RIF assay performed from January 2020 to August 2021 were compared with the results of culture by Mycobacterium Growth Indicator Tube (MGIT) 960. Results: Out of 75 specimens, fluorescent microscopy and liquid culture using MGIT and Xpert assay were positive in 3 (4%), 21 (28%), and 14 (18%), respectively. The sensitivity and specificity of the Xpert MTB/RIF assay were 66.67% and 100%. All smear-positive specimens were positive by culture and Xpert assay. Three specimens were positive by all the tests, i.e., microscopy, culture, and Xpert assay. Fifty-four specimens were negative by microscopy, culture, and Xpert assay. Discordance between the results of culture and Xpert assay was observed in seven specimens which were culture positive and Xpert assay negative. Three (21.42%) out of 21 culture-positive specimens showed monoresistance to rifampicin by Xpert MTB/RIF assay and culture drug susceptibility testing. Conclusion: Xpert MTB/RIF assay showed good sensitivity and specificity compared to liquid culture in genital TB. This test is easy to perform, provides results in 2 h, and can also detect rifampicin resistance, which is a surrogate marker for multidrug-resistant TB. Hence, the Xpert assay can be used under the National TB Elimination Program for early and rapid diagnosis of TB in endometrial specimens to prevent complications like infertility.

Comparison of xpert MTB/RIF assay, line probe assay, and culture in diagnosis of pulmonary tuberculosis on bronchoscopic specimen.

Background: In patients unable to expectorate good quality sputum or with minimal to none sputum production, bronchoscopic specimens may be collected. The objective of the study is to determine the use of Xpert MTB/RIF assay and line probe assay (LPA) in the diagnosis of pulmonary TB (PTB) using specimens collected by bronchoscopy in a tertiary care center. Methods: Bronchoscopy specimens received in the TB laboratory were processed by microscopy, Xpert MTB/RIF assay, LPA, and mycobacteria growth indicator tube (MGIT) culture. Results of MGIT culture are considered gold standard. Results: Of the 173 specimens tested, MTB was detected in 48 (27.74%) samples by any of the above methods. Positivity in bronchoalveolar lavage was 31.4% (44/140) and in bronchial wash was 12.1% (4/33). Detection by microscopy, Xpert assay, and culture was 20 (11.56%), 45 (26.01%), and 38 (21.96%), respectively. Culture detected MTB in three additional specimens compared to Xpert assay. Xpert assay detected MTB in 45 (26%) specimens which include 10 specimens which were negative by culture. LPA detected MTB in 18 (90%) out of 20 smear-positive specimens. RIF resistance was detected in 20 (41.7%) specimens by Xpert and/or MGIT culture drug susceptibility testing (DST). Isoniazid (INH) resistance was detected in 19 specimens by LPA and MGIT culture DST. Conclusion: Bronchoscopy can provide alternative respiratory specimens for diagnosing PTB in patients with difficulty to expectorate sputum. The utility of Xpert MTB/RIF as a rapid, sensitive, and specific test should always be supplemented with culture in difficult-to-obtain and precious respiratory specimens. LPA plays an important role in rapid detection of INH monoresistance.

Comparison of gastric lavage/sputum and stool specimens in the diagnosis of pediatric pulmonary tuberculosis- A pilot study.

BACKGROUND AND OBJECTIVE: Global TB report 2021 mentions 11 % prevalence of pediatric TB, whereas 5.65% of the cases were reported from India in 2020. India features in the list of TB high burden countries, HIV-TB high burden and MDR-TB high burden countries. The diagnosis of pulmonary tuberculosis in children is difficult as they tend to swallow the sputum, invasive techniques of gastric aspirates needs to be followed and the disease itself is paucibacillary. The disease progresses rapidly in young children and hence rapid diagnosis is needed. Obtaining appropriate respiratory samples for diagnosis is difficult especially in primary care settings. Stool sample is easy to obtain and since children swallow sputum, it can be used to diagnose pulmonary tuberculosis. With this background, a pilot study was planned to evaluate the accuracy of the Xpert MTB/RIF assay for the detection of MTB in stool specimens obtained from pediatric pulmonary TB patients confirmed either by gastric lavage(GL) or sputum(SP) Xpert MTB/RIF assay. In addition, the results of microscopy of stool specimen were compared with that of gastric lavage/ sputum (GL/SP) specimen by Ziehl-Neelsen (ZN) and fluorescent light-emitting diode (LED) staining. MATERIAL AND METHODS: A prospective study was carried out on 50 GL/SP Xpert MTB/RIF assay positive children (0-14 years). Stool specimens from these children were processed for Xpert MTB/RIF assay. The GL/SP and stool specimens were processed for ZN and Auramine O fluorescent microscopy as well. RESULTS: Fluorescent staining detected acid fast bacilli (AFB) in 24 GL/SP and 16 stool specimens as compared to 20 GL/SP and 10 stool specimens by ZN staining. Stool Xpert MTB/ RIF assay was positive in 29 out of 50 children. Rifampicin resistance was detected in 13 of the 50 (26%) GL/SP specimens. Of these 13 children, rifampicin resistance was detected in 7 stool specimens, rifampicin indeterminate resistance was detected in one specimen and in the remaining 5 children, M.tuberculosis was not detected in stool. CONCLUSION: Stool is a good non-invasive specimen for the detection of pulmonary TB in children, especially in remote areas, where invasive techniques cannot be performed for sample collection.

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.

Role of CD4 count estimation in the era of HIV-1 viral load among PLHIV.

BACKGROUND: ART has improved the life expectancy of people living with HIV (PLHIV) by suppressing the virus and increasing CD4 count. Some PLHIV shows immune-virological discordant responses i.e. suppressed viral load to the undetectable level but still with immunological failure or good immunological response with virological failure. Immuno-virological response plays a key role to address treatment outcome, regimen change and management for people living with HIV. It is reported that PLHIV with discordant responses were found to be at an increased risk to develop AIDS and non-AIDS events related death. AIMS & OBJECTIVE: To determine immuno-virological discordance amongst PLHIV on ART and its effect on mortality. MATERIAL & METHOD: After getting institutional Ethics committee approval, total 1921 patients were included in the study who were on ART for at least 6 month or more and have at least two CD4 count tests results and were tested from July 2019 to June 2020. Retrospective analysis was done. RESULTS: Total 1921 patients were included in study of which 1383 (72%) showed immuno-virological concordance & 538 (28%) showed immuno-virological discordance. Overall mortality rate among PLHIV was 3.6%. Mortality rate in immuno-virological concordant group was 2.8%. Of immuno-virological discordant population, 505 (26.3%) were virological only responders (VO) with 5.35% mortality rate & 33 (1.7%) were immunological only responders (IO) with 9.09% mortality rate. High number of immunological discordant patients in the present study warrants the further evaluation of these patients with change in appropriate treatment strategy to decrease the mortality among this group. CONCLUSION: This study emphasizes the role of immunological monitoring as well as virological monitoring to improve the life expectancy of PLHIV.

An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit.

BACKGROUND & OBJECTIVES: The conventional techniques used in TB diagnosis like AFB (acid fast bacilli) smear microscopy lack sensitivity and the gold standard, culture test takes time. A test based on multiplex polymerase chain reaction (PCR) targeting the 38 kDa gene and IS6110 insertion sequence, specific to Mycobacterium tuberculosis was developed to further increase the sensitivity of a TB-PCR kit targeting only 38 kDa gene developed earlier in the same laboratory. The multiplex test was validated using sputum samples from pulmonary TB (PTB) cases. The sensitivity and specificity were compared with AFB smear examination and Lowenstein-Jensen (LJ) culture test. METHODS: Multiplex PCR amplifying 340 and 245 bp sequence of 38 kDa gene and IS6110, respectively was standardized and analytical sensitivity was verified. Sputum samples (n=120) obtained from PTB cases were subjected to AFB smear examination, LJ culture and a multiplex as well as single target PCR test. Additionally, 72 non-TB respiratory samples were included in the study as negative controls. RESULTS: Analytical sensitivity of multiplex PCR was found to be 100 fg for 38 kDa gene and 1 fg for IS6110. Multiplex PCR, using both the targets, showed highest sensitivity of 81.7 per cent, followed by 69.2 per cent for L-J culture test and 53.3 per cent for AFB smear when clinical diagnosis was considered as a gold standard. The sensitivity of detection of M. tuberculosis in AFB smear positive and negative samples by multiplex PCR was 93.7 and 67.9 per cent, respectively. Sensitivity of 77.1 per cent observed for the detection of M. tuberculosis with single target PCR increased to 89.2 per cent with multiplex PCR in culture positive samples. Four samples showed positive PCR results only with primers for 38 kDa gene. INTERPRETATION & CONCLUSIONS: Multiplex PCR increased the sensitivity of single target PCR and will be useful in diagnosing paucibacillary smear negative samples. Further, it can also be used to detect samples with M. tuberculosis strains lacking IS6110.

Time to sputum conversion in smear positive pulmonary TB patients on category I DOTS and factors delaying it.

BACKGROUND: Sputum smear positive pulmonary tuberculosis patients expel infectious viable bacilli for a period following commencement of treatment. Patients on Directly Observed Treatment Shortcourse (DOTS) receive intermittent therapy with multidrug regimen. AIMS: To determine the time to sputum smear and culture conversion following initiation of DOTS treatment and study the factors that influence it. METHODS: A prospective study was undertaken at a tertiary care teaching hospital in Mumbai over a one year period on a cohort of 71 sputum smear positive patients on Category I DOTS treatment. Patients were followed up weekly for upto 20 weeks or until they underwent smear and culture conversion whichever was earlier. At each follow up, specimens were collected and processed for microscopy and culture using standard protocol. RESULTS: 60/71 [84.55%] patients completed the study. 56/60 [93.3%] patients underwent sputum smear and culture conversion. The median time to smear and culture conversion was end of 5th week [day 35] and 6 1/2 weeks [day 45] respectively. Univariate and stepwise regression analysis showed that patients who had cavitatory disease, high pretreatment smear grade and a past history of tuberculosis were more likely to undergo delayed or nonconversion [P < 0.05]. CONCLUSION: The time to sputum smear and culture conversion under DOTS is similar to previous antituberculosis regimens. Since viable bacilli continue to be expelled for upto two months, infection control measures should be maintained for such time. Patients with cavitatory disease, high pretreatment smear grade or a past history of tuberculosis need to be monitored more closely.

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.