Can HIV reverse transcriptase activity assay be a low-cost alternative for viral load monitoring in resource-limited settings?

OBJECTIVE: To evaluate the performance and cost of an HIV reverse transcriptase-enzyme activity (HIV-RT) assay in comparison to an HIV-1 RNA assay for routine viral load monitoring in resource limited settings. DESIGN: A cohort-based longitudinal study. SETTING: Two antiretroviral therapy (ART) centres in Karnataka state, South India, providing treatment under the Indian AIDS control programme. PARTICIPANTS: A cohort of 327 HIV-1-infected Indian adult patients initiating first-line ART. OUTCOME MEASURES: Performance and cost of an HIV-RT assay (ExaVir Load V3) in comparison to a gold standard HIV-1 RNA assay (Abbott m2000rt) in a cohort of 327 Indian patients before (WK00) and 4 weeks (WK04) after initiation of first-line therapy. RESULTS: Plasma viral load was determined by an HIV-1 RNA assay and an HIV-RT assay in 629 samples (302 paired samples and 25 single time point samples at WK00) obtained from 327 patients. Overall, a strong correlation of r=0.96 was observed, with good correlation at WK00 (r=0.84) and at WK04 (r=0.77). Bland-Altman analysis of all samples showed a good level of agreement with a mean difference (bias) of 0.22 log10copies/mL. The performance of ExaVir Load V3 was not negatively affected by a nevirapine/efavirenz based antiretroviral regimen. The per test cost of measuring plasma viral load by the Abbott m2000rt and ExaVir Load V3 assays in a basic lab setting was $36.4 and $16.8, respectively. CONCLUSIONS: The strong correlation between the HIV-RT and HIV-1 RNA assays suggests that the HIV-RT assay can be an affordable alternative option for monitoring patients on antiretroviral therapy in resource-limited settings. TRIAL REGISTRATION NUMBER: ISRCTN79261738.

Prevalence of virulence factors and phylogenetic characterization of uropathogenic Escherichia coli causing urinary tract infection in patients with and without diabetes mellitus.

BACKGROUND: We aimed to determine the virulence factor profile and phylogenetic grouping of uropathogenic Escherichia coli (UPEC) causing urinary tract infection (UTI) in patients with and without diabetes mellitus (DM). METHODS: A total of 280 UPEC were collected from the urine samples of patients with DM (n=126) and patients suffering from non-DM UTIs (n=154). All isolates were subjected to phenotypic and genotypic virulence factor profile and phylogenetic grouping. RESULTS: There was no significant difference in phenotypic virulence factors of UPEC causing UTI from patients with and without DM; alpha-haemolysin (DM=52, 41.2%; non-DM=69, 44.8%), mannose resistant haemagglutination (DM=6, 36.2%; non-DM=64, 41.5%), biofilm production (DM=33.3%; non-DM=25.9%), serum resistance (DM=27, 21.4%; non-DM=46, 29.8%), cell surface hydrophobicity (DM=22, 17.1%; non-DM=18, 11.6%) and mannose sensitive hemagglutnation (DM=18, 14.2%; non-DM=29, 18.8%). Among the genotypic virulence markers, papC gene was most prevalent in both patients with DM (n=65, 51.6%) and without DM (n=88, 57.1%), followed by hlyD gene (DM=36, 28.6%; non-DM=52, 33.8%). Only cnf-1 gene was observed to be significantly associated (p<0.05) with the non-DM status (n=35, 22.7%) than DM (n=15, 11.9%). Irrespective of the diabetic status, majority of the UPEC isolates (approximately 50%) belonged to phylogenetic group B2 predominantly harboring the virulence genes papC, hlyD and cnf-1. CONCLUSIONS: This study demonstrates that there may not be a differential selection of virulence properties of UPEC causing UTI in patients with DM and in the general population.

Performance of genotypic tools for prediction of tropism in HIV-1 subtype C V3 loop sequences.

Currently, there is no consensus on the genotypic tools to be used for tropism analysis in HIV-1 subtype C strains. Thus, the aim of the study was to evaluate the performance of the different V3 loop-based genotypic algorithms available. We compiled a dataset of 645 HIV-1 subtype C V3 loop sequences of known coreceptor phenotypes (531 R5-tropic/non-syncytium-inducing and 114 X4-tropic/R5X4-tropic/syncytium-inducing sequences) from the Los Alamos database (http://www.hiv.lanl.gov/) and previously published literature. Coreceptor usage was predicted based on this dataset using different software-based machine-learning algorithms as well as simple classical rules. All the sophisticated machine-learning methods showed a good concordance of above 85%. Geno2Pheno (false-positive rate cutoff of 5-15%) and CoRSeqV3-C were found to have a high predicting capability in determining both HIV-1 subtype C X4-tropic and R5-tropic strains. The current sophisticated genotypic tropism tools based on V3 loop perform well for tropism prediction in HIV-1 subtype C strains and can be used in clinical settings.

Lower prevalence of hlyD, papC and cnf-1 genes in ciprofloxacin-resistant uropathogenic Escherichia coli than their susceptible counterparts isolated from southern India.

OBJECTIVE: The study was conducted to determine the association of the hlyD, papC and cnf-1 virulence genes with drug resistance in uropathogenic Escherichia coli (UPEC) isolated from cases of urinary tract infection (UTI). METHOD: A total of 193 E. coli strains isolated from symptomatic cases of UTI in a tertiary care teaching hospital in Raichur, Northern Karnataka, India were included in the study. The antibiotic susceptibility pattern was determined by Kirby-Bauer's Disk Diffusion method, and the strains resistant to any of the third generation cephalosporins tested were further confirmed for extended-spectrum beta-lactamase (ESBL)-production by an E-strip test. Genotypic virulence markers, namely, hlyD, papC and cnf-1, were detected by the uniplex PCR method and the phylogenetic characterization was performed by a multiplex PCR assay. RESULTS: The majority of the E. coli isolates belonged to the B2 phylogenetic group were significantly associated with ciprofloxacin-sensitivity and non-ESBL production (p<0.05). An increased prevalence of ciprofloxacin-sensitive strains over ciprofloxacin-resistant strains were observed among the UPEC isolates harboring the papC (72.9% vs. 40.2%; p<0.001), hlyD (43.7% vs. 21.6%; p<0.001) and cnf-1 (30.2% vs. 12.3%; p<0.05) genes. The presence of a multivirulent gene in the non-ESBL E. coli strains (44.5%) was significantly higher (p<0.05) than in the ESBL-producing strains (21%). CONCLUSIONS: Among the UPEC isolates, the predominant B2 phylogenetic group was significantly associated with the ciprofloxacin-sensitive strains, as well as with the non-ESBL E. coli strains. The genotypic virulence markers of UPEC were associated with ciprofloxacin-sensitivity, and a significant number of the non-ESBL strains harbored multivirulent genes. The relationship between the presence of the virulence genes and ESBL production was complex and warrants further intensive studies.

HIV-1 coreceptor tropism in India: increasing proportion of X4-tropism in subtype C strains over two decades.

BACKGROUND: Recent studies show an increase in the frequency of X4-tropism in African HIV-1 subtype C (HIV-1C) strains and among Indian children with a longer duration of infection. There is limited availability of comprehensive data on HIV-1 tropism in Indian HIV-1C strains and impact on coreceptor antagonist drug susceptibility. We evaluated coreceptor tropism trends over 2 decades and maraviroc resistance-associated V3 loop substitutions among the Indian HIV-1C strains. METHODS: We performed genotypic tropism testing using Geno2Pheno10% on primary samples from patients (n = 224) and on Indian HIV-1C sequences downloaded from the Los Alamos database (n = 528, 1991-2010). We also studied maraviroc resistance-associated substitutions in R5-tropic HIV-1C (n = 992) and subtype B sequences (n = 576). RESULTS: Among primary samples, 88% belonged to HIV-1C and 11.2% was predicted as X4-tropic, with higher prevalence noted among patients from north-eastern India (19.1%) and significant association with intravenous drug users (P = 0.04). X4-tropism prevalence was higher among antiretroviral therapy-experienced (18.8%) compared with antiretroviral therapy-naive patients (9.1%). Indian database HIV-1C sequences showed X4-tropism at 4%. An increase in the X4 tropism frequency was seen over the years 1991 (1.6%) through 2012 (10%). We found a high frequency of 19T substitution (826/992; 83.3%) among HIV-1C V3 loop compared with subtype B. CONCLUSIONS: The predominance of R5-tropism in Indian HIV-1C strains despite a marginal temporal increase in X4-tropism prevalence highlights the likely effectiveness of coreceptor antagonists in India. Our frequent observation of common maraviroc resistance-associated substitutions among Indian R5-tropic HIV-1C raises the possibility that they may be natural polymorphisms, indicating the need for further elucidation.

High concordance of genotypic coreceptor prediction in plasma-viral RNA and proviral DNA of HIV-1 subtype C: implications for use of whole blood DNA in resource-limited settings.

OBJECTIVES: Genotypic tropism testing (GTT) of HIV is increasingly used prior to the initiation of CCR5 antagonist therapy in HIV-infected individuals. Normally performed on plasma-derived virus, the test is challenging when performed in patients with suppressed viraemia. We aimed to evaluate the performance of cell-associated proviral DNA against plasma-derived viral RNA as the genetic material for GTT in an Indian clinical setting. METHODS: From 52 HIV-1-infected individuals, the env V3 region was successfully amplified and sequenced from both proviral DNA and plasma RNA paired samples having a viral load >2500 copies/mL (n = 42) and from proviral DNA only in 10 antiretroviral therapy (ART)-experienced patients with a viral load <500 copies/mL. GTT was performed using the Geno2Pheno algorithm with the interpretative false positive rate (FPR) cut-off of 10%. RESULTS: Among paired samples, 40 of 42 patients harboured subtype C strains. Plasma RNA tropism prediction revealed X4 tropism in 4 of 42 (9.5%). A high concordance of 97.6% in tropism prediction was noted in simultaneous RNA/DNA samples (38 R5 and 3 X4). Discordance was observed in one sample showing R5 tropism in proviral DNA and X4 tropism in plasma RNA. Comparison of Geno2Pheno FPRs in both the plasma and proviral compartments showed good correlation (overall, r = 0.87; ART-naive patients, r = 0.79; ART-failing patients, r = 0.97). GTT was successfully performed in all 10 whole blood DNA samples having a viral load <500 copies/mL, all showing R5 tropism. CONCLUSIONS: High concordance in tropism prediction from proviral DNA and plasma-viral RNA suggests that prediction of viral tropism using proviral DNA is accurate and feasible in resource-limited clinical settings, particularly in patients with low or suppressed viraemia.

The detection of ESBL-producing Escherichia coli in patients with symptomatic urinary tract infections using different diffusion methods in a rural setting.

OBJECTIVES: This study aimed to determine the prevalence of extended spectrum of beta lactamases (ESBLs), to compare different phenotypic methods for ESBL confirmation and to evaluate the antibiotic resistance patterns among ESBL-producing urinary Escherichia coli. METHODS: Urinary E. coli isolates that were resistant to at least one of the three indicator cephalosporins (cefotaxime, cefpodoxime and ceftazidime) were tested for ESBL production using the double disc synergy test (DDST), the inhibitory potentiated disc diffusion (IPDD) test and the quantitative E-strip method. RESULT: Of the 163 E. coli strains isolated, 80 (49%) were resistant to at least one of the three cephalosporins, and 38 (47.5%) tested positive for ESBLs by the IPDD test and the E-strip test. However, only15 (18.7%) strains tested positive by the DDST. Among the third-generation cephalosporins, cefpodoxime (46.1%) was the best screening indicator, followed by ceftazidime (43%) and cefotaxime (39.9%). Most of the ESBL producers (97.3%) were resistant to three or more drugs, compared with 51.2% of non-ESBL producers. CONCLUSION: Compared with the DDST, the IPDD and E-strip tests appear to be preferable methods for detecting ESBLs, with better sensitivity (100%) and specificilty (97.6%) and positive predictive values (97.3%). ESBL producers showed significantly (p<0.05) higher resistance to tobramycin, co-amoxyclav and amikacin than did non-ESBL producers.

Multi-drug-resistant tuberculosis: the experience of an urban tertiary care hospital in South India using automated BACTEC 460 TB.

The emergence of multi-drug-resistant (MDR) strains has been a major obstacle in the tuberculosis (TB) control programme. In the present study we looked into the prevalence of MDR-TB in an urban tertiary care hospital in South India over four years (2007-2010). During this period, 641 clinical specimens (317 respiratory specimens and 324 non-respiratory specimens) were received for mycobacteriological culture and drug susceptibility testing for first-line drugs, using the BACTEC 460 TB system. Mycobacterium tuberculosis (MTB) was isolated in 34.8% (n = 223) specimens. Of the total 223 MTB isolates 83 (37.2%) were MDR. Forty-two percent of the pulmonary MTB isolates (n = 72) and 20.4% of the extra-pulmonary isolates (n = 10) were MDR. Although we observed a high percentage of drug resistance, the prevalence of MDR was not observed to vary significantly within the four years which suggested good management.

Diabetes mellitus and HIV as co-morbidities in tuberculosis patients of rural south India.

OBJECTIVES: Incidence of tuberculosis (TB) is greatest among patients with impaired immunity. India is experiencing a double epidemic of HIV and diabetes mellitus (DM), both of which are strongly associated with immuno-suppression. This study aimed to discover the prevalence of HIV and DM in both the pulmonary and extra-pulmonary TB patients of rural south India, retrospectively. METHODS: Medical records of 192 microbiologically diagnosed pulmonary TB and 37 extra-pulmonary TB patients were thoroughly studied and data were extracted. The frequency distribution of HIV and DM was evaluated along with other demographic details such as age, sex and occupation in both groups. RESULTS: The mean age of the pulmonary TB patients was 41.11±15.7 years, with significantly higher (p<0.0001) preponderance of DM (31.8%) over HIV (8.9%). 72.13% of the diabetic patients belonged to the age group of 41-60 years. Extra-pulmonary TB patients had a mean age of 34.62±12.9, years with a significantly higher (p<0.006) HIV prevalence of 32.43% over DM (5.4%). 75% of the HIV patients belonged to the age group of 41-60 years. Occupationally, the majority of the pulmonary TB patients were agricultural labourers (25.2%) while the majority of the extra-pulmonary TB patients were housewives or self employed (18.92%). CONCLUSION: Though more importance is being given to HIV-TB coinfection, we cannot overlook DM, which showed a significantly higher prevalence in pulmonary TB patients compared to HIV. The rising prevalence of DM in high TB burden countries may adversely affect TB control.

Prevalence of respiratory syncytial virus infection among hospitalized children presenting with acute lower respiratory tract infections.

OBJECTIVE: To evaluate the prevalence of RSV among hospitalized young children presenting with ALRI in Bangalore, India. METHODS: Respiratory syncytial virus (RSV) antigen detection was performed by direct fluorescent antibody (DFA) staining on 77 nasopharyngeal wash samples collected from hospitalized children below 2 years of age with a diagnosis of acute lower respiratory tract infection (ALRI). RESULTS: Out of 77 samples tested for RSV with DFA, 17 (22.1%) were found RSV-positive with a mean age of 8.24 ± 7.21 months (M:F = 1.8:1). Three children had congenital cardiac disease and one child had a history of prematurity. One child had re-infection within one month of primary infection. RSV-infected children were more likely to have a diagnosis of bronchiolitis than RSV-negative children (p < 0.001). CONCLUSIONS: RSV infection is a significant cause of morbidity among children presenting with ALRI in southern India. In resource-limited settings, DFA can be used as an important tool for rapid detection of RSV and can potentially eliminate prolonged hospitalization and unnecessary use of antibiotics.

Epidemiology of culture isolates from peritoneal dialysis peritonitis patients in southern India using an automated blood culture system to culture peritoneal dialysate.

AIM: Continuous ambulatory peritoneal dialysis (CAPD) is a major form of therapy for chronic end stage renal disease patients, which may lead to CAPD-associated peritonitis. The spectrum of organisms associated with CAPD peritonitis varies geographically. Not much data is available regarding this from southern India. The aim of this study was to characterize the spectrum of organisms associated with CAPD peritonitis in this region and observe the utility of automated blood culture systems to culture peritoneal dialysate. METHODS: Ninety episodes of peritonitis were cultured over a span of 3 years using an automated blood culture system. RESULTS: The yield of culture positivity was 50%. The most predominant organism was found to be coagulase-negative Staphylococcus spp. (21.1%) followed by Enterobacteriaceae (12.2%). Other organisms isolated were non-fermenting Gram-negative bacilli (4.4%), Pseudomonas aeruginosa (3.3%), α-haemolytic Streptococci (3.3%), Candida spp. (2.2%), Staphylococcus aureus (1.1%), ß-haemolytic Streptococci (1.1%) and Micrococci (1.1%). A high degree of resistance to third generation cephalosporins (66.7%) was noted amongst the Gram-negative bacilli. Also, all the Gram-negative bacilli isolated from patients who had prior empirical antibiotic therapy of ceftazidime before arrival at the centre, were resistant to third generation cephalosporins. CONCLUSION: A varied spectrum of organisms isolated from peritoneal dialysate compared to the global scenario was observed. Also, a high degree of third generation cephalosporin resistance was noted amongst the Gram-negative bacilli. Thus, it is suggested that the empirical therapy should be dependent on the local epidemiology.

Ceftriaxone resistant Shigella flexneri, an emerging problem.

Shigellosis is a disease of public health importance in developing countries. It may cause self-limited diarrhea to severe dysentery. Emergence of multi drug resistant (MDR) strains is a growing concern globally. Ceftriaxone and ciprofloxacin are the drugs of choice for MDR cases. Here, we report a case of MDR Shigella flexneri from an immunocompromised patient. The strain was resistant to ceftriaxone [minimum inhibitory concentration (MIC) ≥ 64 µg/ml], limiting the treatment option. Simultaneously, the strain was also found to be resistant to ciprofloxacin (MIC ≥ 4 µg/ml). However, it was susceptible to ceftazidime (MIC 4 µg/ml). This is the first case of ceftriaxone resistant Shigella spp. reported from our hospital.