Phylogenetic Analysis of Spread of Hepatitis C Virus Identified during HIV Outbreak Investigation, Unnao, India.

An HIV outbreak investigation during 2017-2018 in Unnao District, Uttar Pradesh, India, unearthed high prevalence of hepatitis C virus (HCV) antibodies among the study participants. We investigated these HCV infections by analyzing NS5B and core regions. We observed no correlation between HIV-HCV viral loads and clustering of HCV sequences, regardless of HIV serostatus. All HCV isolates belonged to genotype 3a. Monophyletic clustering of isolates in NS5B phylogeny indicates emergence of the outbreak from a single isolate or its closely related descendants. The nucleotide substitution rate for NS5B was 6 × 10-3 and for core was 2 × 10-3 substitutions/site/year. Estimated time to most recent common ancestor of these isolates was 2012, aligning with the timeline of this outbreak, which might be attributable to unsafe injection practices while seeking healthcare. HIV-HCV co-infection underlines the need for integrated testing, surveillance, strengthening of healthcare systems, community empowerment, and molecular analyses as pragmatic public health tools.

Genetic variability in minor capsid protein (L2 gene) of human papillomavirus type 16 among Indian women.

Human papillomavirus type 16 (HPV-16) is the predominant genotype worldwide associated with invasive cervical cancer and hence remains as the focus for diagnostic development and vaccine research. L2, the minor capsid protein forms the packaging unit for the HPV genome along with the L1 protein and is primarily associated with transport of genomic DNA to the nucleus. Unlike L1, L2 is known to elicit cross-neutralizing antibodies and thus becomes a suitable candidate for pan-HPV prophylactic vaccine development. In the present study, a total of 148 cervical HPV-16 isolates from Indian women were analyzed by PCR-directed sequencing, phylogenetic analysis and in silico immunoinformatics tools to determine the L2 variations that may impact the immune response and oncogenesis. Ninety-one SNPs translating to 35 non-synonymous amino acid substitutions were observed, of these 16 substitutions are reported in the Indian isolates for the first time. T245A, L266F, S378V and S384A substitutions were significantly associated with high-grade cervical neoplastic status. Multiple substitutions were observed in samples from high-grade cervical neoplastic status as compared to those from normal cervical status (p = 0.027), specifically from the D3 sub-lineage. It was observed that substitution T85A was part of both, B and T cell epitopes recognized by MHC-I molecules; T245A was common to B and T cell epitopes recognized by MHC-II molecules and S122P/A was common to the region recognized by both MHC-I and MHC-II molecules. These findings reporting L2 protein substitutions have implications on cervical oncogenesis and design of next-generation L2-based HPV vaccines.

Kaposi sarcoma (KS) with primary effusion lymphoma in HIV infected MSM (men having sex with men) co-infected with pulmonary tuberculosis and syphilis: a case report from India.

We describe a case of a 30-year-old MSM recently diagnosed with HIV, immunocompromised with a purplish or brown rash all over the body for 3 to 4 months. The histopathology of the cutaneous lesions and pleural effusion aspirate confirmed the diagnosis of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). While KS is one of the AIDS-defining illnesses seen in immunocompromised patients having low CD4 count, PEL is a rare and distinct subset of AIDS-related lymphoma. Despite the widespread availability of HIV testing, HIV diagnosis gets delayed due to stigma among MSM. This case report emphasizes the importance of early suspicion for symptoms of HIV-associated opportunistic infections in high-risk populations like MSM. The report reiterates the need for an ambient stigma-free environment for improving HIV screening in this high-risk population.

Characterization of major capsid protein (L1) variants of Human papillomavirus type 16 by cervical neoplastic status in Indian women: Phylogenetic and functional analysis.

The etiological role of infection with Human papillomavirus type 16 (HPV16) in cervical cancer is well established. HPV16 variants, classified based on less than 10% nucleotide variations in the major capsid (L1 ORF) are known to contribute to persistent infection leading to cancer development. L1 protein forms the cornerstone of HPV structure and antigenicity. In the present study, HPV16 L1 variants were characterized by cervical lesion grade and variations in sequences were correlated to structure and function. The L1 gene was analyzed in 152 HPV16 positive cervical samples obtained from Indian women using polymerase chain reaction-directed sequencing. Phylogenetic analysis was carried out for lineage typing. Sixty-one SNPs were detected in L1 genes resulting in 20 nonsynonymous amino acid substitutions of which N56T, N92T, L158F, V178G, N181I, K236T, K443Q, K454T, and K475R are reported in Indian isolates for the first time. The substitutions N181T, T353P, and T389S were significantly associated with high-grade cervical disease. The predominance of lineage A (A1-A4, 84.96%) was observed among the isolates, while the D3 sublineage showed significant association with high-grade cervical lesions. No evidence for recombination and the positive selection was obtained. These substitutions, when mapped on three-dimensional structure, revealed that 11 and 4 substitutions are part of experimentally validated B- and T-cell epitopes, of which T266A and N285T were common to both types of epitopes and may impact HPV vaccine efficacy. The variants identified through this study have the potential to serve as translational leads for designing diagnostic probes and vaccines.

Seroprevalence & seroincidence of Orientia tsutsugamushi infection in Gorakhpur, Uttar Pradesh, India: A community-based serosurvey during lean (April-May) & epidemic (October-November) periods for acute encephalitis syndrome.

Background & objectives: In India, acute encephalitis syndrome (AES) cases are frequently reported from Gorakhpur district in Uttar Pradesh. Scrub typhus is one of the predominant aetiological agents for these cases. In order to delineate the extent of the background of scrub typhus seroprevalence and the associated risk factors at community level, serosurveys during both lean and epidemic periods (phase 1 and phase 2, respectively) of AES outbreaks were conducted in this region. Methods: Two community-based serosurveys were conducted during lean (April-May 2016) and epidemic AES (October-November 2016) periods. A total of 1085 and 906 individuals were enrolled during lean and epidemic AES periods, respectively, from different villages reporting recent AES cases. Scrub typhus-seronegative individuals (n=254) during the lean period were tested again during the epidemic period to estimate the incidence of scrub typhus. Results: The seroprevalence of Orientia tsutsugamushi during AES epidemic period [immunoglobulin (Ig) IgG: 70.8%, IgM: 4.4%] was high as compared to that of lean AES period (IgG: 50.6%, P <0.001; IgM: 3.4%). The factors independently associated with O. tsutsugamushi positivity during lean AES period were female gender, illiteracy, not wearing footwear, not taking bath after work whereas increasing age, close contact with animals, source of drinking water and open-air defecation emerged as additional risk factors during the epidemic AES season. IgM positivity was significantly higher among febrile individuals compared to those without fever (7.7 vs. 3.5%, P=0.006). The seroincidence for O. tsutsugamushi was 19.7 per cent, and the subclinical infection rate was 54 per cent. Interpretation & conclusions: The community-based surveys identified endemicity of O. tsutsugamushi and the associated risk factors in Gorakhpur region. The findings will be helpful for planning appropriate interventional strategies to control scrub typhus.

Molecular mechanisms associated with Fluconazole resistance in clinical Candida albicans isolates from India.

Resistance to azole antifungals is a significant problem in Candida albicans. An understanding of resistance at molecular level is essential for the development of strategies to tackle resistance and rationale design of newer antifungals and target-based molecular approaches. This study presents the first evaluation of molecular mechanisms associated with fluconazole resistance in clinical C.albicans isolates from India. Target site (ERG11) alterations were determined by DNA sequencing, whereas real-time PCRs were performed to quantify target and efflux pump genes (CDR1, CDR2, MDR1) in 87 [Fluconazole susceptible (n = 30), susceptible-dose dependent (n = 30) and resistant (n = 27)] C.albicans isolates. Cross-resistance to fluconazole, ketoconazole and itraconazole was observed in 74.1% isolates. Six amino acid substitutions were identified, including 4 (E116D, F145L, E226D, I437V) previously reported ones and 2 (P406L, Q474H) new ones. CDR1 over-expression was seen in 77.7% resistant isolates. CDR2 was exclusively expressed with CDR1 and their concomitant over-expression was associated with azole cross-resistance. MDR1 and ERG11 over-expression did not seem to be associated with resistance. Our results show that drug efflux mediated by Adenosine-5'-triphosphate (ATP)-binding cassette transporters, especially CDR1 is the predominant mechanism of fluconazole resistance and azole cross-resistance in C. albicans and indicate the need for research directed towards developing strategies to tackle efflux mediated resistance to salvage azoles.

Pseudomembranous Type of Oral Candidiasis is Associated with Decreased Salivary Flow Rate and Secretory Immunoglobulin A Levels.

Saliva plays an important role in maintaining microbial homeostasis in the oral cavity, while salivary gland hypofunction predisposes the oral mucosa to pathologic alteration and increases the risk for oral candidiasis. This study sought to determine the salivary flow rate (SFR) and secretory immunoglobulin A (SIgA) levels in HIV-positive and HIV-negative individuals and evaluate their relationship with the determinants of oral candidiasis. Sixty HIV-positive (30 with and 30 without oral candidiasis) and 30 healthy HIV-negative individuals were enrolled. Cotton pellet was weighed pre- and post-saliva collection for the assessment of SFR, while SIgA levels were estimated by commercial ELISA (Diametra, Italy) kit. The mean ± SD, SFR and SIgA levels in HIV-positive individuals with candidiasis, without candidiasis and HIV-negative controls were 0.396 ± 0.290, 0.546 ± 0.355 and 0.534 ± 0.214 ml/min and 115.891 ± 37.621, 136.024 ± 51.075 and 149.418 ± 31.765 µg/ml, respectively. A positive correlation between low CD4 counts (indicator of immunodeficiency) and SIgA was observed in HIV-positive individuals with candidiasis (r = 0.373, p = 0.045). We also report here for the first time the significant decrease in SFR and SIgA levels in individuals presenting with pseudomembranous type of oral candidiasis and Candida albicans infection.

Pneumocystis jirovecii infection and the associated dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations in HIV-positive individuals from Pune, India.

The present study was undertaken to detect Pneumocystis jirovecii infection among HIV-positive patients presenting with symptoms of lower respiratory tract infection and analyze the associated dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations. P. jirovecii infection was detected in 12.6% cases. We did not find DHPS gene mutations at the commonest positions of codon 55 and 57; however, mutation at codon 171 was detected in two cases. No mutations in DHFR gene were detected. The results indicate low prevalence of DHPS and DHFR mutations in Indian P. jirovecii isolates, suggesting that the selective pressure of sulfa drugs on the local strains has probably not reached the levels found in developed nations.

Validation of dried blood spot for serological diagnosis of Hepatitis B and C: a multicentric study.

The present study aimed to determine diagnostic performance of dried blood spot (DBS) for the detection of Hepatitis B surface antigen (HBsAg) and Hepatitis C virus antibodies (anti-HCV) using CLIA at 3 different laboratories across India. DBS can serve as a simple and convenient alternative to plasma/serum for HBsAg detection. However for anti-HCV, site-specific validation of the assay is warranted.

Biofilm production in oral Candida isolates from HIV-positive individuals from Pune, India.

Biofilm formation is implicated as a potential virulence factor in Candida species and carries important clinical repercussions because of their increased resistance to antifungal treatment, ability to withstand host defences and to serve as a reservoir for continuing infections. The present study was undertaken to determine the biofilm production among oral Candida isolates from HIV-positive and HIV-negative individuals from Pune, India. Biofilm formation was determined using the spectrophotometric or microtitre plate method in 182 Candida isolates, of which 154 were from HIV-positive and 28 were from HIV-negative individuals. A total of 63.2% of the Candida isolates were biofilm producers. Significantly increased biofilm forming abilities both qualitatively as well as quantitatively were observed in Candida isolates from HIV-positive individuals (66.2%) compared to isolates from HIV-negative ones (46.4%), (P- 0.041). Eighty-one (59.6%) C. albicans isolates and 34 (73.9%) non -C. albicans Candida (NCAC) showed biofilm positivity. The NCAC showed significantly greater intensity of biofilm formation compared to the C. albicans, P- 0.032. Our results thus show the enhanced biofilm forming abilities of oral Candida isolates from HIV-infected individuals compared to HIV-uninfected ones and highlight the important role played by biofilm formation in the pathogenesis of NCAC isolates.

Prevention of recurrence of bacterial vaginosis using lactobacilli-containing vaginal tablets among women with HIV: a randomized, placebo-controlled, double-blinded phase IV trial.

OBJECTIVES: The effectiveness of lactobacilli-containing vaginal tablets (VT) in bacterial vaginosis (BV) recurrence prevention among women infected with HIV treated with standard oral metronidazole in Pune, India was studied. METHODS: Women infected with HIV with confirmed BV diagnosis (Nugent score ≥7 and Amsel criteria >3) were enrolled in a 12-month, double-blind, randomized, placebo-controlled, phase IV study between 2018 and 2021. After a standard course of oral metronidazole for 7 days (400 mg three times a day), women were randomly assigned to either lactobacilli-containing or placebo VT arms to receive VTs for 4 months. BV recurrence was assessed after the initial cure from BV. RESULTS: Of the 464 women infected with HIV, 80 women with confirmed BV were enrolled. The retention was affected due to the COVID-19 pandemic (6-month retention rates 78%). The cure was seen in 85% and 93.5% of participants from the treatment and placebo arms, respectively, after four VT cycles. BV recurrence was seen in 41.4% and 44.8% in the treatment and placebo arm, respectively, with no significant difference in the two groups. CONCLUSION: The lactobacilli-containing VT was acceptable and safe; however, the addition of VT over standard oral metronidazole did not show any additional benefit in the prevention of BV recurrence in women infected with HIV, indicating the need for long-term randomized trials among them. Registered at Clinical Trials Registry- India, (CTRI) Number: CTRI/2018/04/013298.

HIV-1 exploits Hes-1 expression during pre-existing HPV-16 infection for cancer progression.

High Risk Human Papilloma Viruses (HR-HPV) persistently infect women with Human Immunodeficiency Virus-1 (HIV-1). HPV-16 escapes immune surveillance in HIV-1 positive women receiving combined antiretroviral therapy (cART). HIV-1 Tat and HPV E6/E7 proteins exploit Notch signaling. Notch-1, a developmentally conserved protein, influences cell fate from birth to death. Notch-1 and its downstream targets, Hes-1 and Hey-1 contribute to invasive and aggressive cancers. Cervical cancer cells utilize Notch-1 and hyper-express CXCR4, a co-receptor of HIV-1. Accumulating evidence shows that HIV-1 affects cell cycle progression in pre-existing HPV infection. Additionally, Tat binds Notch-1 receptor for activation and influences cell proliferation. Oncogenic viruses may interfere or converge together to favor tumor growth. The molecular dialogue during HIV-1/HPV-16+ co-infections in the context of Notch-1 signaling has not been explored thus far. This in vitro study was designed with cell lines (HPV-ve C33A and HPV-16+ CaSki) which were transfected with plasmids (pLEGFPN1 encoding HIV-1 Tat and pNL4-3 encoding HIV-1 [full HIV-1 genome]). HIV-1 Tat and HIV-1 inhibited Notch-1expression, with differential effects on EGFR. Notch-1 inhibition nullified Cyclin D expression with p21 induction and increased G2-M cell population in CaSki cells. On the contrary, HIV-1 infection shuts down p21 expression through interaction of Notch-1 downstream genes Hes-1-EGFR and Cyclin D for G2-M arrest, DDR response and cancer progression. This work lays foundations for future research and interventions, and therefore is necessary. Our results describe for the first time how HIV-1 Tat cancers have an aggressive nature due to the interplay between Notch-1 and EGFR signaling. Notch-1 inhibitor, DAPT used in organ cancer treatment may help rescue HIV-1 induced cancers. Graphical abstract: The illustration shows how HIV interacts with HPV-16 to induce Notch 1 suppression for cancer progression (Created with BioRender.com). Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00809-y.

Genetic variations in the long control region of human papillomavirus type 16 isolates from India: implications for cervical carcinogenesis.

Introduction. Infection with high-risk human papillomavirus (HPV) types, specifically HPV type 16 (HPV16), is considered to be the most important risk factor in the development of cervical intraepithelial neoplasia and cancer. The long control region (LCR) is a noncoding region that comprises approximately 10 % of the HPV genome and contains regulatory elements for viral transcription and replication. Sequence variations in LCR may impact on the replication efficiency and oncogenic potential of the virus.Gap statement. Studies documenting variations in LCR of HPV16 isolates pertaining to cervical neoplastic status in India are limited.Aim. The present study was designed to characterize variations in the LCR of Indian isolates of HPV16 and study their association with cervical disease grades.Methodology. The LCR was amplified and sequenced from HPV16 positive cervical samples belonging to different cervical disease grades. Sequences were aligned to identify variations and potential transcription factor binding sites (TFbs) were predicted using the JASPAR database in addition to phylogenetic studies.Results. Among the 163 HPV16 isolates analysed, 47 different nucleotide variations were detected in the LCR, of which 25 are reported for first time in Indian isolates. Point mutations were detected in 35/54 (64.8 %) samples with normal cervical status, 44/50 (88 %) samples with low-grade cervical disease and 53/59 (89.8 %) samples with high-grade cervical disease. Variations T6586C, G6657A and T6850G were significantly associated with high-grade cervical status. Thirteen LCR variations were detected in the binding sites for CEBPB, ETS1, JUN, MYB, NFIL3, PHOX2A and SOX9 transcription factors.Conclusion. The present study helped to identify unique variations in the LCRs of HPV16 Indian isolates. The variations in the A4 sub-lineage were significantly associated with high-grade disease status. The isolates belonging to the A4 and D3 sub-lineages harboured mutations in putative TFbs, implying a potential impact on viral replication and progression to cervical cancer.

Diagnostic performance of oral swab specimen for SARS-CoV-2 detection with rapid point-of-care lateral flow antigen test.

We evaluated the performance of oral swab specimen both health-care worker (HCW) collected and self-collected for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection with rapid antigen test (RAT) as compared to reverse transcriptase polymerase chain reaction (RT-PCR). Of the 529 participants enrolled, 121 (22.8%) were RT-PCR positive. Among the RT-PCR positives, 62 (51.2%) were RAT positive using oral swab. When compared with RT-PCR, RAT with oral swab had sensitivity and specificity of 63.3 and 96.8% respectively among symptomatic individuals. No statistically significant difference was observed in RAT positivity with HCW collection and self-collection, p = 0.606. Ct values were significantly lower in RT-PCR and RAT positive samples (ORF gene: 18.85 ± 4.36; E gene: 18.72 ± 4.84) as compared to RT-PCR positive and RAT negative samples (ORF gene: 26.98 ± 7.09; E gene: 26.97 ± 7.07), p < 0.0001. Our study demonstrated moderate sensitivity of RAT with oral swab in symptomatic individuals. Oral swab was the preferred sampling by almost all participants in terms of convenience and comfort as compared to nasopharyngeal swab. Oral swabs have utility for SARS-CoV-2 antigen detection among symptomatic individuals residing in remote rural areas and can serve as an initial screening tool during COVID-19 spikes when cases rise exponentially and laboratory capacities for RT-PCR testing become overwhelmed.

Diagnostic performance of patient self-collected oral swab (tongue and cheek) in comparison with healthcare worker-collected nasopharyngeal swab for severe acute respiratory syndrome coronavirus-2 detection.

The present study was conducted to compare the performance of patient self-collected oral swab (OS) with healthcare worker (HCW)-collected nasopharyngeal swab (NPS) for SARS-CoV-2 detection by reverse transcription polymerase chain reaction (RT-PCR) in real-world setting. Paired OS and NPS were collected from 485 consecutive individuals presenting with symptoms of coronavirus disease-19 (COVID-19) or asymptomatic contacts of COVID-19 cases. Both specimens were processed for RT-PCR and cycle threshold (Ct) value for each test was obtained. Positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA) and kappa were calculated for OS RT-PCR compared with NPS RT-PCR as reference. A total of 116/485 (23.9%) participants were positive by NPS RT-PCR. OS had PPA of 71.6%, NPA of 98.8%, OPA of 92.4% and kappa of 0.771. Almost all participants (483/485, 99.6%) reported OS as a convenient and comfortable sample for SARS-CoV-2 testing over NPS. All participants with Ct values <25 and majority (90.8%) with Ct values <30 were detected by OS. To conclude, OS self-sampling was preferred in comparison with NPS due the ease and comfort during collection. The performance of OS RT-PCR for SARS-CoV-2 detection, however, was sub-optimal in comparison with NPS RT-PCR.

Adherence to buccal epithelial cells, enzymatic and hemolytic activities of Candida isolates from HIV-infected individuals.

Candida spp., along with other microorganisms are usually found in the normal flora of the human oral mucosa. However in HIV-infected individuals these yeasts can become opportunistic pathogens and lead to mucosal infections. The transformation from a harmless commensal to a virulent pathogen under conditions of dysfunctional host defense system is attributable to an extensive repertoire of selectively expressed virulence determinants. The present study was undertaken to analyze the production of four important virulence factors viz. adherence to buccal epithelial cells (BEC), proteinase activity, phospholipase activity and hemolysis, and to evaluate the correlation between these virulence factors in 65 Candida isolates from HIV-infected individuals with oral candidiasis. A total of 95.3%, 67.7%, 41.5% and 100% of the Candida isolates showed adherence to BEC, and proteinase, phospholipase and hemolytic activities, respectively. Production of proteinase and phospholipase enzymes was seen in 89.7% and 59.0% of C. albicans isolates and 34.6% and 15.4% of non-Candida albicans Candida (NAC) isolates, respectively. C. albicans showed significantly greater level of virulence factor expression with regards to adherence to BEC (P < 0.001), phospholipase production (P < 0.044) and hemolysis (P = 0.037) as compared to NAC. A correlative relationship between proteinase activity and adherence to BEC, as well as phospholipase production was noted.

Ocular manifestations and refractive errors among people living with HIV in Pune, India: a cross-sectional study.

OBJECTIVE: We aimed to determine the ocular manifestation and refractive error prevalences among people living with HIV (PLHIV) in Pune, India. METHODS: This cross-sectional study included HIV-infected adults attending a National AIDS Research Institute clinic. Ophthalmologic examination included visual acuity estimation, refraction, orthoptic evaluation, slit lamp and fundus examination, and photography. RESULTS: In total, 441 HIV-infected individuals were enrolled. The participants' median age was 44 (interquartile range 38-49) years and 227 (51.5%) were men. Refractive errors occurred in 132 (29.9%) individuals. Ocular manifestations were present in 93 (21.1%) participants and the most frequent was cataract in 59 (13.4%) participants. Multivariate logistic regression analysis showed that participants who were illiterate (adjusted odds ratio [AOR] 2.80, 95% confidence interval [CI] 1.47-5.33) and those aged greater than 40 years (AOR 5.59, 95% CI 2.69-11.61) were more likely to have ocular manifestations. The odds of having ocular manifestations were greater in participants with treatment substitution or changes (AOR 2.11, 95% CI 1.16-3.82). CONCLUSIONS: Cataract and refractive errors were prevalent among PLHIV. PLHIV should be encouraged to have regular ophthalmic checkups. Individuals with lower education levels and older age should be counseled regarding eye care and timely reporting of ocular symptoms.

Characterisation of colonisation properties of vaginal lactobacilli from healthy Indian women: Implications for identification of potential probiotic candidates.

The ability of probiotic bacteria to colonise the target site is considered of utmost importance and used for preliminary screening of potential probiotic candidates. Thirty-eight vaginal Lactobacillus strains isolated from healthy women and characterised by 16S RNA sequencing were assessed for colonisation characteristics including adherence to human vaginal epithelial cells, auto-aggregation and hydrophobicity. No significant difference in adherence (P = 0.384), auto-aggregation (P = 0.282) and hydrophobicity (P = 0.084) abilities between Lactobacillus species was observed, however, significant difference in colonisation characteristics between strains of the same species was noted (P < 0.001). We identified seven Lactobacillus strains that may serve as best candidates for vaginal probiotic development.