Mapping the vaginal microbiota variations in women from a community clinic in Mumbai, India.

PURPOSE: The vaginal microbiome contributes significantly to women's reproductive health and fluctuates due to various physiological and pathological factors. The study's objective is to map the vaginal microbiome of non-pregnant women and evaluate variations based on various potential factors influencing vaginal milieu. METHODS: Fifty-two sexually active, non-pregnant women between 18 and 45 years were recruited from a community clinic and clinical history was recorded. Vaginal swabs were collected to assess the vaginal microbiome by sequencing the V3-V4 region of the 16S rRNA using the Illumina HiSeq platform, followed by data analysis with QIIME 2. Vaginal milieu was assessed by Nugent score and profiling cytokines in the cervico-vaginal lavage. RESULTS: Lactobacillus iners (34.3%) were the most abundant species in all women. Significant changes in abundance of genera (Lactobacillus, Prevotella and Anaerococcus), expression of pro-inflammatory cytokine IFN-γ and changes in alpha and beta diversity was observed in women having asymptomatic bacterial vaginosis (BV). Differences in beta diversity were seen between healthy women and women exhibiting presence of Candida spp. Variations in the abundance of genera (Lactobacillus, Bifidobacterium, Porphyromonas) were observed in women who had delivery less than twelve months back, probably as more of these women (50%, 53.7%) had higher abnormal Nugent score. CONCLUSION: Lactobacillus iners was the most prevalent vaginal species in women from a Mumbai community clinic. Maximum variations in the vaginal microbiome characterized by a perturbation of the Lactobacillus predominant vaginal microbiota are seen in those women who have asymptomatic BV and childbirth within last twelve months.

A novel gut microbiome-immune axis influencing pathology in HCMV infected infants with neonatal cholestasis.

The interplay of active HCMV infection with gut dysbiosis in the immunopathology of cholestasis in neonates and infants remains unexplored. In this study, we evaluated gut microbiome profiles and immune dysfunction in a cohort of HCMV infected cholestatic infants (IgM positive, N = 21; IgM negative, N = 25) compared to healthy infants, N = 10. HCMV infected IgM positive individuals exhibited increased clinical severity in terms of liver dysfunction, altered CD4+: CD8+ ratio, and elevated Granzyme B levels in cellular immune subsets. Gut microbiome analysis revealed distinct and differential diversity and composition within infected groups aligned with clinical severity reflected through the increased abundance of Gammaproteobacteria, reduced Bifidobacteria, and a unique signature mapping to the HCMV infected IgM negative group. Correlation analyses revealed associations between Bifidobacterium breve, Gammaproteobacteria, Firmicutes, Clostridia, Finegoldia magna, Veillonella dispar, and Granzyme B expressing immune cell subsets. Our study describes a novel gut microbiome-immune axis that may influence disease severity in cholestatic infants with active HCMV infection.

Integrated immune monitoring of HCMV infection in pregnant women with complications and its association with adverse pregnancy outcomes.

Human Cytomegalovirus (HCMV) infection is associated with bad obstetric history (BOH) and adverse pregnancy outcomes (APO). Here, we characterized antiviral humoral profiles, systemic and virus specific cellular immune responses concurrently in pregnant women (n = 67) with complications including BOH and associated these signatures with pregnancy outcomes. Infection status was determined using nested blood PCR, seropositivity and IgG avidity by ELISA. Systemic and HCMV specific (pp65) cellular immune responses were evaluated by flow cytometry. Seropositivity was determined for other TORCH pathogens (n = 33) on samples with recorded pregnancy outcomes. This approach was more sensitive in detecting HCMV infection. Blood PCR positive participants, irrespective of their IgG avidity status, had higher cytotoxic potential in circulating CD8+ T cells (p < 0.05) suggesting that infection associated cellular dysfunction was uncoupled with avidity maturation of antiviral humoral responses. Also, impaired anamnestic degranulation of HCMV-pp65-specific T cells compared to HCMV blood PCR negative participants (p < 0.05) was observed. APO correlated with HCMV blood PCR positivity but not serostatus (p = 0.0039). Most HCMV IgM positive participants (5/6) were HCMV blood PCR positive with APO. None were found to be IgM positive for other TORCH pathogens. Multiple TORCH seropositivity however was significantly enriched in the APO group (p = 0.024). Generation of HCMV specific high avidity IgG antibodies had no bearing on APO (p = 0.9999). Our study highlights the utility of an integrated screening approach for antenatal HCMV infection in the context of BOH, where infection is associated with systemic and virus specific cellular immune dysfunction as well as APO.

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

Hepatic interferon γ and tumor necrosis factor a expression in infants with neonatal cholestasis and cytomegalovirus infection.

AIM OF THE STUDY: To determine the hepatic interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) levels in infants with neonatal cholestasis (NC) and associated cytomegalovirus (CMV) infection. MATERIAL AND METHODS: This study was conducted in 21 infants with NC over a period of 6 months from June 2017 to December 2017 to determine the hepatic IFN-γ and TNF-α levels in infants with NC and associated CMV infection. RESULTS: IFN-γ levels were positive in 16 (80%), low positive in 3 (16%) and negative in 1 (5%) patients. High positive and positive TNF-α levels were seen in 9 (56.3%) patients with positive liver CMV PCR and low positive levels were seen in 7 (43.7%) patients with positive liver CMV PCR (odds ratio [OR] = 2.6). Positive IFN-γ was present in 13 (81.3%) patients with positive liver CMV PCR and low positive or negative IFN-γ was seen in 3 (18.7%) patients with positive liver CMV PCR (OR = 2.2). Six (60%) patients with positive or high positive TNF-α levels in liver tissue had biliary atresia (BA) whereas 7 (77.7%) with low positive TNF-α levels had non-BA neonatal hepatitis (OR = 5.25). Six (37.5%) patients with positive IFN-γ had BA whereas 2 (50%) patients with low positive or negative IFN-γ had BA (OR = 0.6). CONCLUSIONS: There is high prevalence of CMV in liver tissues in patients with NC and elevated TNF-α and IFN-γ levels are seen in these patients. Elevated TNF-α is also seen in patients with BA. The association of elevated TNF-α, BA and CMV infection needs to be evaluated further.

Outcome of hearts with cold ischemic time greater than 300 minutes. A case-matched study.

OBJECTIVE: Expansion of potential donor pool may be facilitated by using cardiac allografts with long ischemic time. Early graft failure and potential relation to transplant coronary artery disease remains a concern. We sought to evaluate outcomes of heart transplantation in recipients of donor allografts with prolonged ischemia time. METHODS: The study group consisted of 46 (mean age, 52 years) consecutive patients at UCLA from 1994 to 2002 that underwent heart transplantation with ischemia time > 300 min. This group was compared to a case-matched control group of 46 (mean age, 51 years) patients identified from our database during this time frame for the following factors: UNOS status, congenital heart disease diagnosis, preop inotropes, pretransplantation creatinine > 1.5 and recipient age. Primary endpoint was mortality and secondary were rejection rate and transplant coronary artery disease. Allografts were perfused and stored in cold University of Wisconsin solution. RESULTS: Mean donor ages of the study and case-matched control group were 34+/-15 and 34+/-14 years, respectively. Mean ischemia times were 388 (range, 301-600 min) and 173 (range, 96-236 min), respectively. The death incidence rate per 100 transplants per year was 9% for the study group and 7.4% for the matched group (P = 0.50). Thirty-day mortality for the study and case-matched groups were 4.3 and 2.1%, respectively (P = 0.9). Late mortality was 16.5 and 18.5%, respectively (P = 0.9). The risk of death after 30 days was 7.5 and 5.8%, respectively (P = 0.5, log-rank). One-year incidence of acute cellular rejection in the study and case-matched groups were 2 and 4.5% (P = 0.36), respectively. One-year incidence of transplant coronary artery disease in the study and case-matched groups were 4.3 and 5.4%, respectively (P = 0.68). CONCLUSIONS: Donor hearts with ischemia time greater than 300 min provide comparable early and intermediate outcomes given judicious and careful donor and recipient matching and our current techniques of myocardial preservation and modified reperfusion.