Immune profiling reveals umbilical cord blood mononuclear cells from South India display an IL-8 dominant, CXCL-10 deficient polyfunctional monocyte response to pathogen-associated molecular patterns (PAMPs) that is distinct from adult blood cells.

Neonate responses to pathogen-associated molecular patterns (PAMPS) differ from adults; such understanding is poor in Indian neonates, despite recognised significant infectious risk. Immune profiling analysis was undertaken of ten secreted mediators contextualised with cellular source induced by six PAMPs in umbilical cord (CB; n=21) and adult-blood (PBMC, n=14) from a tertiary care hospital in South India. Differential cytokine expression analysis (minimum log2-fold difference; adj p-value<0.05) identified bacterial PAMPs induced higher concentrations of IL-1ß, IL-10, TNF-α in adults versus IL-8, GM-CSF, IFN-γ and IL-2 in CB. CB responded to poly I:C and SARS-CoV-2 lysate with a dominant IL-8 response, whereas, in PBMC, CXCL-10 dominated poly I:C, but not SARS-CoV-2, responses, highlighting potential IL-8 importance, in absence of Type I Interferons, in antiviral CB immunity. Candida albicans was the only PAMP to uniformly induce higher secretion of effectors in CB. The predominant source of IL-8/IL-6/TNF-α/IL-1ß in both CB and PBMC was polyfunctional monocytes and IFN-γ /IL-2/IL-17 from innate lymphocytes. Correlation matrix analyses revealed IL-8 to be the most differentially regulated, correlating positively in CB versus negatively in PBMC with IL-6, GM-CSF, IFN-γ, IL-2, consistent with more negatively regulated cytokine modules in adults, potentially linked to higher anti-inflammatory IL-10. Cord and adult blood from India respond robustly to PAMPs with unique effector combinations. These data provide a strong foundation to monitor, explore, mechanisms that regulate such immunity during the life course, an area of significant global health importance given infection-related infant mortality incidence.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Risk factors for recognized and unrecognized SARS-CoV-2 infection: a seroepidemiologic analysis of the Prospective Urban Rural Epidemiology (PURE) study.

There are limited data on individual risk factors for SARS-CoV-2 infection (including unrecognized infection). In this seroepidemiologic substudy of an ongoing prospective cohort study of community-dwelling adults, participants were thoroughly characterized pre-pandemic. The SARS-CoV-2 infection was ascertained by serology. Among 8,719 participants from 11 high-, middle-, and low-income countries, 3,009 (35%) were seropositive for SARS-CoV-2. Characteristics independently associated with seropositivity were younger age (odds ratio, OR; 95% confidence interval, CI, per five-year increase: 0.95; 0.91-0.98) and body mass index >25 kg/m2 (OR, 95% CI: 1.16, 1.01-1.34). Smoking (as compared with never smoking, OR, 95% CI: 0.83, 0.70-0.97) and COVID-19 vaccination (OR, 95% CI: 0.70, 0.60-0.82) were associated with a reduced risk of seropositivity. Among seropositive participants, 83% were unaware of having been infected with SARS-CoV-2. Seropositivity and a lack of awareness of infection were more common in lower-income countries. The COVID-19 vaccination reduces the risk of SARS-CoV-2 infection (including recognized and unrecognized infections). Overweight or obesity is an independent risk factor for SARS-CoV-2 infection. Infection and lack of infection awareness are more common in lower-income countries.IMPORTANCEIn this large, international study, evidence of SARS-CoV-2 infection was obtained by testing blood specimens from 8,719 community-dwelling adults from 11 countries. The key findings are that (i) the large majority (83%) of community-dwelling adults from several high-, middle-, and low-income countries with blood test evidence of SARS-CoV-2 infection were unaware of this infection-especially in lower-income countries; and (ii) overweight/obesity predisposes to SARS-CoV-2 infection, while COVID-19 vaccination is associated with a reduced risk of SARS-CoV-2 infection. These observations are not attributable to other individual characteristics, highlighting the importance of the COVID-19 vaccination to prevent not only severe infection but possibly any infection. Further research is needed to understand the mechanisms by which overweight/obesity might increase the risk of SARS-CoV-2 infection.

BCG revaccination in adults enhances pro-inflammatory markers of trained immunity along with anti-inflammatory pathways.

This study characterized mechanisms of Bacille Calmette-Guérin (BCG) revaccination-induced trained immunity (TI) in India. Adults, BCG vaccinated at birth, were sampled longitudinally before and after a second BCG dose. BCG revaccination significantly elevated tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 in HLA-DR+CD16-CD14hi monocytes, demonstrating induction of TI. Mycobacteria-specific CD4+ T cell interferon (IFN) γ, IL-2, and TNF-α were significantly higher in re-vaccinees and correlated positively with HLA-DR+CD16-CD14hi TI responses. This, however, did not translate into increased mycobacterial growth control, measured by mycobacterial growth inhibition assay (MGIA). Post revaccination, elevated secreted TNF-α, IL-1ß, and IL-6 to "heterologous" fungal, bacterial, and enhanced CXCL-10 and IFNα to viral stimuli were also observed concomitant with increased anti-inflammatory cytokine, IL-1RA. RNA sequencing after revaccination highlighted a BCG and LPS induced signature which included upregulated IL17 and TNF pathway genes and downregulated key inflammatory genes: CXCL11, CCL24, HLADRA, CTSS, CTSC. Our data highlight a balanced immune response comprising pro- and anti-inflammatory mediators to be a feature of BCG revaccination-induced immunity.

Polyfunctional CD4 T-cells correlating with neutralising antibody is a hallmark of COVISHIELDTM and COVAXIN® induced immunity in COVID-19 exposed Indians.

Detailed characterisation of immune responses induced by COVID-19 vaccines rolled out in India: COVISHIELDTM (CS) and COVAXIN® (CO) in a pre-exposed population is only recently being discovered. We addressed this issue in subjects who received their primary series of vaccination between November 2021 and January 2022. Both vaccines are capable of strongly boosting Wuhan Spike-specific neutralising antibody, polyfunctional Th1 cytokine producing CD4+ T-cells and single IFN-γ + CD8+ T-cells. Consistent with inherent differences in vaccine platform, the vector-based CS vaccine-induced immunity was of greater magnitude, breadth, targeting Delta and Omicron variants compared to the whole-virion inactivated vaccine CO, with CS vaccinees showing persistent CD8+ T-cells responses until 3 months post primary vaccination. This study provides detailed evidence on the magnitude and quality of CS and CO vaccine induced responses in subjects with pre-existing SARS-CoV-2 immunity in India, thereby mitigating vaccine hesitancy arguments in such a population, which remains a global health challenge.

Evidence for the heterologous benefits of prior BCG vaccination on COVISHIELD™ vaccine-induced immune responses in SARS-CoV-2 seronegative young Indian adults.

This proof-of-concept study tested if prior BCG revaccination can qualitatively and quantitively enhance antibody and T-cell responses induced by Oxford/AstraZeneca ChAdOx1nCoV-19 or COVISHIELD™, an efficacious and the most widely distributed vaccine in India. We compared COVISHIELD™ induced longitudinal immune responses in 21 BCG re-vaccinees (BCG-RV) and 13 BCG-non-revaccinees (BCG-NRV), all of whom were BCG vaccinated at birth; latent tuberculosis negative and SARS-CoV-2 seronegative prior to COVISHIELD™ vaccination. Compared to BCG-NRV, BCG-RV displayed significantly higher and persistent spike-specific neutralizing (n) Ab titers and polyfunctional CD4+ and CD8+ T-cells for eight months post COVISHIELD™ booster, including distinct CD4+IFN-γ+ and CD4+IFN-γ- effector memory (EM) subsets co-expressing IL-2, TNF-α and activation induced markers (AIM) CD154/CD137 as well as CD8+IFN-γ+ EM,TEMRA (T cell EM expressing RA) subset combinations co-expressing TNF-α and AIM CD137/CD69. Additionally, elevated nAb and T-cell responses to the Delta mutant in BCG-RV highlighted greater immune response breadth. Mechanistically, these BCG adjuvant effects were associated with elevated markers of trained immunity, including higher IL-1ß and TNF-α expression in CD14+HLA-DR+monocytes and changes in chromatin accessibility highlighting BCG-induced epigenetic changes. This study provides first in-depth analysis of both antibody and memory T-cell responses induced by COVISHIELD™ in SARS-CoV-2 seronegative young adults in India with strong evidence of a BCG-induced booster effect and therefore a rational basis to validate BCG, a low-cost and globally available vaccine, as an adjuvant to enhance heterologous adaptive immune responses to current and emerging COVID-19 vaccines.

BCG revaccination qualitatively and quantitatively enhances SARS-CoV-2 spike-specific neutralizing antibody and T cell responses induced by the COVISHIELD™ vaccine in SARS-CoV-2 seronegative young Indian adults.

This study tested if prior BCG revaccination can further boost immune responses subsequently induced by a widely distributed and otherwise efficacious Oxford/AstraZeneca ChAdOx1nCoV-19 vaccine, referred to as COVISHIELD™, in India. We compared COVISHIELD™ induced longitudinal immune responses in 21 BCG re-vaccinees (BCG-RV) and 13 BCG-non-revaccinees (BCG-NRV), all of whom were BCG vaccinated at birth and latent tuberculosis negative, after COVISHIELD™ prime and boost with baseline samples that were collected pre-pandemic and pre-BCG revaccination. Compared to BCG-NRV, BCG-RV displayed significantly higher magnitude of spike-specific Ab and T cell responses, including a greater proportion of high responders; better quality polyfunctional CD4 and CD8 T cells that persisted and a more robust Ab and T cell response to the Delta mutant of SARS-CoV-2 highlighting greater breadth. Mechanistically, BCG adjuvant effects on COVISHIELD™ induced adaptive responses was associated with more robust innate responses to pathogen-associated-molecular-patterns through TNF-α and IL-1ß secretion. This study provides first in-depth analysis of immune responses induced by COVISHIELD™ in India and highlights the potential of using a cheap and globally available vaccine, BCG, as an adjuvant to enhance heterologous adaptive immune responses induced by COVIDSHIELD™ and other emerging vaccines.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

The Evolution of Regulatory Elements in the Emerging Promoter-Variant Strains of HIV-1 Subtype C.

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not the coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but no significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a coinfection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.

Innate Immune Cytokine Profiling and Biomarker Identification for Outcome in Dengue Patients.

Background: Early biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and "cytokine storm" associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue. Results: Dengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised. Conclusion: Our findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ+CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85-0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.

Molecular detection of virulence markers to identify diarrhoeagenic Escherichia coli isolated from Mula-Mutha river, Pune District, India.

In this study presence of virulence genes in multidrug resistant Escherichia coli isolated from Mula-Mutha river, Pune, India was undertaken. The objective was to understand whether the isolates were of diarrhoeagenic or of environmental origin. This was essential since the river flows through urban and rural parts of Pune and its water is used not only for industrial and agricultural purposes but also for domestic usage. One hundred and two multidrug E. coli isolates were selected from our previous study which detected genes coding for antibiotic resistance as well as identified integrons associated with multidrug resistance. Isolates were subjected to multiplex PCR to detect presence of virulence genes, set1A, set1B, sen astA, aggA, aafA, pet, stx1 and stx. Sequencing was performed to confirm the amplified PCR product. Seven of the 102 E. coli isolates showed gene set1A alone identifying them as Enteroaggregative E. coli. Thus, the findings revealed that majority of drug resistant E. coli were environmental in origin. The presence of antibiotic resistant genes, integrons in the environment as well as diarrhoeagenic E. coli isolates is a warning and calls for efficient public health measures to ensure that untreated sewage and industrial waste does not enter the Mula-Mutha river.

A century of BCG: Impact on tuberculosis control and beyond.

BCG turns 100 this year and while it might not be the perfect vaccine, it has certainly contributed significantly towards eradication and prevention of spread of tuberculosis (TB). The search for newer and better vaccines for TB is an ongoing endeavor and latest results from trials of candidate TB vaccines such as M72AS01 look promising. However, recent encouraging data from BCG revaccination trials in adults combined with studies on mucosal and intravenous routes of BCG vaccination in non-human primate models have renewed interest in BCG for TB prevention. In addition, several well-demonstrated non-specific effects of BCG, for example, prevention of viral and respiratory infections, give BCG an added advantage. Also, BCG vaccination is currently being widely tested in human clinical trials to determine whether it protects against SARS-CoV-2 infection and/or death with detailed analyses and outcomes from several ongoing trials across the world awaited. Through this review, we attempt to bring together information on various aspects of the BCG-induced immune response, its efficacy in TB control, comparison with other candidate TB vaccines and strategies to improve its efficiency including revaccination and alternate routes of administration. Finally, we discuss the future relevance of BCG use especially in light of its several heterologous benefits.

Moraxella catarrhalis: A Cause of Concern with Emerging Resistance and Presence of BRO Beta-Lactamase Gene-Report from a Tertiary Care Hospital in South India.

BACKGROUND: Found as a commensal in the upper respiratory tract, Gram-negative diplococcus Moraxella catarrhalis did not hold much importance as an infectious agent for long. The emergence of the first antibiotic-resistant strain of M. catarrhalis was noted in 1977 in Sweden. This has gradually spread worldwide over the years to more than 95% of the strains showing resistance to penicillin now. Penicillin resistance is mediated by the production of beta-lactamases encoded by bro-1 and bro-2 genes that code for beta-lactamases BRO-1 and BRO-2, respectively. The purpose of this study was to explore the trends of antibiotic resistance, the presence of bro genes, and clinical correlation of these findings with the rise in M. catarrhalis was noted in 1977 in Sweden. This has gradually spread worldwide over the years to more than 95% of the strains showing resistance to penicillin now. Penicillin resistance is mediated by the production of beta-lactamases encoded by bro-1 and bro-2 genes that code for beta-lactamases BRO-1 and BRO-2, respectively. The purpose of this study was to explore the trends of antibiotic resistance, the presence of bro genes, and clinical correlation of these findings with the rise in. METHODS: Strains of M. catarrhalis was noted in 1977 in Sweden. This has gradually spread worldwide over the years to more than 95% of the strains showing resistance to penicillin now. Penicillin resistance is mediated by the production of beta-lactamases encoded by bro-1 and bro-2 genes that code for beta-lactamases BRO-1 and BRO-2, respectively. The purpose of this study was to explore the trends of antibiotic resistance, the presence of bro genes, and clinical correlation of these findings with the rise in. RESULTS: Fourteen strains of M. catarrhalis was noted in 1977 in Sweden. This has gradually spread worldwide over the years to more than 95% of the strains showing resistance to penicillin now. Penicillin resistance is mediated by the production of beta-lactamases encoded by bro-1 and bro-2 genes that code for beta-lactamases BRO-1 and BRO-2, respectively. The purpose of this study was to explore the trends of antibiotic resistance, the presence of bro genes, and clinical correlation of these findings with the rise in. CONCLUSION: The increase in antibiotic resistance and beta-lactamase production in M. catarrhalis is a cause of concern. The emerging resistance pattern emphasises the need for an appropriate antibiotic stewardship program in clinical practice. Importance should be given to the monitoring of the trends of antibiotic susceptibility and their usage to prevent the emergence of outbreaks with resistant strains and treatment failures.M. catarrhalis was noted in 1977 in Sweden. This has gradually spread worldwide over the years to more than 95% of the strains showing resistance to penicillin now. Penicillin resistance is mediated by the production of beta-lactamases encoded by bro-1 and bro-2 genes that code for beta-lactamases BRO-1 and BRO-2, respectively. The purpose of this study was to explore the trends of antibiotic resistance, the presence of bro genes, and clinical correlation of these findings with the rise in.

Effect of Structured Physical Activity on Inflammation and Immune Activation Profile of Antiretroviral Therapy-Experienced Children Living With HIV.

AIM: To compare the markers of inflammation and immune activation in virally suppressed HIV-infected children on antiretroviral therapy, who practiced regular structured exercise comprising running and yoga to those who did not over a 2-year period. METHODS: This retrospective cohort study included 72 children aged 8 to 16 years divided into 2 groups, exercisers (n = 36) and the nonexercisers (n = 36) based on their intentional physical activity. The analyses were carried out at baseline and after 2 years (Y2) for the soluble biomarkers of inflammation and immune activation (tumor necrosis factor alpha, interleukin-6, interleukin-10, interferon gamma, sCD14, and sCD163). In addition, cell-associated biomarker (CD38), lipopolysaccharides, and the gene expression of interleukin-2 and brain-derived neurotrophic factor were also measured at Y2. RESULTS: Reduction in levels of sCD14 (effect size [ES], -0.6; 95% confidence interval [CI], -1.08 to -0.14), tumor necrosis factor alpha (ES, -0.7; 95% CI, -1.18 to -0.23), interferon gamma (ES, -0.7; 95% CI, -1.17 to -0.22), and interleukin-10 (ES, -0.6; 95% CI, -1.08 to -0.14) was observed among exercisers as compared with nonexercisers at Y2. In addition, CD38+ expressing CD4+ T cells were found to be lower among exercisers (P = .01) at Y2. However, the differences in levels of interleukin-6, sCD163, lipopolysaccharides, interleukin-2, and brain-derived neurotrophic factor were not significantly different among the 2 groups. CONCLUSION: The study result suggests that regular structured physical activity improves the inflammatory profile of antiretroviral therapy-treated HIV-infected children.

Evaluation of LAMP-based assays for carbapenemase genes.

Purpose. Rapid and accurate detection of carbapenem resistance is a critical requirement for the selection of appropriate therapy and initiation of infection control measures. Although several tests are available, their use is limited by one or more factors. Phenotypic tests are lengthy, have variable sensitivity and specificity and do not generally identify the carbapenemase. Molecular assays overcome many of these issues but cost can be a barrier to adoption, particularly in low-resource settings. To address the need for affordable, molecular tools, we have assessed the performance characteristics of loop-mediated isothermal amplification (LAMP)-based assays for the major carbapenemase genes, blaNDM, blaKPC, blaOXA-48, blaOXA-23 blaVIM and blaIMP.Methodology. The assays were validated using 1849 Gram-negative Indian clinical isolates obtained from seven hospitals and diagnostic centres.Results. The assays had diagnostic sensitivities of 98.14 %, 98.92 %, 100 %, 98.48 %, and diagnostic specificities of 98.94 %, 99.61 %, 97.42 %, 99.38 % for blaNDM, blaOXA-48, blaOXA-23 and blaVIM, respectively. Due to a low number of isolates positive for blaKPC and blaIMP, the performance characteristics of assays for these two genes could not be suitably evaluated.Conclusion. The performance characteristics suggest suitability for diagnostic and surveillance purposes.

Surface colonization and subsequent development of infections with multi drug resistant organisms in a neonatal intensive care unit.

BACKGROUND: This study analyzes colonization of the neonates in a NICU and incidence of these colonized infants developing infections due to the colonizers. METHODS: Over a 12 month period, samples (surface swabs and rectal swabs) were obtained from all the infants admitted to NICU. The samples were cultured and examined for the presence of colonizers and especially for multi-drug resistant organisms. RESULTS: From the total 533 patients, 473 (89%) neonates acquired colonizers and 60 (11%) did not. Of the 473 (89%) colonized infants, 57 (12%) developed infections of whom 33 (58%) were infected from the same organism as the colonizer and 24 (42%) neonates developed an infection that was different from the colonizer. 416 (88%) infants did not develop any infection inspite of being colonized. CONCLUSIONS: The total numbers of babies contracting infection were more in the colonized group than the non-colonized. Other factors like gestational age and preterm may have played a role in development of infection in addition to colonization in these babies. Screening for the presence of MDRO colonization may be of limited use in predicting infections in the colonized individual. However, knowledge of their presence results in implementation of strict infection control practices. This along with judicious uses of antimicrobials effectively reduces infections from colonized bacteria and more importantly prevent their spread.

Viral evolution in the cell-associated HIV-1 DNA during early ART can lead to drug resistance and virological failure in children.

Using cell-associated DNA and cell-free RNA of human immunodeficiency virus type-1 (HIV-1), we investigated the role of drug-resistant viral variants that emerged during early antiretroviral therapy (ART) in determining virological outcome. This case-control study compared virologic nonresponder children (two viral loads [VLs] ≥ 200 copies/mL within 2 years of ART) and responder children (two VLs < 200 copies/mL after six months of ART) infected with HIV-1 initiated on nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based ART. The partial reverse-transcriptase gene of HIV-1 in cell-associated DNA was genotyped using next-generation sequencing (NGS; Illumina; threshold 0.5%; at baseline and month six of ART) and in cell-free RNA (concurrently and at virological failure; VL > 1000 copies/mL at ≥ 12 months of ART) using the Sanger method. Among 30 nonresponders and 37 responders, baseline differences were insignificant while adherence, VL, and drug resistance mutations (DRMs) observed at month six differed significantly ( P ≥ 0.05). At month six, NGS estimated a higher number of DRMs compared with Sanger (50% vs 33%; P = 0.001). Among the nonresponders carrying a resistant virus (86.6%) at virological failure, 26% harbored clinically relevant low-frequency DRMs in the cell-associated DNA at month six (0.5%-20%; K103N, G190A, Y181C, and M184I). Plasma VL of > 3 log 10 copies/mL (AOR, 30.4; 95% CI, 3.3-281; P = 0.003) and treatment-relevant DRMs detected in the cell-associated DNA at month six (AOR, 24.2; 95% CI, 2.6-221; P = 0.005) were independently associated with increased risk for early virological failure. Our findings suggest that treatment-relevant DRMs acquired in cell-associated DNA during the first six months of ART can predict virological failure in children initiated on NNRTI-based ART.

Emergence of new genotypes and lineages of dengue viruses during the 2012-15 epidemics in southern India.

OBJECTIVES: To genotypically characterize dengue virus (DENV) isolates among dengue-infected children from 2012-13/2014-15 outbreaks in southern India. METHODS: Children hospitalized with suspected dengue were tested for dengue RT-PCR targeting Capsid-preMembrane (C-prM) and Envelope (Env) regions. Following virologic confirmation (n=612), a representative selection of DENV isolates (n=99) were sequenced for C-prM, aligned using ClustalW and subjected to phylogenetic analysis by maximum-likelihood method in MEGA6. RESULTS: In 2012-13 (n=113), DENV-3 (44, 38.9%) and DENV-2 (43, 38.1%) predominated; DENV-1 (22, 19.5%) and DENV-4 (1, 0.9%) were less common. The pattern changed in 2014-15 (n=499), when DENV-1 (329, 65.7%) predominated, followed by DENV-2 (97, 21.2%), DENV-3 (36, 6.7%) and DENV-4 (10, 2.0%). Multiple-serotype co-infections occurred in 2.7% and 5.4% in 2012-13 and 2014-15, respectively. Genotype III (GIII) of DENV-1 predominated (85.7%) in 2012-13, ceding to GI predominance (80.8%) in 2014-15. Among DENV-2, 71.9% (23/32) showed distinct clustering suggesting a new lineage, 'GIVc'. All tested DENV-4 were GIC, whose clustering pattern showed the emergence of two distinct clades. CONCLUSIONS: New genotypic/lineage variations in DENV-1 and DENV-2 may have influenced the magnitude and severity of dengue epidemics in southern India during this period. These findings emphasize the role of active surveillance of DENV serotypes/genotypes in aiding outbreak control and vaccine studies.

Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.

Background: Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Methods: Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. Results: We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.  Conclusions: We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.

Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.