Serum procalcitonin and C-reactive protein as indices of early sepsis and mortality in North Indian pediatric burn injuries: a prospective evaluation and literature review.

BACKGROUND: Delays in diagnosing sepsis in children afflicted with thermal injuries can result in high morbidity and mortality. Our study evaluated the role of the biomarkers Procalcitonin (PCT) and C-reactive protein (CRP) as predictors of early sepsis and mortality, respectively, in this group of patients. METHODS: This was a prospective evaluation of 90 pediatric burn cases treated at a tertiary care burn center in Northern India. Patients, aged 1-16 years, presenting within 24 hours of being burned, with >10% body surface area of burn injury were included in the study. Levels of PCT and CRP were measured on days 1, 3, 5, and 7. Patients were followed until discharge, 30th post-burn day, or death, whichever occurred first. RESULTS: Sepsis was clinically present in 49 of 90 (54.4%) cases with a median 30% total body surface area (TBSA) of burns. Mortality was seen in 31 of 90 (34.4%) cases with a median of 35% TBSA burns. High PCT and CRP were seen in the sepsis group, particularly on days 3, 5, and 7. PCT was also significantly higher in the mortality group (days 1 and 3). CONCLUSIONS: While PCT was a good early predictor of sepsis and mortality in children with burns, CRP was reliable as a predictor of sepsis only. Both markers, however, can serve as adjuncts to culture sensitivity reports for diagnosing early onset sepsis and initiation of antibiotic therapy in appropriate patients.

Application of LAMP assay for detection of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in ICU admitted sepsis patients: A rapid and cost-effective diagnostic tool.

Carbapenem-resistant significant members of Acinetobacter calcoaceticus-Acinetobacter baumannii (CR-SM-ACB) complex have emerged as an important cause of sepsis, especially in ICUs. This study demonstrates the application of loop-mediated-isothermal-amplification (LAMP) assay for detection of CR-SM-ACB-complex from patients with sepsis. Whole-blood and culture-broths(CB) collected from patients with culture-positive sepsis were subjected to LAMP and compared with PCR, and RealAmp. Vitek-2 system and conventional PCR results were used as confirmatory references. The sensitivity and specificity of LAMP(97 % & 100 %) and RealAmp(100 % & 100 %) for detection of CR-SM-ACB-complex from CB were better than PCR(87 % & 100 %). Diagnostic accuracy of LAMP, RealAmp, and PCR for detection of SM-ACB-complex from CB was 98.5 %, 100 %, and 88.5 % respectively. Turnaround time of Culture, LAMP, PCR, and RealAmp was 28-53, 6-20, 9-23, and 6-20hours, respectively. LAMP is a simple, inexpensive tool that can be applied directly to positive CB and may be customized to detect emerging pathogens and locally-prevalent resistance genes and to optimize antimicrobial use.

Varicella zoster virus outbreak in a long-term care unit of a tertiary care hospital in northern India.

Nosocomial outbreak of varicella zoster virus (VZV) has been reported when susceptible individuals encounter a case of chicken pox or shingles. A suspected VZV outbreak was investigated in a 50-bedded in-patient facility of Physical Medicine and Rehabilitation in a tertiary care multispecialty hospital. A 30-year-old female patient admitted with Pott's spine was clinically diagnosed with chicken pox on 31 December 2022. The following week, four more cases were identified in the same ward. All cases were diagnosed as laboratory-confirmed varicella zoster infection by PCR. Primary case was a housekeeping staff who was clinically diagnosed with chicken pox 3 weeks prior (9 December 2022). He returned to work on eighth day of infection (17 December 2022) after apparent clinical recovery but before the lesions had crusted over. Thirty-one HCWs were identified as contacts a and three had no evidence of immunity. Two of these susceptible HCWs had onset of chickenpox shortly after first dose of VZV vaccination was inoculated. All cases recovered after treatment with no reported complications. VZV infection is highly contagious in healthcare settings with susceptible populations. Prompt identification of cases and implementation of infection prevention and control measures like patient isolation and vaccination are essential for the containment of outbreaks.

Evaluation of risk factors associated with hard-to-heal wound infection in a tertiary care hospital.

OBJECTIVE: The incidence of hard-to-heal wound infection, especially as a result of multidrug-resistant Gram-negative organisms, has increased in recent years. The reason for the increase is multifactorial and the ability of these pathogenic isolates to form biofilms is one of the important risk factors in wound infection. This study aimed to evaluate the risk factors associated with such cases. METHOD: This prospective analytical study, conducted over a period of two months, included pus or tissue samples from hospital inpatients with Gram-negative hard-to-heal wound infection. The samples were processed with conventional microbiological techniques. Patient demographic details and the presence of various risk factors were recorded. Biofilm production was detected by tissue culture plate method in the laboratory. The data were analysed using SPSS version 21 (IBM Ltd., US). RESULTS: The experimental cohort comprised 200 patients. Klebsiella spp. was the most common identified organism, followed by Escherichia coli and Pseudomonas spp. Carbapenem resistance was observed in 106 (53%) strains. Almost 66% of the strains showed biofilm formation. On evaluation of associated risk factors, age (p=0.043), presence of biofilms (p=0.0001), diabetes (p=0.002), hypertension (p=0.02) and medical device use (p=0.008) had significant association, whereas sex, previous surgery and prior antibiotic use had no significant impact on the chronicity of the wound. CONCLUSION: In this study, chronicity of wounds was observed to be associated with multiple risk factors, especially the biofilm-forming ability of the strain. Biofilms are difficult to eradicate and additional measures, such as physical debridement, are important for resolving chronicity. Knowledge about specific risk factors would also allow clinicians a better understanding of the healing process and drive appropriate wound care interventions. DECLARATION OF INTEREST: A grant was received from the Indian Council of Medical Research (ICMR) for this work (grant ID: 2017-02686). The authors have no conflicts of interest to declare.

Polydispersity-mediated high efficacy of an in-situ aqueous nanosuspension of PPEF.3HCl in methicillin resistant Staphylococcus aureus sepsis model.

Recently, World Health Organization declared antimicrobial resistance as the third greatest threat to human health. Absence of known cross-resistance, new class, new target, and a new mode of action are few major strategies being undertaken by researches to combat multidrug resistant pathogen. PPEF.3HCl, a bisbenzimidazole was developed as highly potent antibacterial agent against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, targeting topoisomerase IA. The present work encompasses a radical on-site generation of In-situ nanosuspension of PPEF.3HCl with enhanced efficacy against methicillin resistant S. aureus in septicemia model. We have generated instantaneously a PPEF.3HCl nanosuspension (IsPPEF.3HCl-NS) by mixing optimized monophasic PPEF.3HCl preconcentrate in propylene glycol into an aqueous medium comprising tween 80 as stabilizer. The IsPPEF.3HCl-NS showed precipitation efficiency of > 90 %, average particle size < 500 nm, retained upto 5 h, a negative zeta potential and bi/trimodal particle size distribution. Differential scanning calorimetry, X-ray diffraction confirmed partial amorphization and transmission electron microscopy revealed spherical particles. IsPPEF.3HCl-NS was non-hemolytic and exhibited good stability in serum. More significantly, it exhibited a âˆ¼ 1.6-fold increase in macrophage uptake compared to free PPEF.3HCl in the RAW 264.7 macrophage cell line. Confocal microscopy revealed accumulation of IsPPEF.3HCl-NS within the lysosomal compartment and cell cytosol, proposing high efficacy. In terms of antimicrobial efficacy, IsPPEF.3HCl-NS outperforms free PPEF.3HCl against clinical methicillin sensitive and resistant S. aureus strains. In a pivotal experiment, IsPPEF.3HCl-NS exhibited over 83 % survival at 8 mg/kg.bw and an impressive reduction of âˆ¼ 4-5 log-fold in bacterial load, primarily in the kidney, liver and spleen of septicemia mice. IsPPEF.3HCl-NS prepared by the In-situ approach, coupled with enhanced intramacrophage delivery and superior efficacy, positions IsPPEF.3HCl-NS as a pioneering and highly promising formulation in the battle against antimicrobial resistance.

Risk factors and outcome associated with the acquisition of MDR linezolid-resistant Enterococcus faecium: a report from tertiary care centre.

OBJECTIVE: The aim of the study was to determine the resistance profile of linezolid-resistant Enterococcus faecium (LREfm) and to investigate risk factors and outcomes associated with LREfm infections. MATERIAL AND METHODS: A prospective case-control study was undertaken (2019 to 2022) and included 202 patients with LREfm infections (cases) and 200 controls with LSEfm infections. Clinical data was prospectively collected and analysed for risk factors and outcomes. Antimicrobial susceptibility was performed, and resistance profile was studied using WHOnet. RESULTS: Risk factors associated with LREfm infection were site of infection UTI (OR 5.87, 95% CI 2.59-13.29, p ≤ 0.001), prior use of carbapenem (OR 2.85 95% CI 1.62-5.02, p ≤ 0.001) and linezolid (OR 10.13, 95% CI 4.13-24.82, p ≤ 0.001), use of central line (OR 5.54, 95% CI 2.35-13.09, p ≤ 0.001), urinary catheter (OR 0.29, 95% CI 0.12-0.70, p ≤ 0.001) and ventilation (OR 14.87, 95% CI 7.86-28.11, p ≤ 0.007). The hospital stay 8-14 days (< 0.001) prior to infection and the mortality rate (p = 0.003) were also significantly high among patients with LREfm infections. Linezolid and vancomycin resistance coexisted; further, MDR, XDR and PDR phenotypes were significantly higher among LREfm. CONCLUSION: This study provided insight into epidemiology of MDR LREfm in a setting where linezolid use is high. The main drivers of infections with LREfm are multiple, including use of carbapenems and linezolid. Invasive procedures and increased hospital stay facilitate spread through breach in infection control practises. As therapeutic options are limited, ongoing surveillance of LREfm and VRE is critical to guide appropriate use of linezolid and infection control policies.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Procalcitonin for Detecting Culture-Positive Sepsis in Neonates: A Prospective, Multicenter Study.

INTRODUCTION: It is unclear if serum procalcitonin (PCT) estimated at sepsis suspicion can help detect culture-positive sepsis in neonates. We evaluated the diagnostic performance of PCT in culture-positive sepsis in neonates. METHODS: This was a prospective study (February 2016 to September 2020) conducted in four level-3 units in India. We enrolled neonates suspected of sepsis in the first 28 days of life. Neonates with birth weight <750 g, asphyxia, shock, and major malformations were excluded. Blood for PCT assay was drawn along with the blood culture at the time of suspicion of sepsis and before antibiotic initiation. The investigators labeled the neonates as having culture-positive sepsis or "no sepsis" based on the culture reports and clinical course. PCT assay was performed by electrochemiluminescence immunoassay, and the clinicians were masked to the PCT levels while assigning the label of sepsis. Primary outcomes were the sensitivity, specificity, and likelihood ratios to identify culture-positive sepsis. RESULTS: The mean birth weight (SD) and median gestation (IQR) were 2,113 (727) g and 36 (32-38) weeks, respectively. Of the 1,204 neonates with eligible cultures, 155 (12.9%) had culture-positive sepsis. Most (79.4%) were culture-positive within 72 h of birth. The sensitivity, specificity, and positive and negative likelihood ratios at 2 ng/mL PCT threshold were 52.3% (95% confidence interval: 44.1-60.3), 64.5% (60.7-68.1), 1.47 (1.23-1.76), and 0.74 (0.62-0.88), respectively. Adding PCT to assessing neonates with 12.9% pretest probability of sepsis generated posttest probabilities of 18% and 10% for positive and negative test results, respectively. CONCLUSION: Serum PCT did not reliably identify culture-positive sepsis in neonates.

Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens.

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with -6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

Clinico-epidemiological profile of COVID-19 patients admitted during third wave of pandemic in a tertiary care hospital in New Delhi, India.

Even nearly two years after the first reported case, the novel coronavirus (SARS-CoV-2) continues to ebb and flow around the world. A retrospective cohort study was carried out to determine the clinico-epidemiological profile and outcome of the cases. The study analyzed secondary data from 827 patients who presented to our center with COVID-19-related illnesses between December 15, 2021, and February 15, 2022 (third wave in India). There was a significant difference in the vaccination status of patients treated at home and those admitted, with 87.9% having received two doses compared to 74% in the second group being unvaccinated. Patients who were isolated at home recovered at a rate of 99.4%, while hospitalized patients died at a rate of 26.5%. Vaccination reduces the severity of COVID-19; however, constant vigilance for new variants, precautionary measures, and increased vaccination drives are critical moving forward.   *Other members of the Safdarjung Hospital COVID-19 working group: B. Lal (Medicine), Harish Sachdeva (Anaesthesiology), Santvana Kohli (Anaesthesiology), Amandeep Jaswal (Anaesthesiology), Sumitra Bachani (Obstetrics and Gynecology), Ajay Kumar (Pediatrics), Rohit Kumar (Pulmonary Medicine), Vidya Sagar Chaturvedi (Surgery), Vinod Chaitanya (Medicine).

Bacteriological profile of paediatric skin and soft tissue infections and their antibiogram at a tertiary care teaching hospital in north India.

The increasing complexity of wound care in pediatric patients along with delay in the initiation of treatment predisposes these patients to many complications such as pressure ulcers, non-healing surgical wounds and skin damage. A retrospective study was conducted over a period of five years to gain insight into the etiology of pediatric wound infection. A total of 2819 wound culture positive cases were included. Most samples were from the burn ward (30.15%) followed by the general surgery ward (20.46%). Overall, the most common isolate was Staphylococcus aureus (39.73%) followed by Pseudomonas spp (19.12%). The prevalence of MRSA (Methicillin resistant S. aureus), CRE (Carbapenem-resistant Enterobacteriaceae), CRP (Carbapenem resistant Pseudomonas), CRAB (Carbapenem resistant Acinetobacter baumannii) was 47.3%, 62.5%, 70.5%, 96.4%, respectively. The high degree of resistance in children highlights the importance of regular surveillance for identification of common pathogens and optimization of antimicrobial treatment for multidrug resistant organisms.

Short communication: Virological and B cell profiles of chikungunya and Dengue virus co-infections in Delhi during 2017-2019.

With explosive epidemics of chikungunya in India since 2004, chikungunya virus (CHIKV) now co-circulates in geographical areas where Dengue virus (DENV) is already endemic and thus provides opportunity for the same mosquito to be infected with both viruses. Although there are excellent studies that have addressed the clinical of mono and co-infection, we have little to no knowledge on the current viral sequences that pre-dominate co-infections, and the B cell response elicited. In this study, we analyzed febrile patients that were confirmed to have DENV-CHIKV co-infections and asked the following questions: 1) what is the frequency of co-infections found in a single cycle of transmission; 2) what are the viral sequences associated with them; 3) what does the antibody secreting cell / plasmablast response look like in patients that are co-infected with both viruses. We report those co-infections occur at a frequency of 6.7% in the transmission cycle, and while DENV-3 is now frequently detected, we do not see a serotype bias in the patients that are co-infected with ESCA strain of CHIKV. Moreover, the effector B cell response (plasmablasts) observed are specific to both infecting viruses indicating no overt bias. Further studies to associate whether any of these properties have a bearing on clinical disease manifestation will be both timely and important.

Health-care-associated bloodstream and urinary tract infections in a network of hospitals in India: a multicentre, hospital-based, prospective surveillance study.

BACKGROUND: Health-care-associated infections (HAIs) cause significant morbidity and mortality globally, including in low-income and middle-income countries (LMICs). Networks of hospitals implementing standardised HAI surveillance can provide valuable data on HAI burden, and identify and monitor HAI prevention gaps. Hospitals in many LMICs use HAI case definitions developed for higher-resourced settings, which require human resources and laboratory and imaging tests that are often not available. METHODS: A network of 26 tertiary-level hospitals in India was created to implement HAI surveillance and prevention activities. Existing HAI case definitions were modified to facilitate standardised, resource-appropriate surveillance across hospitals. Hospitals identified health-care-associated bloodstream infections and urinary tract infections (UTIs) and reported clinical and microbiological data to the network for analysis. FINDINGS: 26 network hospitals reported 2622 health-care-associated bloodstream infections and 737 health-care-associated UTIs from 89 intensive care units (ICUs) between May 1, 2017, and Oct 31, 2018. Central line-associated bloodstream infection rates were highest in neonatal ICUs (>20 per 1000 central line days). Catheter-associated UTI rates were highest in paediatric medical ICUs (4·5 per 1000 urinary catheter days). Klebsiella spp (24·8%) were the most frequent organism in bloodstream infections and Candida spp (29·4%) in UTIs. Carbapenem resistance was common in Gram-negative infections, occurring in 72% of bloodstream infections and 76% of UTIs caused by Klebsiella spp, 77% of bloodstream infections and 76% of UTIs caused by Acinetobacter spp, and 64% of bloodstream infections and 72% of UTIs caused by Pseudomonas spp. INTERPRETATION: The first standardised HAI surveillance network in India has succeeded in implementing locally adapted and context-appropriate protocols consistently across hospitals and has been able to identify a large number of HAIs. Network data show high HAI and antimicrobial resistance rates in tertiary hospitals, showing the importance of implementing multimodal HAI prevention and antimicrobial resistance containment strategies. FUNDING: US Centers for Disease Control and Prevention cooperative agreement with All India Institute of Medical Sciences, New Delhi. TRANSLATION: For the Hindi translation of the abstract see Supplementary Materials section.

SARS-CoV-2 infection in children with chronic kidney disease.

BACKGROUND: Information on the course of SARS-CoV-2 infection in children with chronic kidney disease (CKD) is limited. METHODS: We retrospectively reviewed the presentation and outcomes of SARS-CoV-2 infection in patients with CKD followed at any of the four pediatric nephrology centers in New Delhi from April 2020 to June 2021. Outcomes, including cardiopulmonary and renal complications, were reported in relation to underlying disease category and illness severity at presentation. RESULTS: Underlying illness in 88 patients included nephrotic syndrome (50%), other CKD stages 1-4 (18.2%), CKD 5D (17%), and CKD 5T (14.8%). Thirty-two of 61 patients with symptomatic COVID-19 and 9/27 asymptomatic patients were admitted for median 10 (interquartile range 7-15) days. Seventeen (19.3%) patients developed moderate or severe COVID-19. Systemic complications, observed in 30 (34.1%), included acute kidney injury (AKI, 34.2%), COVID-19 pneumonia (15.9%), unrelated pulmonary disease (2.3%), and shock (4.5%). Nineteen (21.6%) had severe complications (AKI stage 2-3, encephalopathy, respiratory failure, shock). Eight (11%) of twelve (16.4%) patients with severe AKI required dialysis. Three (3.4%) patients, two with steroid-resistant nephrotic syndrome in relapse and one with CKD 1-4, died due to respiratory failure. Univariate logistic regression indicated that patients presenting with nephrotic syndrome in relapse or moderate to severe COVID-19 were at risk of AKI (respective odds ratio, 95%CI: 3.62, 1.01-12.99; 4.58, 1.06-19.86) and/or severe complications (respective odds ratio, 95%CI: 5.92, 1.99-17.66; 61.2, 6.99-536.01). CONCLUSIONS: Children with CKD presenting with moderate-to-severe COVID-19 or in nephrotic syndrome relapse are at risk of severe complications, including severe AKI and mortality. A higher resolution version of the Graphical abstract is available as Supplementary information.

Self-reported survey on infection prevention and control structures in healthcare facilities part of a national level healthcare associated infection surveillance network in India, 2019.

BACKGROUND: Healthcare associated infections (HAIs) are prevalent and difficult to treat worldwide. Most HAIs can be prevented by effective implementation of Infection Prevention and Control (IPC) measures. A survey was conducted to assess the existing IPC practices across a network of Indian Hospitals using the World Health Organization designed self-assessment IPC Assessment Framework (IPCAF) tool. METHODS: This was a cross sectional observation study. Thirty-two tertiary care public and private facilities, part of the existing Indian HAI surveillance network was included. Data collected was analyzed by a central team at All India Institute of Medical Sciences, New Delhi, a tertiary care hospital of India. The WHO questionnaire tool was used to understand the capacity and efforts to implement IPC practices across the network. RESULTS: The overall median score of IPCAF across the network was 620. Based on the final IPCAF score of the facilities; 13% hospitals had basic IPC practices, 28% hospitals had intermediate and 59% hospitals had advanced IPC practices. The component multimodal strategies had the broadest range of score while the component IPC guidelines had the narrowest one. CONCLUSIONS: Quality improvement training for IPC nurses and healthcare professionals are needed to be provided to health facilities.

Validation of Pefloxacin for detection of fluoroquinolone (FQ) resistance among Salmonella Typhi with special reference to GyrB mutations.

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml-1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella.Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species.Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations.Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005-2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30µg), ciprofloxacin (5µg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi.Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml-1) and non-susceptible (≥0.125 µg ml-1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type.Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus-baumannii Complex and Associated Carbapenem Resistance.

Background: Acinetobacter calcoaceticus-baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S-23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/µl and 1 ng/µl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species. Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

Evaluating the efficacy of a multistrain probiotic supplementation for prevention of neonatal sepsis in 0-2-month-old low birth weight infants in India-the "ProSPoNS" Study protocol for a phase III, multicentric, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Progress has been made in the reduction of under-five mortality in India; however, neonatal mortality is reducing at a slower rate. Efforts are required to bring down neonatal mortality in order to attain the Sustainable Development Goal-3. Prevention of sepsis among the high-risk, vulnerable low birth weight neonates by a newer intervention with probiotic supplementation is promising. METHODS: A phase III, multicenter, randomized, double-blind, placebo-controlled study is being conducted at six sites in India. A total of 6144 healthy low birth weight (LBW) infants fulfilling the eligibility criteria would be enrolled within the first week of life, after obtaining written informed consent from the parents of the infant. Randomization in 1:1 ratio, stratified by site, sex, and birth weight, would be done through an interactive web response system (IWRS) using a standard web browser and email service. Vivomixx®, a probiotic containing a mix of 8 strains of bacteria, in a suspension form standardized to deliver 10 billion CFU/ml, or an organoleptically similar placebo would be fed to enrolled infants in a 1-ml/day dose for 30 days. The follow-up of enrolled infants for 60 days would take place as per a pre-specified schedule for recording morbidities and outcome assessments at the six participating sites. Screening for morbidities would be conducted by trained field workers in the community, and sick infants would be referred to designated clinics/hospitals. A physician would examine the referred infants presenting with complaints and clinical signs, and blood samples would be collected from sick infants for diagnosis of neonatal sepsis by performing sepsis screen and blood culture. Appropriate treatment would be provided as per hospital protocol. The study would be implemented as per the MRC guideline for the management of Global Health Trials in accordance with ICH-GCP and Indian Regulatory guidelines. A contract research organization would be engaged for comprehensive monitoring and quality assurance. The final analysis would be conducted in a blinded manner as per the statistical analysis plan (SAP) to estimate the primary outcomes of sepsis, possible serious bacterial infection (PSBI), and secondary outcomes. The codes will be broken after DMC permission. The protocol has been reviewed by the Research Ethics Committee of the Liverpool School of Tropical Medicine (REC-LSTM), from Research Ethics Committees of the six subject recruitment participating sites. DISCUSSION: This adequately powered and well-designed trial would conclusively answer the question whether probiotics can prevent neonatal sepsis in the high-risk group of low birth weight infants as indicated by a pilot study in 1340 LBW infants, evidence from systematic reviews of hospital-based studies, and a primary study on healthy newborns in Orissa. Results of the study would be generalizable to India and other low-middle-income countries. TRIAL REGISTRATION: Clinical Trial Registry of India (CTRI) CTRI/2019/05/019197 . Registered on 16 May 2019.

Bacterial Colonization of Vagina in Indian Women During Labor and Its Association With Puerperal and Neonatal Sepsis: A Tertiary Hospital Study.

Objective The aim was to find the prevalence of colonization of vagina with aerobic bacteria among low-risk Indian women in active labor and its association with early-onset neonatal sepsis (EONS) and puerperal sepsis. Methods The study was conducted prospectively from October 2018 to March 2020 in a tertiary hospital in New Delhi, India. Low-risk pregnant women (N=920) in active labor with intact membranes were recruited. High vaginal swabs were collected, cultured by standard methods to detect aerobic bacteria. The primary outcomes were the development of puerperal sepsis and EONS. Results In a total of 920 low-risk subjects, vaginal colonization was found in 484 (52.6%), coagulase-negative Staphylococcus being the predominant colonizer (13.2%) followed by Escherichia coli (8.9%). Multigravida women were at 1.4 times higher risk of colonization than primigravida (odds ratio [OR] 1.399; 95% CI 1.064, 1.84). Women whose sample was collected at the first vaginal examination were at 0.34 times lower risk of colonization as compared to women with more than one vaginal examination (OR 0.34; 95% CI 0.241, 0.481). The incidence of colonization increased with progressive vaginal examinations (p<0.001). None of the colonized women and their neonates developed puerperal sepsis or EONS, respectively. Conclusion Vaginal colonization of aerobic bacteria in active labor is not associated with an increased risk of puerperal sepsis or EONS.

Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India.

Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance. Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools. Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to >128 µg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87-90, del263-264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; ß-lactamase genes blaNDM-1-like , blaOXA-48-like , and blaCTX-M-like were detected in eight, five, and nine isolates, respectively. Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.