An integrated method for targeted Oxford Nanopore sequencing and automated bioinformatics for the simultaneous detection of bacteria, fungi, and ARG.

AIMS: The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). METHODS AND RESULTS: The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n = 12), clinical isolates (n = 114), and blood samples from patients with suspected bloodstream infections (n = 33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering ≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of >80%. For ARGs, reads with percent identity of >90% and >60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. CONCLUSION: NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.

Coagulase-negative staphylococci: Emerging pathogen in central nervous system shunt infection.

Central nervous system (CNS) shunts are commonly used to treat patients with hydrocephalus. Its placement is associated with increased risk of infection. The study was intended to evaluate infection rate associated with CNS shunt surgeries and identify risk factors for shunt infection. The frequency and characterisation of aetiological agents along with their antibiotic resistance pattern were also studied. A prospective study of 86 patients who underwent 97 surgeries over a period of 18 months was conducted. One hundred seventy-six cerebrospinal fluid samples and 44 shunt tips obtained were processed using standard microbiological techniques. Of 86 patients, 39 (45.35%) operated for shunt revision were infected while 47 patients operated for shunt insertion were not found to be infected. Methicillin-resistant Staphylococcus epidermidis was the predominant isolate. 57.58% isolates of Staphylococci were found to be biofilm producers. Mortality of 15% was observed among infected patients. Shunt infection remains a serious issue in the patients undergoing shunt surgery. Accurate diagnosis, treatment and prevention of infection are essential in such patients.

Drug resistance profiles of Mycobacterium tuberculosis isolates to first line anti-tuberculous drugs: A five years study.

BACKGROUND: Drug resistance is a major problem in the treatment of tuberculosis (TB). An estimate of drug resistance is extremely important in the epidemiology and control of TB. However, an assessment of the magnitude of drug resistance in TB is not very well described globally and data remains scantier for India. In view of this, we reviewed our data over last five years. MATERIALS AND METHODS: Six hundred and seventy-three Mycobacterium tuberculosis isolates were subjected to drug susceptibility against primary anti-tuberculosis drugs by economic variant proportion method. All isolates resistant to isoniazid and rifampicin were taken as multi-drug resistant (MDR). RESULTS: Out of the 673 strains tested, 95 (14.11%) showed monoresistance, 365 (54.23%) strains were found to be resistant to more than one drug. A total of 118 (17.53%) strains were found to be resistant to all the four drugs tested. MDR was seen with 320 (47.54%) isolates. This study observed maximum resistance with rifampicin (74.4%) followed by streptomycin (70.0%), isoniazid (53.2%), and ethambutol (21.7%). CONCLUSION: While this information may not reflect true prevalence of drug resistance in the region, this may help in further planning long term surveillance studies to know the trend of drug resistance in this area.

Change in antimicrobial resistance pattern of Salmonella Typhi in central India.

BACKGROUND & OBJECTIVES: Multidrug resistant Salmonella Typhi infections have been reported worldwide in the last decade. In a study from central India in 1991 multidrug resistant S. Typhi were reported to account for 91 per cent of patients of enteric fever. The present study was planned to study the antimicrobial susceptibility pattern, phage types and biotypes of strains of S. Typhi prevalent in this region. METHODS: A total of 54 isolates of Salmonella were recovered from 1468 blood samples of patients suspected to have enteric fever and admitted in the Government Medical College and Hospital, Nagpur. Antimicrobial susceptibility pattern of these isolates was studied by disc diffusion test. Phage typing and biotyping of the first 33 strains was done at the National Salmonella Phage Typing Center, New Delhi. Minimum inhibitory concentration of chloramphenicol was determined by agar dilution method. RESULTS: Of 54 isolates of Salmonella, 51(94%) were S. Typhi and 3 (6%) were S. paratyphi A serotype. Of the 30 isolates of S. Typhi subjected to phage typing, two phage types E1 23 (77%) and A 7 (23%) were found to be prevalent in this region. Multidrug resistance was observed in 12 (22%) strains of S. Typhi. Thirty five (68%) strains were sensitive to chloramphenicol, ampicillin, gentamicin, cotrimoxazole, cefotaxime and ciprofloxacin. Resistance to two antibiotics was observed in 4 (8%) strains. Cefotaxime resistance was observed in one isolate and gentamycin resistance in two, while none of the isolates was found to be ciprofloxacin resistant. INTERPRETATION & CONCLUSION: Multidrug resistance in S. Typhi has decreased from that reported in 1991 though there is a still a small percentage of strains which continue to be multidrug resistant.