Metagenomic next-generation sequencing in clinical microbiology Jobin John Jacob, Balaji Veeraraghavan, Karthick Vasudevan Indian Journal of Medical Microbiology 2019 37(2):133-140

Pneumococcal conjugate vaccine rollout in India: Expectations and challenges Rosemol Varghese, Balaji Veeraraghavan, Yuvraj Jeyaraman, Girish Kumar, Narendra Kumar Arora, S Balasubramanian Indian Journal of Medical Microbiology 2019 37(2):141-146 India is one among the four Asian countries with the greatest number of deaths due to pneumococcal infection among children under 5 years. pneumococcal conjugate vaccine (PCV) has been introduced in a phased manner in five major Indian states. Ambiguity remains in choosing the appropriate type of PCV and optimum schedule with maximum effectiveness specific for each country. Here, we discuss the evidences with respect to serotype coverage, immunogenicity, reactogenicity and dosage schedule for introduction of PCV13 in India. In addition, the expected PCV impact and the challenges are detailed. PCV13 is expected to provide >75% serotype coverage for invasive pneumococcal disease (IPD) serotypes in Indian children combined with the replacement by nonvaccine serotypes which is unpredictable due to lack of complete data. Nasopharyngeal (NP) surveillance is easy, feasible and can replace IPD surveillance in resource-poor settings. Continuous IPD as well as NP surveillance in all the regions are necessary to assess the impact of PCV in India.

Current strategy for local- to global-level molecular epidemiological characterisation of global antimicrobial resistance surveillance system pathogens Dhiviya Prabaa Muthuirulandi Sethuvel, Naveen Kumar Devanga Ragupathi, Yamuna Devi Bakthavatchalam, Saranya Vijayakumar, Rosemol Varghese, Chaitra Shankar, Jobin John Jacob, Karthick Vasudevan, Divyaa Elangovan, Veeraraghavan Balaji Indian Journal of Medical Microbiology 2019 37(2):147-162 The prime goal of molecular epidemiology is to identify the origin and evolution of pathogens, which can potentially influence the public health worldwide. Traditional methods provide limited information which is not sufficient for outbreak investigation and studying transmission dynamics. The recent advancement of next-generation sequencing had a major impact on molecular epidemiological studies. Currently, whole-genome sequencing (WGS) has become the gold standard typing method, especially for clinically significant pathogens. Here, we aimed to describe the application of appropriate molecular typing methods for global antimicrobial resistance surveillance system pathogens based on the level of discrimination and epidemiological settings. This shows that sequence-based methods such as multi-locus sequence typing (MLST) are widely used due to cost-effectiveness and database accessibility. However, WGS is the only method of choice for studying Escherichia coli and Shigella spp. WGS is shown to have higher discrimination than other methods in typing Klebsiella pneumoniae, Acinetobacter baumannii and Salmonella spp. due to its changing accessory genome content. For Gram positives such as Streptococcus pneumoniae, WGS would be preferable to understand the evolution of the strains. Similarly, for Staphylococcus aureus, combination of MLST, staphylococcal protein A or SCCmec typing along with WGS could be the choice for epidemiological typing of hospital- and community-acquired strains. This review highlights that combinations of different typing methods should be used to get complete information since no one standalone method is sufficient to study the varying genome diversity.

Experience of Indian association of medical microbiology external quality assurance scheme centre, New Delhi: Challenges and quality assessment of clinical microbiology laboratories Chand Wattal, Jaswinder Kaur Oberoi, Neeraj Goel, Sanghamitra Datta, Reena Raveendran, KJ Prasad Indian Journal of Medical Microbiology 2019 37(2):163-172 Introduction: EQAS program at New Delhi under IAMM was started in January 2014 across North and North east regions of India with 217 participants, which grew up to 540 by 2018. Materials and Methods: In 2014, 4 analytes per year were sent for 3 exercises, i.e. smear culture and serology. 2018 onwards PT analytes were increased from 4 to 12 and comparative performance of techniques analysed. Results: Out of the 22 smears sent for gram staining, ZN staining, Kinyoun staining and Albert staining, completely correct results ranged between 29.55% - 79.9%, 94.3% - 99.2%, 35.5% & 93.8%, respectively. Correct results for culture isolate identification & susceptibility testing and serology exercises varied between 70 & 92.4% and 73.1 & 98.59%, respectively. In the year 2018, 470 responses were received for bacterial culture identification & antibiotic susceptibility testing out of which manual and automated systems were used by 54% & 46% and 52.5% & 47.5% participants, respectively. Techniques used in BBV assays for HBsAg, HCV & HIV found all methods like ELISA, ELFA, CLIA and Card Test performing similarly. The major challenges in running the EQA program included requirement of large amount of specimens for PT item preparation, stability in hot and humid conditions and timely delivery of PT challenges in remote parts of the country. Conclusion: A large number of the participating laboratories (77%) had an overall score of >80% for all exercises, demonstrating acceptable baseline performance of EQAS registered laboratories. However, continued EQAS participation could further improve the quality of results.

RNA-seq analysis reveals resistome genes and signalling pathway associated with vancomycin-intermediate Staphylococcus aureus Devika Subramanian, Jeyakumar Natarajan Indian Journal of Medical Microbiology 2019 37(2):173-185 Context: Vancomycin-intermediate Staphylococcus aureus remains one of the most prevalent multidrug-resistant pathogens causing healthcare infections that are difficult to treat. Aims: This study uses a comprehensive computational analysis to systematically investigate various gene expression profiles of resistant and sensitive S. aureus strains on exposure to antibiotics. Settings and Design: The transcriptional changes leading to the development of multiple antibiotic resistance were examined by an integrative analysis of nine differential expression experiments under selected conditions of vancomycin-intermediate and -sensitive strains for four different antibiotics using publicly available RNA-Seq datasets. Materials and Methods: For each antibiotic, three experimental conditions for expression analysis were selected to identify those genes that are particularly involved in the development of resistance. The results were further scrutinised to generate a resistome that can be analysed for their role in the development or adaptation to antibiotic resistance. Results: The 99 genes in the resistome are then compiled to create a multiple drug resistome of 25 known and novel genes identified to play a part in antibiotic resistance. The inclusion of agr genes and associated virulence factors in the identified resistome supports the role of agr quorum sensing system in multiple drug resistance. In addition, enrichment analysis also identified the kyoto encyclopedia of genes and genomes (KEGG) pathways – quorum sensing and two-component system pathways – in the resistome gene set. Conclusion: Further studies on understanding the role of the identified molecular targets such as SAA6008_00181, SAA6008_01127, agrA, agrC and coa in adapting to the pressure of antibiotics at sub-inhibitory concentrations can help in learning the molecular mechanisms causing resistance to the pathogens as well as finding other potential therapeutics.

Clinico-microbiological analysis of toxigenic clostridium difficile from hospitalised patients in a tertiary care hospital, Mangalore, Karnataka, India Sherin Justin, Beena Antony Indian Journal of Medical Microbiology 2019 37(2):186-191 Purpose: Prevalence of Clostridium difficile, an anaerobic, Gram-positive, spore-forming bacillus, is very much underestimated in India. The present study was intended to assess the burden of toxigenic C. difficile in hospitalised patients with clinically significant diarrhoea and analysis of their clinical picture. Materials and Methods: This cross-sectional study was conducted in a tertiary care teaching hospital, South India, from January 2012 to December 2014. Stool samples were collected consecutively from 563 inpatients from various wards. The prevalence of toxigenic C. difficile was determined by toxigenic culture and a two-step algorithm. The clinical spectrum of these patients was also analysed. Associated pathogens were identified using standard procedures. Statistical analysis was done by frequency, percentage, Chi-square test and z-test. Results: Out of the 563 stool samples analysed, the prevalence of toxigenic C. difficile was 12.79% and that of non-toxigenic C. difficile was 10.83%. The prevalence of toxigenic C. difficile among oncology patients was highly significant (HS). Antibiotic treatment, prolonged hospital stay and underlying diseases/conditions were the risk factors which were HS, and fever was the significant clinical feature among the patients. Escherichia coli was the predominant associated pathogen isolated (18.47%). Conclusion: The presence of toxigenic C. difficile in our locality is a matter of concern. Constant supervision, appropriate treatment and preventive measures are crucial in controlling C. difficile infection.

Assessment of efficacy of palm polymerase chain reaction with microscopy, rapid diagnostic test and conventional polymerase chain reaction for diagnosis of malaria Paras Mahale, Rajas Warke, Mira Ramaiya, Deepa Balasubramanian, Suvin Shetty, Ranjit Mankeshwar, Abhay Chowdhary Indian Journal of Medical Microbiology 2019 37(2):192-197 Purpose: Sensitive, specific, rapid and cost-effective technique for malaria diagnosis is need of the hour. Microscopy has been the gold standard for malaria diagnosis, but its interpersonnel variability and lack of sensitivity make it subjective test. Conventional polymerase chain reaction (cPCR) has proven to be sensitive technique, but costly and time-consuming. Considering these factors, we have compared microscopy and cPCR with newly derives ultra-fast, portable PCR machine called Palm PCR. Materials and Methods: Palm PCR is arranged with three heat blocks precisely made for three stages of PCR cycles with 34 min for 1100 bp Plasmodium genus outer primer to amplify and 10 min each for Plasmodium falciparum and Plasmodium vivax inner primers of 120 bp and 205 bp, respectively. A total of 191 suspected samples were processed and evaluated using receiver operating characteristic (ROC) curve analysis. Results: The area under ROC curve analysis for Palm PCR with reference standard microscopy for P. falciparum, P. vivax and Plasmodium was 0.8969, 0.9121 and 0.9116, respectively, and with reference standard cPCR was 1.0 for all of them. ROC curve area close of suggests that Palm PCR can be as significant as cPCR in malaria diagnosis. In fact, ultra-rapid amplification with same precision makes Palm PCR better technique than cPCR. Conclusion: Palm PCR is sensitive, rapid and works on battery with simple laboratory facility requirements. Portable electrophoresis and transilluminator combined with Palm PCR could be implemented as an important diagnostic tool in resource-limited and rural areas. Similar studies with wider parameters in rural areas will help us evaluate and maybe establish Palm PCR as PCR platform of choice for such specific set-ups.

An emerging threat of ceftriaxone-resistant non-typhoidal salmonella in South India: Incidence and molecular profile Agila Kumari Pragasam, Shalini Anandan, James John, Ayyanraj Neeravi, Vignesh Narasimman, Dhiviya Prabaa Muthuirulandi Sethuvel, Divyaa Elangovan, Balaji Veeraraghavan Indian Journal of Medical Microbiology 2019 37(2):198-202 Background: Non-typhoidal Salmonella (NTS) infection is a serious public health problem globally. Although NTS infections are self-limited, antimicrobial therapy is recommended for severe infections and immunocompromised patients. Antimicrobial resistance (AMR) in these pathogens further limits its therapeutic options. Here, we report an incidence of ceftriaxone resistance in NTS over the past 9 years in a southern Indian region. Materials and Methods: Molecular mechanisms of resistance in ceftriaxone-resistant NTS have been tested by both phenotypic and molecular methods. Minimum inhibitory concentration was determined by the E-test and broth microdilution method. AMR gene markers of β-lactamases such as AmpCs (blaMOX, blaCMY, blaDHA, blaFOX, blaACC and blaACT) and extended-spectrum β-lactamases (ESBLs) (blaSHV, blaTEM, blaVEB, blaPER, blaCTXM-1like,blaCTXM-2like, blaCTXM-8like, blaCTXM-9like and blaCTXM-25like) were screened. The presence of IncH12 and IncI1 plasmid was also analysed. Results: The study reports a 5% prevalence of ceftriaxone resistance in NTS. The most common serogroup was Salmonella Group B followed by Salmonella Group E and Salmonella group C1/C2. The occurrence of blaCTX-M-1, blaTEM, blaCMY and blaSHV genes was observed in 54%, 54%, 48% and 3% of the isolates, respectively. Interestingly, few isolates carried dual resistance genes (ESBLs and AmpCs). IncH12 and IncI1 plasmid was identified in isolates carrying ESBL and AmpC genes, respectively. Conclusion: This study shows that ceftriaxone resistance is mainly mediated by β-lactamases such as ESBL and AmpC. As the incidence of ceftriaxone resistance is rising gradually over the years, it is imperative to monitor the AMR in this species.

Review of a 7-year record of the bacteriological profile of airway secretions of children with cystic fibrosis in North India Vikas Gautam, Parinitha Kaza, Joseph L Mathew, Varpreet Kaur, Megha Sharma, Pallab Ray Indian Journal of Medical Microbiology 2019 37(2):203-209 Background: Cystic fibrosis (CF) is now a recognised entity in India, with prevalence rates between 1/10,000 and 1/50,000. However, no data were available with regard to the profile of respiratory pathogens in the Indian setting. Materials and Methods: The records of respiratory secretion bacterial cultures of children with CF in a tertiary care hospital in North India from January 2010 to December 2016 were reviewed. Culture data were evaluated; the organisms were noted and their antimicrobial susceptibilities were analysed. The microbiological profile and antimicrobial susceptibility pattern of CF patients were evaluated. Results: A total of 445 samples from 146 children were processed, of which 246 (55%) samples showed bacterial growth. Mixed infections 48 (19.5%) were common in older children. Children aged 3–6 months (62.5%) showed the highest culture positivity. The most commonly isolated organisms were Pseudomonas aeruginosa (52.6%) and Staphylococcus aureus. Children with initial cultures positive for P. aeruginosa had 55% of their subsequent cultures showing polymicrobial infections. P. aeruginosa was most susceptible to ciprofloxacin (89%) and piperacillin-tazobactum (88%). Among the staphylococcal isolates, 38% were methicillin-resistant S. aureus (MRSA). The percentage of MRSA increased from 66% in 2010 to 75% in 2012, followed by a decline to 24% in 2016. Conclusions: The pattern of airway colonisation in the Indian setting is different from the Caucasian population, and P. aeruginosa and Burkholderia cepacia complex appear early. Colonisation with P. aeruginosa benefits from therapy. In case of infection, care must be taken while initiating empiric therapy. It should be based on local antibiograms to prevent the emergence of resistant microbes.

Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae PA Remya, M Shanthi, Uma Sekar Indian Journal of Medical Microbiology 2019 37(2):210-218 Purpose: This study was undertaken to characterise the virulence factors in clinical strains of Klebsiella pneumoniae and analyse their association with various infections caused and also to determine the association between virulence factors and antimicrobial resistance profile. Materials and Methods: A total number of 370 clinically significant, non-duplicate isolates of K. pneumoniae isolated from both hospitalised patients and patients attending clinics were included in this study. Polymerase chain reaction (PCR) was carried out for the detection of various virulence genes such as mucoviscosity-associated gene A (magA), gene associated with allantoin metabolism (allS), Klebsiella ferric iron uptake(Kfu), capsule-associated gene A (K2A), regulator of mucoid phenotype A (rmpA), enterobactin (entB), yersiniabactin (YbtS), aerobactin, Fimbrial adhesin (FimH) and uridine-diphosphate galacturonate 4-epimerase (uge). Antimicrobial susceptibility testing and PCR-based detection of beta-lactamase-encoding genes such as extended-spectrum beta-lactamases, AmpCs and carbapenemases were performed. Univariate analysis was done to find the association between virulence genes and mortality. Results: The siderophore, entB, was present in most (90.5%) of the isolates. Of the 370 isolates, 345 carried multiple virulence genes; 15 harboured single virulence genes and 10 did not harbour any of the studied virulence genes. The most common combination of occurrence was entB and FimH. A mortality rate of 12.75% (38/298) was observed among hospitalised patients. None of the virulence genes had any significant association with mortality. Conclusion: Pathogenic K. pneumoniae can harbour single to multiple virulence genes. Invasive infection with even a single virulence gene-harbouring K. pneumoniae can lead to poor outcomes. Both multidrug-resistant (MDR) and non-MDR K. pneumoniae can harbour a variety of virulence genes. None of the virulence genes have a significant association with mortality.