Shalini Anandan

Rapid Screening for Carbapenem Resistant Organisms: Current Results and Future Approaches

Carbapenem producing Enterobacteriaceae (CPE) is a major public health threat. A total of 120 carbapenem resistant E.coli (n=32) and K.pneumoniae (n=88) from blood stream infections were screened for the presence of carbapenem resistant genes KPC, NDM, IMP, VIM, and OXA-48 like using both conventional multiplex PCR and Xpert(®) Carba-R test. Additionally 26 faeces samples were directly screened with Xpert(®) Carba-R test. Of the tested isolates, 40% (n=48) of NDM and 39.2% (n=47/) of OXA-48-like were identified. Co-production of OXA-48 and NDM was seen in 15 (12.5%) isolates. In Xpert(®) Carba-R test, only NDM was identified in 55% (n=66) of tested isolates. Of the tested faeces samples, 12 were identified as carbapenemase producers: nine with NDM, two with the co-production of NDM and VIM and in Klebsiella spp (n=1), NDM and KPC co-production was seen. However, Xpert(®) Carba-R test fails to detect OXA-48 like as compared with multiplex PCR. The sensitivity, specificity, PPV, NPV of Xpert(®) Carba-R test was 100%, 77%, 96% and 100% respectively. Incorporation of OXA-48 like specific sequence in the panel of Xpert(®) Carba-R test may improve its sensitivity and maximize the coverage of assay.

Laboratory detection and clinical implication of oxacillinase-48 like carbapenemase: The hidden threat

Carbapenemase producing Gram-negative pathogen is of great concern for physician. The challenging aspects are treatment option and infection control. Monitoring of respective carbapenemase resistance mechanism is necessary to prevent the outbreaks. Currently, the rapid emergence of oxacillinase (OXA-48) like is alarming. Increasing frequency of OXA-48 is seen than the classical carbapenemase (KPC, NDM, IMP, and VIM) across the world. The blaOXA-48 gene is commonly identified in Escherichia coli and Klebsiella pneumoniae. The transferrable plasmid of OXA-48 is associated with rapid spread and inter-species dissemination. In general, OXA-48-like enzymes weakly hydrolyzes both carbapenem and broad spectrum cephalosporins. Except OXA-163, which effectively hydrolyze cephalosporin. This poor hydrolytic profile obscures the detection of OXA-48-like. It may go undetected in routine diagnosis and complicates the treatment option. Co-production of OXA-48-like with CTX-M-15 and other carbapenemase (NDM, VIM) leads to the emergence of multidrug resistant strains.

Haemophilus parainfluenzae: report of an unusual cause of neonatal sepsis and a literature review

Haemphilus parainfluenzae, an unusual cause of early-onset neonatal sepsis, is rarely reported. Risk factors for this serious infection include prolonged rupture of membranes, choriamnionitis, and prematurity. A high index of suspicion, proper culture techniques, and rapid species identification are needed to diagnose H. parainfluenzae sepsis. We present the first documented case from India with a review of the literature.

Biofilm Formation and Motility Depend on the Nature of the Acinetobacter baumannii Clinical Isolates

Acinetobacter baumannii is a nosocomial pathogen involved in various infections ranging from minor soft-tissue infections to more severe infections such as ventilator-associated pneumonia and bacteremia. The severity and the type of infections depend on the genetic and phenotypic variations of the strains. In this study, we compared the extent of biofilm formation and motility displayed by 60 multidrug-resistant A. baumannii clinical strains isolated from blood and sputum samples from patients from Southern India. Our results showed that isolates from the sputum samples formed significantly more robust biofilm compared to the blood isolates. On the other hand, we observed that the blood isolates were more motile than the sputum isolates. To the best of our knowledge, this is the first study that systematically evaluated the correlation between these two phenotypic traits and the nature of the isolates.

Draft genome sequence of blaTEM-1-mediated cephalosporin-resistant Salmonella enterica serovar Typhi from bloodstream infection

Enteric fever is a major cause of concern in developing countries across the globe. The primary choice of antibiotics remains fluoroquinolones, followed by cephalosporins. Resistance to third-generation cephalosporins is rarely reported in Salmonella enterica serovar Typhi. This study reports the whole genome sequence of an S. Typhi isolate resistant to cefixime [minimum inhibitory concentration (MIC)=512μg/mL] by microbroth dilution. Interestingly, the isolate was negative for the cephalosporin resistance gene blaCTX-M by PCR, which is a known mechanism for higher cephalosporin resistance. The isolate was further subjected to next-generation sequencing that identified blaTEM-1B and blaDHA-1 genes in association with qnrB4 and sul1. blaTEM is a known gene coding for β-lactam resistance. In certain cases, overexpression of blaTEM was reported to result in cephalosporin resistance. This suggests that the high cefixime MIC would have been contributed by overexpression of blaTEM-1B. The blaTEM-1B gene was found to be associated with a promoter Px with -35 and -10 regions as TTAATA and TAAAGT, respectively. The promoter regions were unique, but the -10 region was similar to that found in Pa/Pb (previously reported promoter for blaTEM) with a single nucleotide change. In addition, an IncN plasmid was identified, which is usually reported in association with the most prevalent extended-spectrum β-lactamase (ESBL), metallo- and non-metallo-carbapenemase, and plasmid-mediated quinolone resistance (PMQR) genes. Plasmids such as IncN might possibly confer resistance and enhance spread. It is imperative to continuously monitor the drug resistance profile and evolving genetic elements.

Group A rotavirus gastroenteritis in older children and adults at a hospital in southern India.

There is limited data on the spectrum and prevalence of rotavirus genotypes in older children and adults in Asia. This pilot study conducted between November 2012 and April 2013 tested for rotavirus in older children (>12 years of age), and adults with gastroenteritis from southern India. Stool samples from patients who were hospitalized or attended out-patient units with diarrhea were screened for rotavirus using Premier™ Rotaclone(®). Confirmatory testing was by another antigen detection sandwich, in-house ELISA, based on capture by a polyclonal serum and VP6 PCR. Genotyping for VP7 and VP4 was done using hemi-nested PCRs for G- and P-types circulating in India. A total of 626 stool samples from older children and adults were screened and 52 (8.4%) were initially positive for rotavirus by antigen detection. A high proportion of samples (27/51) were found to be false positives on confirmatory testing. Of the 23 samples for which genotyping results were obtained, G1P[8] was the most common genotype. There was one each of G1P[6], G1P[4] and two strains of G9P[4] while one sample showed mixed genotypes of G2 and G9P[4]. This study shows that group A rotavirus is found in 3.8% of diarrheal specimens in older children and adults with gastroenteritis in southern India and that common genotypes circulate in children and adults.

Determination of MIC distribution of arbekacin, cefminox, fosfomycin, biapenem and other antibiotics against gram-negative clinical isolates in South India: a prospective study.

Objectives To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates. Methods We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method. Results Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested. Conclusion Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.

Effectiveness of 7.5 percent povidone iodine in comparison to 1 percent clotrimazole with lignocaine in the treatment of otomycosis.

Objectives. Otomycosis is a common ENT disease frequenting the tropics. Its recurrent nature poses a great challenge to the treating physician. In spite of a number of antifungals in the market, the frequent nature of this disease warrants repeated use of these drugs, contributing to drug resistance and financial burden on the rural population. Our primary aims were to evaluate the effectiveness of povidone iodine in the treatment of otomycosis and to identify the most common fungal isolate in our population. Study Design and Setting. A single blinded prospective longitudinal study was done over a period of 12 months in a tertiary referral center. 34 patients in the age group 15–70 years clinically diagnosed with otomycosis were included in this study. These individuals were divided into two groups selected randomly. One arm received 7.5% povidone iodine otic drops and the other 1% Clotrimazole and lignocaine drops. Evaluation was based on resolution of symptoms and signs after treatment. Result. Both arms showed improvements which were comparable thus suggesting the role of povidone iodine in the management of otomycosis. Conclusion. Povidone iodine is an effective antifungal in the treatment of otomycosis.

Evaluation of IgM ELISA using a sonicate and a lipopolysaccharide antigen for the serodiagnosis of melioidosis

Melioidosis, caused by Burkholderia pseudomallei, has variable manifestations. The disease can present as an acute or a chronic form or localized or disseminated or can remain latent for many years. Acute septicaemic melioidosis has a high fatality rate when untreated and therefore, an early diagnosis is critical. Lack of testing facilities and of an awareness of the manifestations of the disease makes it likely that it is underreported in India. A sonicate and a lipopolysaccharide (LPS) antigen were evaluated by an IgM enzyme immunoassay in patients with culture-confirmed melioidosis (n = 29), fever of unknown origin (n = 214) and healthy controls (n = 109). Patients with melioidosis had significantly higher optical density values than both control categories, but the sensitivity of both tests was low (25% for sonicate, 62% for LPS). These data highlight the problems with serodiagnosis in endemic settings, where high cut-off values are required for specificity, and result in low sensitivity.

A Pilot Study on Carbapenemase Detection: Do We See the Same Level of Agreement as with the CLSI Observations

INTRODUCTION Rapid identification of carbapenemase producing organisms is of great importance for timely detection, treatment and implementation of control measures to prevent the spread. The Modified Hodge Test (MHT) and Carba NP test is recommended by CLSI for the detection of carbapenemases in Enterobacteriaceae. However, MHT may give false positive results or fail to detect metallo β-lactamases (MBLs). In the US, MHT is the most widely used test for detection of carbapenemases and has been found to have a sensitivity and specificity of >90% for bla KPC producers. However, in India, the prevalence of bla NDM is higher than bla KPC producers. AIM To evaluate the usefulness of CarbaNP in an Indian setting. MATERIALS AND METHODS A total of 260 isolates of carbapenem resistant E.coli (n=57), Klebsiella spp. (n=85), Pseudomonas aeruginosa (n=60), and Acinetobacter baumannii (58) isolated from clinical specimens between 2012-2014 at the Christian Medical College, Vellore were included in the study. All the carbapenem resistant isolates were subjected to CarbaNP, MHT and multiplex PCR for detection of carbapenemase genes. RESULTS CarbaNP was found to be positive in 88% (n=50/57), 81% (n=69/51), 38% (n=23/60) and 81% (n=47/58) for E.coli, Klebsiella spp., P. aeruginosa, and A. baumannii respectively. While in MHT it showed, 89% (n=51/57) and 81 % (n=69/85) for E.coli and Klebsiella spp. respectively. In P.aeruginosa, synergy testing of imipenem plus cloxacillin showed that, 65% of CarbaNP negatives were ampC producers. Overall, the sensitivity and specificity of CarbaNP was found to be 94% and 100 for bla NDM; 77% and 100 % for bla OXA-48 like producers and 81% and 100% for CarbAcinetoNP respectively. CONCLUSION This observation was more than what was reported in CLSI guidelines. Therefore, it is advisable to evaluate an assay for better laboratory diagnosis at respective regions.