Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India.

Background: Surveillance of antimicrobial resistance (AMR) is of great importance. Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens and emergence of resistance in these have increased the morbidity and mortality rates. This surveillance study was initiated by the Government of India ‑ Indian Council of Medical Research. The aim of this study is to determine the antimicrobial susceptibility profile and to characterise the enzyme mediated antimicrobial resistance such as extended spectrum beta‑lactamases (ESBLs) and carbapenemases among multidrug‑resistant (MDR) P. aeruginosa and A. baumannii. Materials and Methods: A multi‑centric study was conducted from January 2014 to December 2015 with a total number of 240 MDR P. aeruginosa and 312 MDR A. baumannii isolated from blood, cerebrospinal fluid, respiratory, pus, urine and intra‑abdominal infections. Kirby–Bauer disc diffusion was done to determine the antimicrobial susceptibility profile. Further, MDR isolates were characterised by multiplex polymerase chain reaction to determine the resistance genes for ESBLs and carbapenemases. Results: Among the ESBLs, blaVEB (23%), blaTEM (5%) and blaSHV (0.4%) in P. aeruginosa and blaPER (54%), blaTEM (16%) and blaSHV (1%) in A. baumannii were the most prevalent. Likewise, blaVIM (37%), blaNDM (14%), blaGES (8%) and blaIMP (2%) in P. aeruginosa and blaOXA‑23like (98%), blaOXA‑58like (2%), blaNDM (22%) and blaVIM (3%) in A. baumannii were found to be the most prevalent carbapenemases. blaOXA‑51like gene, intrinsic to A. baumannii was present in all the isolates tested. Conclusion: The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid‑mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.

Isolation of Campylobacter from human stool samples.

Context: Campylobacter is an undetected cause of diarrhoea especially under 5 years of age in most of the countries. Isolation of this organism is diffi cult, expensive and cumbersome. Aims: Our objective of this study was to isolate this pathogen from the stool specimens on routinely available blood containing laboratory media using the candle jar for creating the microaerophilic atmosphere in our setup. Settings and Designs: A descriptive study. Materials and Methods: A total of 50 stool samples were inoculated onto selective and non-selective media with and without fi ltration using a 0.45 μm membrane. The inoculated media were simultaneously incubated in microaerophilic conditions using the Anoxomat as well as in candle jars at temperatures 37°C and 42°C. The culture isolates were confi rmed by standard phenotypic tests. A simplex polymerase chain reaction (PCR) targeting the 16S ribosomal deoxyribonucleic acid of Campylobacter was performed on the deoxyribonucleic acid (DNA) of the culture isolates as well as on the DNA extracted from the stool fi ltrates. Statistical Analysis: Data was expressed as a proportion. Results: Campylobacter could be isolated in 5 out of 50 stool samples using both the Anoxomat as well as the candle jar. Furthermore, we did not fi nd any difference between the isolation using the selective and blood containing media as well as the different incubation temperatures. All the fi ve were confi rmed phenotypically and genotypically to be Campylobacter jejuni. The PCR results corroborated with that of the culture. Conclusions: Isolation by culture was as sensitive as that of the PCR.

Unusual manifestation of Salmonella enterica serotype enteritidis infection in a case of langerhans cell histiocytosis.

Nontyphoidal Salmonella (NTS) are established foodborne pathogens, most commonly reported in cases of gastroenteritis. These pathogens are however, increasingly being implicated in cases of bacteraemia and other extraintestinal manifestations. We report a case of a scalp abscess due to Salmonella enterica serotype Enteritidis, which is a serotype of NTS, in a child suffering from a haematologic malignancy. The child was on steroid and anticancer chemotherapy and developed the abscess secondary to bacteraemia with Salmonella Enteritidis. The abscess was drained and resolved following a course of intravenous antibiotic treatment.

Early diagnosis of typhoid fever by nested PCR for flagellin gene of Salmonella enterica serotype Typhi.

Background & objectives: Typhoid fever caused by Salmonella Typhi continues to be a major health problem in spite of the use of antibiotics and the development of newer antibacterial drugs. Inability to make an early laboratory diagnosis and resort to empirical therapy, often lead to increased morbidity and mortality in cases of typhoid fever. This study was aimed to optimize a nested PCR for early diagnosis of typhoid fever and using it as a diagnostic tool in culture negative cases of suspected typhoid fever. Methods: Eighty patients with clinical diagnosis of typhoid fever and 40 controls were included in the study. The blood samples collected were subjected to culture, Widal and nested PCR targeting the flagellin gene of S. Typhi. Results: The sensitivity of PCR on blood was found to be 100 per cent whereas the specificity was 76.9 per cent. The positive predictive value (PPV) of PCR was calculated to be 76.9 per cent with an accuracy of 86 per cent. None of the 40 control samples gave a positive PCR. Interpretation & conclusions: Due to its high sensitivity and specificity nested PCR can be used as a useful tool to diagnose clinically suspected, culture negative cases of typhoid fever.

Fatal meningitis caused by vancomycin-resistant enterococci: Report of two cases from south India.

Vancomycin-resistant enterococci rarely cause meningitis and present a therapeutic challenge. Antimicrobial susceptibility testing was done for strains of Enterococcus species isolated from CSF samples of patients with meningitis by phenotypic methods. Multiplex polymerase chain reaction was performed to determine the genetic basis of vancomycin resistance of such isolates. We report here two cases of enterococcal meningitis caused by vancomycin-resistant Enterococcus species. One of the isolates was identified as Enterococcus faecalis and the other as Enterococcus gallinarum. We also report the simultaneous presence of vanC1 and vanA resistance genes in the strain of E. gallinarum. To the best of our knowledge, this is the first report of vanA resistance gene in an isolate of E. gallinarum from the Indian subcontinent. This is also the first Indian report of vancomycin-resistant Enterococcus causing meningitis.

Molecular description of plasmid-mediated AmpC β-lactamases among nosocomial isolates of Escherichia coli & Klebsiella pneumoniae from six different hospitals in India.

Background & objectives: Plasmid mediated AmpC β-lactamase (PMABL) resistance in Escherichia coli and Klebsiella spp. is an emerging problem worldwide. Phenotypic methods are commonly used for detection of PMABL production in Gram-negative isolates, but molecular data about the prevalence of plasmid-mediated AmpC-type resistance at the national level are needed. Hence, a prospective study was undertaken to determine the occurrence of PMABL gene and its types among clinical isolates of E. coli and K. pneumoniae obtained from six different hospitals in India. Methods: A total of 241 nosocomial isolates of K. pneumoniae (n=109) and E.coli (n=132) from six geographically distant hospitals in India were included. These were screened for cefoxitin resistance. AmpC disk test and modified three dimensional extraction test were used for phenotypic detection of PMABL production. Molecular types were determined by a multiplex PCR. Results: Among the 241 isolates, 187 (77.5%) were found to be cefoxitin resistant (K. pneumoniae n=83, E. coli n=104). AmpC activity was detectable in 153 (63.4%) isolates, (K. pneumoniae n=69, E. coli n=84). By PCR, the plasmid encoded AmpC genes were found in 92 (38.1%) isolates and the molecular types of the genes detected predominantly were DHA, CIT followed by MOX and ACC types. Interpretation & conclusions: A high percentage of plasmid-encoded AmpC enzymes was noted in E. coli and K. pneumonia isolates obtained from different parts of the country. Phenotypic methods alone may not reflect the true number of PMABL producers. Genotypic methods need to be employed in national surveillance studies.

Drug resistance in malaria.

Antimalarial chemotherapy is an important component of all malaria control programmes throughout the world. This is especially so in light of the fact that there are no antimalarial vaccines which are available for clinical use at present. Emergence and spread of malaria parasites which are resistant to many of the available antimalarials today is, therefore, a major cause for concern. Till date, resistance to all groups of antimalarials excluding artemisinin has been reported. In recent years, in vitro resistance to even artemisinin has been described. While resistance to antibacterial agents has come to prominence as a clinical problem in recent years, antiparasitic resistance in general and antimalarial resistance in particular has not received much attention, especially in the Indian scenario. The present review deals with commonly used antimalarial drugs and the mechanisms of resistance to them. Various methods of detecting antimalarial resistance and avoiding the same have also been dealt with. Newer parasite targets which can be used in developing newer antimalarial agents and antimalarials obtained from plants have also been mentioned.

AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India

AmpC â-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC â-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC â -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57 percent) strains were resistant to 3GC, among which 63(47 percent) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7 percent) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9 percent (51/63) of screen positive isolates were detected by Amp C disc test and 93.6 percent (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9 percent) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.

Persistent diarrhea: Risk factors and outcome.

Objective. To identify risk factors associated with Persistent diarrhea (PD) and deaths due to PD. Methods. This prospective case control study included 60 children with PD (cases) and 60 children (controls) with acute diarrhoea (AD). Detailed history, examination and appropriate investigations were done for all children. Crude Odds ratio was calculated for each risk factor by univariate analysis and adjusted odds ratio was calculated by multivariate logistic regression. Results. Prior antibiotic use, steroid use, anemia, vitamin A deficiency, malnutrition, LRI, UTI, oral candidiasis, and hyponatremia, were statistically significant risk factors by univariate analysis. Prior antibiotic use, vitamin A deficiency, malnutrition and LRI were independently associated with PD by multivariate logistic regression analysis. The risk factors for mortality were stool frequency more than 10 times per day, severe malnutrition, oral candidiasis, hypoalbuminemia and HIV positivity. Conclusions. The presence of these risk factors should alert the clinician to take appropriate measures, to decrease the mortality.

Comparison of an in-house latex agglutination test with IgM ELISA and MAT in the diagnosis of leptospirosis.

The laboratory diagnosis of leptospirosis is fraught with several problems. Isolation of Leptospira by culture has a low sensitivity and the microscopic agglutination test (MAT) is time consuming To overcome these problems, a rapid latex agglutination test (LAT) has been standardized for the detection of antileptospiral antibodies in serum samples from suspected cases of leptospirosis. We compared the efficiency of the LAT to a commercially available IgM ELISA and MAT. A total of 150 serum samples were tested by LAT, IgM ELISA, and MAT. The positivity was 26.7%, 26% and 24% respectively. The sensitivity and specificity of LAT as compared to MAT was 90.62 and 91.96% respectively. Even though LAT and ELISA showed similar results, its rapidity and simplicity made latex agglutination test more suitable as a rapid screening test.

Asymptomatic colonization of upper respiratory tract by potential bacterial pathogens.

Objective. To screen for asymptomatic respiratory carriage of S. pneumoniae, H. influenzae and Group A Streptococcus (GAS) in children attending JIPMER, correlate carriage rate with different socio-demographic factors and to detect antimicrobial resistance among the isolates. Methods. Throat swabs were collected from both in patients and out patients (≤12 yr of age) and processed. Bacteria were identified by standard techniques. Susceptibility to commonly used antimicrobial agents was determined by Kirby Bauer disc diffusion technique. Results. Overall carriage rate of respiratory pathogens was 30% with S. pneumoniae, H. influenzae and GAS accounting for 22%, 5% and 4.5% respectively. Three patients had >1 organism. Antibiotic resistance was highest in S. pneumoniae with 66.7% of strains resistant to penicillin. MDR strains were also encountered. Erythromycin resistance was observed in both H. influenzae (28.4%) and GAS (22%).No statistically significant association was found between the carriage rate of these organisms and different socio-demographic factors. Conclusions. S. pneumoniae carriage rate was comparatively higher in the community and its antimicrobial resistance is an issue to address.

Neonatal melioidosis: A case report from India.

Melioidosis, caused by Burkholderia pseudomallei , is an infectious disease of major public health importance in Southeast Asia and Australia. We report, for the first time from the Indian subcontinent, a case of melioidosis in a neonate, its clinical presentation, microbiological diagnosis, possible mode of transmission and outcome. A pre-term female baby developed respiratory distress soon after birth. The child was febrile, had tachypnea, grunting, normal heart rate with a low pulse volume and poor peripheral perfusion. Chest X-ray revealed right-sided bronchopneumonia. B. pseudomallei was isolated from the blood culture of the neonate collected aseptically. The neonate was successfully treated with meropenem.

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria.

Background & objectives: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-β-lactamases (MBLs) and AmpC β-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs. Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffi i) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby- Bauer disc diffusion method. Modifi ed Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC β-lactamases, respectively. Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modifi ed Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test. One P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase. Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection of MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria.

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishman's stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishman's thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.

Screening for methicillin-resistant Staphylococcus aureus carriers among patients and health care workers of a tertiary care hospital in south India.

A total of 200 subjects were screened for carriage of methicillin-resistant Staphylococcus aureus (MRSA) at different sites using oxacillin blood agar and mannitol salt agar with oxacillin. Overall carriage rate was 8.5%, with the highest rate in inpatients (15.6%) while the lowest was seen in health care workers (1.8%). The commonest site of colonization was the anterior nares. Oxacillin blood agar was found to be superior to mannitol salt agar with oxacillin for the isolation of MRSA. Male sex and prolonged hospital stay were found to be the major risk factors for MRSA colonization.