Characteristics, outcome and risk factors for mortality of paediatric patients with ICU-acquired candidemia in India: A multicentre prospective study.

BACKGROUND: The epidemiology, clinical profile and outcome of paediatric candidemia vary considerably by age, healthcare settings and prevalent Candida species. Despite these differences, few comprehensive studies are undertaken. This nationwide study addresses this knowledge gap. METHODS: 487 children who contracted ICU-acquired candidemia at 23 Indian tertiary care centres were assessed for 398 variables spanning demography, clinical characteristics, microbiology, treatment and outcome. RESULTS: Both neonates (5.0 days; range = 3.0-9.5) and non-neonatal children (7.0 days; range = 3.0-13.0) developed candidemia early after ICU admission. Majority of neonates were premature (63.7%) with low birthweight (57.1%). Perinatal asphyxia (7.3%), pneumonia (8.2%), congenital heart disease (8.4%) and invasive procedures were common comorbidities, and antibiotic use (94.1%) was widespread. C tropicalis (24.7%) and C albicans (20.7%) dominated both age groups. Antifungal treatment (66.5%) and removal of central catheters (44.8%) lagged behind. Overall resistance was low; however, emergence of resistant C krusei and C auris needs attention. The 30-day crude mortality was 27.8% (neonates) and 29.4% (non-neonates). Logistic regression identified admission to public sector ICUs (OR = 5.64), mechanical ventilation (OR = 2.82), corticosteroid therapy (OR = 8.89) and antifungal therapy (OR = 0.22) as independent predictors of 30-day crude mortality in neonates. Similarly, admission to public sector ICUs (OR = 3.62), mechanical ventilation (OR = 3.13), exposure to carbapenems (OR = 2.18) and azole antifungal therapy (OR = 0.48) were independent predictors for non-neonates. CONCLUSIONS: Our findings reveal a distinct epidemiology, including early infection with a different spectrum of Candida species, calling for appropriate intervention strategies to reduce candidemia morbidity and mortality. Independent factors identified in our regression models can help tackle these challenges.

Investigational approach to adenoviral conjunctivitis: comparison of three diagnostic tests using a Bayesian latent class model.

INTRODUCTION: Highly contagious adenoviral conjunctivitis represents 15-70% of all conjunctivitis worldwide. Human adenovirus (hAdV) serotypes 3,4,7,8,19 and 37 contributes to 89% of all adenoviral conjunctivitis. Accurate and rapid diagnosis of adenoviral infections at serotype level could prevent misdiagnosis, spread of disease, unnecessary antibiotic use and increased treatment costs. METHODOLOGY: Sixty-two suspected viral conjunctivitis cases were recruited from November2013-January2015. Swabs collected from inferior palpebral conjunctiva and processed for viral culture (Hep2 cell line), immunofluorescence assay (IFA) and polymerase chain reaction (PCR) (targeting hexon gene). Serotype 3,4,7,8,19 and 37 identification was carried out with an optimized multiplex-PCR (based on hypervariable region of hexon gene) and confirmed by sequence analysis. Bayesian Latent Class Model (LCM) analysis was used to compare sensitivity and specificity of three tests. RESULTS: Adenovirus was detected in 54.8% (34/62) of cases by combination of all three methods. Culture was positive in 23/34 cases (67.6%). PCR and IFA detected adenovirus in 24 (70.5%) and 21 (61.7%) cases respectively. LCM analysis revealed, sensitivity and specificity of PCR, Culture and IFA was 77.8% and 92.4%; 72.2% and 90.8%; 67.6% and 92.9% respectively. Serotyping by multiplex-PCR showed, two cases each were hAdV3 and hAdV4, 18 hAdV8 and two remained unidentified. Results of Multiplex-PCR and sequence analysis showed 100% concordance Conclusion: LCM analysis revealed, PCR is the most appropriate method for identification. Multiplex-PCR is a simple and rapid method (serotypes identification within two days); owing its short turnaround time and accuracy, it can be used as a diagnostic tool for surveillance of adenoviral keratoconjunctivitis.

Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria.

PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-ß-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

Inhibition of quorum sensing-controlled biofilm formation in Pseudomonas aeruginosa by quorum-sensing inhibitors.

Antimicrobial therapy against extensively drug-resistant (XDR) P. aeruginosa biofilms is less efficient compared to the treatment of equal bacterial counts of free-floating planktonic cells, which has become a serious threat in hospital environment. P. aeruginosa regulate their cooperative activities and physiological processes through a cell to cell chemical communication process called Quorum sensing (QS). This attracted our interest to synthesize, and to chemically characterize two anti-QS compounds, N-(4-{4-fluoroanilno} butanoyl) -l-homoserine lactone (FABHL) and N-(4-{4-chlororoanilno} butanoyl) -l-homoserine lactone (CABHL) to inhibit biofilm formation via disabling the QS circuits. Structural and morphological properties of these compounds were characterized by 1H Nuclear Magnetic Resonance (NMR), 13C NMR and High-resolution mass spectrometry (HRMS). Two biofilm forming XDR P. aeruginosa isolates were included in this study. Anti-biofilm property of FABHL or CABHL was confirmed by biofilm formation assay and it was shown to occur without affecting the bacterial growth. Anti-QS property of FABHL or CABHL was determined by evaluating the expression levels of QS genes (lasR and rhlR) by quantitative real time PCR (qRT-PCR). Although, FABHL and CABHL downregulates the expression levels of QS genes, lasR expression was significantly reduced. Molecular modeling studies revealed that the binding energy of FABHL and CABHL with LasR protein was -4.27 and -4.51, respectively. Hence, the synthesized compounds have the potential to serve as a potent anti-biofilm agent via disabling the QS systems. Lethality of FABHL and CABHL against PBMCs was assessed by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphynyl tetrazolium bromide (MTT) assay. Cell viability was observed for both the compounds.

Global DNA Methylation Level among Ciprofloxacin-Resistant Clinical Isolates of Escherichia coli.

INTRODUCTION: Fluoroquinolone resistant clinical isolates belonging to the family Enterobacteriaceae is a major public health concern in India. Data analysis in JIPMER hospital revealed 10% rise in fluoroquinolone resistance within a span of three years suggestive of the possible involvement of mechanism/s other than QRDR capable of imparting fluoroquinolone resistance. DNA methylation regulates gene expression. Moreover, methylated cytosine is a mutational hotspot. Thus, DNA methylation can alter bacterial gene expression profile as well as facilitate the bacteria in accumulating mutations possibly leading to increased antimicrobial resistance. Therefore, the present study was carried out to identify the potential involvement of DNA methylation in ciprofloxacin resistance. AIM: To elucidate and compare the methylation level of genomic and plasmid DNA among clinical isolates of E. coli sensitive and resistant to ciprofloxacin. MATERIALS AND METHODS: The study included 40 clinical E. coli isolates of which, 30 were ciprofloxacin-resistant and 10 were sensitive to ciprofloxacin. Genomic DNA (gDNA) and plasmid DNA were extracted and quantified. Methylation levels were elucidated using 5-mC DNA ELISA kit (Zymoresearch, California, USA) as per kit protocol and guidelines. STATISTICAL ANALYSIS: Spearman correlation 2-tailed test. A p-value <0.05 was considered significant. RESULTS: The MIC values of sensitive and resistant strains against ciprofloxacin ranged from 0.125 µg/mL - 0.75 µg/mL and 8 µg/mL - >256 µg/mL respectively. No difference was found in plasmid DNA methylation level but, the gDNA methylation level of the resistant strains significantly differed from that of the sensitive strains. Based on Spearman correlation test gDNA methylation level of bacteria was found to be inversely proportional to its MIC against ciprofloxacin with p= -0.956 (p-value < 0.0001). CONCLUSION: The influence of DNA methylation over plasmid-mediated quinolone resistance needs to be further confirmed by bisulphite DNA sequencing of the plasmid-borne genes. Extensive usage of ciprofloxacin has led to rise in ciprofloxacin resistance possibly induced by DNA methylation. Thus rational usage of ciprofloxacin in a clinical setting is essential to combat the further development of ciprofloxacin resistance. Hypomethylated genes and adenine methylation needs to identified to fill up gaps in knowledge concerning the involvement of DNA methylation in fluoroquinolone resistance exhibited by E. coli.

High incidence of plasmid-mediated quinolone resistance genes among ciprofloxacin-resistant clinical isolates of Enterobacteriaceae at a tertiary care hospital in Puducherry, India.

Background. Plasmid-mediated quinolone resistance (PMQR) has received considerable attention recently. Data analysis in Jawaharlal Institute of Postgraduate Medical Education & Research (JIPMER) revealed 75% of the Enterobacteriaceae isolates to be ciprofloxacin-resistant in 2012. Few reports regarding the prevalence of PMQR are available from India. Hence, the present study was carried out to ascertain the prevalence of PMQR genes among clinical isolates of ciprofloxacin-resistant Enterobacteriaceae in JIPMER. Methods. The study included 642 ciprofloxacin-resistant clinical Enterobacteriaceae isolates. JIPMER hospital's annual consumption data for fluoroquinolones were retrieved from the Department of Pharmacy. The test isolates were screened for the presence of qnr A, B, D, S and aac(6')-Ib-cr genes. PMQR-positive isolates alone were tested for the presence of class I (intI1) and class II (intI2) integrons. Randomly selected PCR amplicons were sequenced and analysed using MEGA software. A total of 30 PMQR strains chosen at random were assessed for the transferability of the PMQR genes. Results. A majority of the strains exhibited high MIC values with 106 strains exhibiting MIC values >256 µg/mL. The aac(6')-Ib-cr gene had the highest prevalence at 64% (414) while, qnrB and qnrS genes were present in 15% (97) and 10% (64) of the isolates respectively. None of the strains were positive for qnrA and qnrD. All PMQR-positive isolates were screened for class I (intI1) and class II (intI2) integrons. Class I integron was found to be predominant among the test isolates with a few of them carrying both the classes of integrons. Transferability of PMQR genes to transconjugants was identified. Conclusion. The incidence of PMQR genes in the tertiary-care setup of the JIPMER hospital was found to be high which could be probably due to the increased prescription of fluoroquinolones. Thus, there is a need for rational usage of fluoroquinolones.

Serodiagnosis of Scrub Typhus at a Tertiary Care Hospital from Southern India.

INTRODUCTION: Scrub typhus, a zoonotic disease is one of the most covert emerging and re-emerging Rickettsial infections. There is an upsurge in the incidence of the disease worldwide with ever-changing habitat. Clinical diagnosis of scrub typhus is challenging as the signs and symptoms of scrub typhus are similar to other febrile illnesses. In developing countries, among the various laboratory tests to diagnose scrub typhus, Weil-Felix test is commonly performed despite its low sensitivity. The Immunofluorescence antibody (IFA) test has its limitations in terms of cost and expertise required. The present study was conducted to determine the seropositivity of IgM ELISA for scrub typhus in clinically suspected cases. MATERIALS AND METHODS: Weil-Felix test and IgM ELISA were performed using clinically suspected cases of scrub typhus using commercially available kits. RESULTS: Out of 482 samples tested, 109 were positive by both Weil-Felix test and IgM ELISA. One hundred and sixteen samples which were negative by Weil-Felix test reacted positive by IgM ELISA. Fourteen samples which were positive by Weil-Felix test were negative by ELISA. CONCLUSION: Owing to the limitations of the Weil-Felix test and IFA, commercially available recombinant IgM ELISA which has a good sensitivity and specificity may be an alternative in laboratories with moderate set up.

Relationship between Antimicrobial Consumption and the Incidence of Antimicrobial Resistance in Escherichia coli and Klebsiella pneumoniae Isolates.

INTRODUCTION: Gram negative organisms are one of the major causes of nosocomial diseases. Development of resistance to antibiotics by these organisms increases their risk in clinical treatment of patients. It also affects morbidity and mortality hence needs to be monitored and controlled. AIM: The aim of the present study was to analyse the correlation between consumption of parenteral antibiotics and the rates of antimicrobial resistance among the Escherichia coli and Klebsiella pneumoniae isolates collected during Dec 2010 - Jun 2013 from JIPMER hospital. MATERIALS AND METHODS: Consumption data of parenteral antibiotics in J01 category of Anatomical Therapeutic Chemical (ATC) in JIPMER was obtained and expressed in Defined Daily Doses (DDD) per 1000 inhabitants. Valid consumption and resistance data during the period Dec 2010 to Jun 2013 were obtained at 6 month intervals and were correlated to draw a relationship between antimicrobial consumption and its impact on drug resistance for Escherichia coli and Klebsiella pneumoniae. RESULTS: Escherichia coli isolates showed high resistance for increased use of gentamycin and ciprofloxacin. Increase in antibiotic consumption increases the resistance for Escherichia coli except for amikacin. Among the Klebsiella isolates, meropenem and gentamycin showed high correlations followed by ceftazidime, amikacin, ceftriaxone and ciprofloxacin. CONCLUSION: In summary, a statistically significant association was noticed between consumption of the studied antimicrobials and resistance of Escherichia coli isolates, except for amikacin and ceftazidime. In the case of Klebsiella pneumoniae, there was a statistically significant association between the resistance rates and consumption of gentamycin, ceftazidime and meropenem. Further, a linear relationship was noted between antimicrobial consumption and resistant isolates of Escherichia coli and Klebsiella pneumoniae, except for Escherichia coli resistance to amikacin.

Detection of Salmonella in Blood by PCR using iroB gene.

BACKGROUND: Salmonella, a genus of more than 2500 serological variants (serovars), includes many organisms that can cause human disease. Enteric fever remains an important public health problem in developing countries. Non typhoidal Salmonella generally produce a self limited gastroenteritis in healthy individuals whereas in extremes of age and immunocompromised cause severe fatal disease. The protean manifestations make this disease a true diagnostic challenge. AIM: The present study was carried out to optimize PCR for detecting the Salmonella genus using iroB gene and evaluate its use in the rapid diagnosis of typhoid and non typhoidal salmonellosis. MATERIALS AND METHODS: The study was carried out between August 2009 and July 2011 on blood samples from patients attending JIPMER hospital, Pondicherry, India with clinical suspicion of enteric fever and salmonellosis. Whole blood was used DNA extraction and conventional PCR done with iroB and fliC primers. Blood culture and Widal test were performed for all the patients. STATISTICAL ANALYSIS: Performed using Fischer's exact test with Graphpad Instat 3. RESULTS: PCR results were compared with blood culture. Sensitivity and specificity of PCR with fliC gene are 95.6% and 93.3% respectively. Sensitivity and specificity of PCR with iroB gene are 96.6% and 93.3% respectively CONCLUSION: With iroB gene, additional cases of Salmonella Paratyphi A and non typhoidal Salmonella were detected when compared to fliC gene.

Third-generation cephalosporin-resistant Vibrio cholerae, India.

Vibrio cholerae resistance to third-generation cephalosporins is rarely reported. We detected a strain that was negative for extended-spectrum ß-lactamase and positive for the AmpC disk test, modified Hodge test, and EDTA disk synergy test and harbored the blaDHA-1 and blaNDM-1 genes. The antimicrobial drug susceptibility profile of V. cholerae should be monitored.

Risk factors and predictors of mortality in culture proven neonatal sepsis.

OBJECTIVE: To determine the risk factors associated with culture-proven neonatal sepsis and identify predictors of mortality among them. METHODS: This prospective observational study was conducted in a tertiary care teaching hospital in South India over a period of 2 y. All admitted inborn and out born neonates with clinically suspected sepsis were included in the study. Blood culture was done for all neonates. Various factors associated with sepsis and mortality were identified. Chi-square test or Fisher's exact test was used to compare two groups. The results of these tests were confirmed with logistic regression analysis. RESULTS: Of the 120 neonates, only 50 (41.6%) had a positive blood culture. Premature rupture of membranes (PROM) >24 h, Apgar score <6 at 5 min, birth weight ≤1.5 kg and mechanical ventilation were found to be the independent risk factors for culture-proven sepsis based on logistic regression analysis. Twenty-one (42%) of the 50 neonates with culture-proven sepsis died, while only 15 (21.4%) of the 70 neonates who were blood culture negative died (Relative risk, 1.69; 95% confidence interval, 1.13 to 2.53; P value 0.0263). Birth weight ≤1.5 kg, shock and lethargy were proved to be independent predictors of mortality. CONCLUSIONS: The mortality rate was significantly high in neonates with a culture-proven sepsis compared to those with a negative blood culture. PROM >24 h, Apgar score <6 at 5 min, birth weight ≤1.5 kg and mechanical ventilation were independent risk factors for culture-proven sepsis, while lethargy, shock and birth weight ≤1.5 kg were independent predictors of mortality.

Neonatal sepsis in a tertiary care hospital in South India: bacteriological profile and antibiotic sensitivity pattern.

OBJECTIVE: To identify the common bacterial pathogens associated with neonatal sepsis and their antibiotic susceptibility pattern. METHODS: This prospective observational cohort study was done in a tertiary care teaching hospital located in South India for a period of 2 years. All the admitted inborn and out born neonates during this study period were screened for sepsis based on the clinical features and septic screening. All infants satisfying the criteria for sepsis were subjected to blood culture. Growths, if any, were noted and standard antibiotic sensitivity testing was done by Kirby-Bauer disk diffusion method. The Chi-square test or Fisher's exact test was used to compare two groups. RESULTS: Out of the 120 clinically suspected and positive screening test cases of neonatal sepsis, 41.6% (50 of 120) were culture-proven cases of neonatal sepsis. Klebsiella pneumoniae was isolated from 66% of culture positive cases followed by Coagulase-negative staphylococci in 12% of cases. Klebsiella pneumoniae was resistant to most of the antibiotics tested except amikacin and meropenem. Of the total 33 Klebsiella pneumoniae isolates, 16 (32.0%) were ESBL producers. The prevalence of ESBL producing Klebsiella pneumoniae during two month outbreak and rest of the study period was 83.3% (15 of 18) and 20% (3 of 15) respectively (P value 0.0010). CONCLUSIONS: Klebsiella pneumoniae was the most common agent causing both early-onset and late-onset sepsis and significantly associated with sepsis in inborn babies. Amikacin should be used along with the third-generation cephalosporins for empirical treatment of gram-negative neonatal sepsis.

Streptomyces bacteremia in a patient with actinomycotic mycetoma.

A 29-year-old woman presented with multiple painful swelling with discharging sinuses over the scalp. Histopathological examination of the biopsy tissue was suggestive of actinomycotic mycetoma. Streptomyces spp. was isolated from blood culture. The patient was successfully treated with trimethoprim-sulfamethoxazole and crystalline penicillin. This case is reported because of the rare occurrence of bacteremia by Streptomyces spp. secondary to subcutaneous actinomycotic mycetoma. Moreover, an interesting association between successive two pregnancies and occurrence of mycetoma of the scalp was observed in this case.

Clinical and serological evaluation of Leptospirosis in Puducherry, India.

BACKGROUND: Leptospirosis is a zoonotic disease with a worldwide distribution. There is a paucity of available data about prevalence of this disease in Pondicherry. Our aim was to investigate the seropositivity rate of leptospirosis in suspected cases and also to identify the predominant serogroups present by performing Microscopic Agglutination Test (MAT). The other aim of this study was to compare the results of a commercially available IgM ELISA with that of MAT. METHODOLOGY: A total of 110 blood samples from patients suspected of leptospirosis were sent for diagnosis. These samples were subjected to IgM ELISA and the microscopic agglutination test (MAT). MAT was done using a panel of 12 Leptospira serovars. RESULTS: MAT analysis of the 110 samples showed 40 (36%) to be positive. Antibodies were predominantly seen against serogroup Leptospira Icterohemorrhagiae (27%), followed by Pomona (17%), and Pyrogenes (12%). IgM ELISA done on these samples showed a positivity of 37% compared to MAT. CONCLUSION: This study reveals that the MAT test can be standardized in a diagnostic laboratory and used in conjunction with an IgM ELISA.

Leptospirosis in a case of non-Hodgkin lymphoma.

Leptospiral meningitis, which is an important feature of anicteric leptospirosis, is generally underdiagnosed. Serological tests are not very useful in diagnosis of leptospiral meningitis. Early detection by molecular tests such as PCR and prompt institution of therapy would be life-saving.

Immunoblot analysis of sera in uncomplicated typhoid fever & with typhoid ileal perforation.

BACKGROUND & OBJECTIVE: Ileal perforation is a serious complication of typhoid fever. The exact reasons for the development of perforation in only a few of those infected with Salmonella Typhi is unknown, and it is likely that immunological factors are involved. Therefore we undertook this study to compare the antibody profile in patients with uncomplicated typhoid fever with those having ileal perforation by immunoblotting. METHODS: Two groups of patients were included in the study. Group II comprised patients with uncomplicated typhoid fever (n=47), and group I with typhoid ileal perforation (n=33). The flagellar (H), lipopolysaccharide (LPS) and outer membrane protein (OMP) antigens of Salmonella Typhi were extracted and used to test patient sera for antibodies by immunoblotting RESULTS: Immunoblotting using S. Typhi antigens enabled the detection of S. Typhi antibodies in the two groups of patients. A significant difference was seen in the response of these two groups of patients with respect to antibodies to flagella, lipopolysaccharide and outer membrane proteins. Antibodies to flagella were more pronounced among patients with uncomplicated typhoid fever, while anti-OMP antibodies were significantly associated with typhoid ileal perforation. INTERPRETATION & CONCLUSION: A comparison of antibodies in patients with uncomplicated typhoid fever and with ileal perforation revealed the differences in the antibody profiles of the two groups. Our study suggests that the difference in antibody response may in some way play a role in the pathogenesis of typhoid ileal perforation which can also potentially be exploited to develop suitable diagnostic tests.

A case of fatal acute pyogenic meningitis in a neonate caused by extended-spectrum beta-lactamase producing Salmonella group B.

Salmonellosis constitutes an important public health problem throughout the world. In severe infections like meningitis and septicemia, antibiotic treatment is essential. Extended-spectrum cephalosporins are preferentially used to treat salmonellosis in children. Treatment failures due to in-vivo acquisition of an extended-spectrum beta-lactamase (ESBL) gene in nontyphoidal salmonellae are now well established. A 45-day-old male baby presented to the pediatric intensive care unit with a history of fever, poor feeding, two episodes of seizures of 3 days duration and recurrent apnoea. At admission, cerebrospinal fluid, stool and blood cultures were done and Salmonella enterica serovar Typhimurium was isolated from all the samples. The stool isolate was confirmed to be ESBL producing. The baby expired due to acute pyogenic meningitis.

A case report and review of the literature: ciprofloxacin resistant Salmonella enterica serovar Typhi in India.

BACKGROUND: Enteric fever is a major public health problem in India. The current treatment of choice is the fluoroquinolones. METHODS: The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by E-test, HIMEDIA HiComb MIC test and agar dilution. RESULTS: An isolate of Salmonella enterica serovar Typhi (S. Typhi) from a case of enteric fever gave a ciprofloxacin MIC of 64 microg/ml. CONCLUSIONS: To our knowledge there have been no reports of such high-level resistance to ciprofloxacin in S. Typhi from southern India. HIMEDIA HiComb MIC test method is an alternative to the E-test. Ciprofloxacin resistant typhoid fever responds to treatment with ceftriaxone.