Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Profile of urinary tract infections in paediatric patients.

Background & objectives: this cross-sectional study was conducted at a tertiary care centre in Puducherry, south India, with the aim of finding the profile of the paediatric urinary tract infection (UTI), bacterial pathogens involved, and also to observe vesicoureteric reflux (VUR) and renal scarring in these patients. Methods: A total of 524 paediatric patients ≤13 yr, suspected to have UTI, were included in the study. Urine samples were collected, processed for uropathogen isolation and antibiotic susceptibility test was performed as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Thirty two culture proven children with UTI underwent micturating cysto-urethrography (MCU) and dimercaptosuccinic acid (DMSA) scanning was done for 69 children. Results: of the 524 children, 186 (35.4%) had culture proven UTI with 105 (56.4%) being infants, 50 (27.4%) between 1-5 yr, 30 (16.12%) between 5-13 yr and 129 (69.35%) males. Posterior urethral valve (PUV) was noted in three, hydronephrosis in one, VUR in 18 and renal scarring in 33. VUR as well as renal scarring were more in males >1 yr of age. A significant association (P=0.0054) was noted with a combined sensitivity and specificity of these investigations being 83 and 90 per cent, respectively of the MCU and DMSA scans for detecting VUR. Escherichia coli was the most common pathogen isolated, sensitive to nitrofurantoin, followed by cefoperazone-sulbactam, aminoglycosides and meropenem. Interpretation & conclusions: Our results indicate that UTI varies with age and gender and extensive evaluation is required in boys under one year of age with UTI. This study also highlights the better efficacy of aminoglycosides, cefoperazone-sulbactam and nitrofurantoin in vitro compared with meropenem in gram-negative uropathogens.

Clinical and Microbiological Profiles of Shigellosis in Children.

Shigellosis presents with varied clinical features are dictated by the species involved, virulence factors of the strain, and the host immune status. We studied the species, virulence genes, and antibiotic susceptibility pattern of the Shigella strains isolated from 33 children aged less than 12 years, with clinical features of shigellosis. Identification and antibiotic sensitivity of Shigella species were done using disc diffusion and E-test. Multiplex PCR was done for the detection of virulence genes (ipaH, ial, set1A, set1B, sen, and stx) and ESBL genes. Parents of the children were interviewed using structured questionnaire to assess the severity of the disease; 26 (79%) of the isolates were Shigella flexneri. Ciprofloxacin and ceftriaxone resistance was seen in 23 (69%) and 3 (9%) Shigella isolates respectively. Two ceftriaxone-resistant strains were found to harbour blaCTX gene and the third blaTEM gene. Virulence gene ipaH was detected in 100% of strains while ial, sen, set1A, and set1B were detected in 85%, 61%, 48%, and 48% respectively.

Fungal profile of funguria cases at a tertiary care hospital in southern India.

Background & objectives: Candida albicans is the most common aetiological agent in funguria cases commonly observed in hospitalized patients. But a few reports are available from India where non-albicans Candida species have accounted for >50 per cent of urinary Candida isolates. We undertook this study to know the fungal profile amongst funguria cases. Methods: A total of 123 consecutive fungal isolates obtained from clinically suspected cases of urinary tract infection from April to September, 2013, were included. Yeast species was identified by standard phenotypic methods. Antifungal susceptibility testing of yeast was performed for fluconazole (25 μg) by disc diffusion method as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: The male to female ratio was 0.92. The mean age of patients was 42.7 ± 18.9 yr. C. tropicalis (58.5%) was the most common fungal agent followed by C. albicans (30.1%). Only one isolate of C. tropicalis was resistant to fluconazole. Interpretation & conclusions: C. tropicalis and C. albicans were the predominant fungal pathogens responsible for urinary tract infection. Less resistance to fluconazole observed in the study may be due to restricted use of fluconazole in this area.

Clinical Spectrum and Predictive Risk Factors of Major Infections in Hospitalized Children with Nephrotic Syndrome.

This observational study was conducted with the primary objective of studying the incidence of major infections in nephrotic syndrome (NS), while the secondary objectives were to evaluate the risk factors for and the etiological spectrum of major infections. Eighty six children up to 13 years of age fulfilling the International Study of Kidney Disease in Children (ISKDC) criteria for NS, who required 101 hospital admissions were recruited from November 2010 to July 2012. Major infections were defined as those that are disseminated, affecting deep organs, requiring hospitalization or potentially life-threatening. The incidence of major infections was 36.6%. Among the major infections, peritonitis and pneumonia together accounted for 72.9%, while urinary tract infections and cellulitis accounted for 16.2%. On logistic regression, severe ascites and more severe clinical types of NS independently predicted major infections, while serum cholesterol >400 mg/dL was the sole predictor of peritonitis.

Antibiotic resistance pattern among common bacterial uropathogens with a special reference to ciprofloxacin resistant Escherichia coli.

Background & objectives: The resistance of bacteria causing urinary tract infection (UTI) to commonly prescribed antibiotics is increasing both in developing as well as in developed countries. Resistance has emerged even to more potent antimicrobial agents. The present study was undertaken to report the current antibiotic resistance pattern among common bacterial uropathogens isolated in a tertiary care hospital in south India, with a special reference to ciprofloxacin. Methods: A total of 19,050 consecutive urine samples were cultured and pathogens isolated were identified by standard methods. Antibiotic susceptibility was done by Kirby Bauer disk diffusion method. The clinical and demographic profile of the patients was noted. Results: Of the 19,050 samples, 62 per cent were sterile, 26.01 per cent showed significant growth, 2.3 per cent showed insignificant growth and 9.6 per cent were found contaminated. Significant association (P<0.001) of prior use of antibiotics in males, UTI in adults, gynaecological surgery in females, obstructive uropathy in males and complicated UTI in females with the occurrence of UTI with ciprofloxacin resistant Escherichia coli was noted. Significant association was noted in females with prior antibiotics, with prior urological surgery and in males with prior complicated UTI. There was no significant association with diabetes mellitus with the occurrence of UTI with ciprofloxacin resistant E. coli. Fluoroquinolone resistance was found to increase with age. Interpretations & conclusions: Ciprofloxacin resistance has emerged due to its frequent use. This resistance was seen more in the in-patients, elderly males and females. Also the resistance to other antibiotics was also high. Increasing antibiotic resistance trends indicate that it is imperative to rationalize the use of antimicrobials in the community and also use these conservatively.

Increasing Antimicrobial Resistance of Vibrio cholerae O1 Biotype El Tor Strains Isolated in a Tertiary-care Centre in India.

The antimicrobial susceptibility patterns are on constant change with the recent emergence of multidrugresistant strains of most bacteria. Results of recent studies in India showed that most isolates of Vibrio cholerae O1 were resistant to the commonly-used antibiotics. The study was conducted to determine the antibiotic susceptibility patterns of V. cholerae O1 isolated during 2008-2010 at the hospital of the Jawaharlal Nehru Institute of Post Graduate Medical Education and Research, Puducherry, India. In total, 154 strains of V. cholerae O1 from 2,658 stool specimens were reported during January 2008–December 2010—34 in 2008, 2 in 2009, and 118 in 2010. The isolates of V. cholerae O1 were subjected to antimicrobial susceptibility testing using the Kirby-Bauer method. The antibiotic disks tested were tetracycline (30 μg), furazolidone (100 μg), ampicillin (10 μg), ceftriaxone (30 μg), and ciprofloxacin (5 μg). Escherichia coli ATCC 25922 was used as the control organism. The minimum inhibitory concentrations (MICs) of ceftriaxone, ciprofloxacin, and tetracycline were determined using the agar dilution method for all the strains. The E-test method was used for the strains which had either intermediate resistance or were resistant to the antibiotics by the agar dilution method. The results of the agar dilution corroborated the results of the E-test. The MIC of ceftriaxone in 151 strains was <2 μg/mL while it was 16 μg/mL in three strains; the latter three strains were resistant to ceftriaxone by the disc-diffusion test. The MIC of ciprofloxacin in 150 strains was <0.5 μg/mL while the MIC of tetracycline was <1 μg/mL. In the remaining four strains, the MIC of tetracycline was >32 μg/mL, and the MIC of ciprofloxacin was >8 μg/mL. These four strains were resistant to both tetracycline and ciprofloxacin by the disc-diffusion test and were exclusive of the three ceftriaxone-resistant strains. The majority of the isolates were obtained from children aged 0-5 year(s)—70.3% (83 of 118) and 41.2% (14 of 34) were reported in 2010 and 2008 respectively. Since treating severe cases of cholera with antibiotics is important, the continuing spread of resistance in V. cholerae to the most important agents of therapy is a matter of concern. Also, chemoprophylaxis with antimicrobial agents is likely to become even more difficult.