Antimicrobial resistance, virulence profiles and molecular subtypes of Salmonella enterica serovars Typhi and Paratyphi A blood isolates from Kolkata, India during 2009-2013.

Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A (S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non-IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (blaTEM-1, catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology.

Validation of a new serology-based dipstick test for rapid diagnosis of typhoid fever.

Currently, no reliable diagnostic test is available for typhoid fever. One serology-based dipstick test, developed indigenously, was validated in this study. Preserved sera from 336 fever patients with known culture results for Salmonella Typhi were blindly tested by the Widal test and the new assay. Analytical sensitivities, specificities, and efficiencies for the new assay versus the Widal test were 68.8% versus 62.5%, 71.1% versus 37.1%, and 70.5% versus 43.2%, respectively (p < 0.001), considering S. Typhi-positive samples as gold standards. Thereafter, fresh sera from 102 hospital-attending children with clinical typhoid fever (including 20 confirmed nontyphoidal cases as control) were tested by both methods and analyzed statistically. The diagnostic sensitivity, specificity, and efficiency were 51.2%, 85%, and 57.8% for the new assay, and 43.9%, 65%, and 48% for the Widal test, respectively. Overall performance ability of the new assay was not better than the Widal test (p > 0.5). Further improvement of the new point-of-care typhoid assay is recommended before implementation in the field setup.

Zinc, vitamin A, and micronutrient supplementation in children with diarrhea: a randomized controlled clinical trial of combination therapy versus monotherapy.

OBJECTIVE: To compare the clinical efficacy of supplementation of zinc, zinc plus vitamin A, and zinc plus combination of micronutrients and vitamins (iron, copper, selenium, vitamin B(12), folate, and vitamin A) on acute diarrhea in children. STUDY DESIGN: This was a double-blind, randomized, placebo-controlled trial. Children aged 6 to 24 months with diarrhea and moderate dehydration were randomized to receive zinc plus placebo vitamin A (group 1), zinc plus other micronutrients plus vitamin A (group 2), zinc plus vitamin A (group 3), or placebo (group 4) as an adjunct to oral rehydration solution. Duration, volume of diarrhea, and consumption of oral rehydration solution were compared as outcome variables within the supplemented groups and with the placebo group. RESULTS: The 167 study subjects included 41 in group 1, 39 in group 2, 44 in group 3, and 43 in group 4. All 3 supplemented groups demonstrated a significant reduction in outcome variables (P < .0001) compared with the placebo group. Group 3 had the lowest reduction of outcome variables and group 2 had a speedy recovery, but differences among the supplemented groups were not statistically significant. CONCLUSIONS: Supplementation with a combination of micronutrients and vitamins was not superior to zinc alone, confirming the clinical benefit of zinc in children with diarrhea.

Randomised controlled clinical trial of Lactobacillus sporogenes (Bacillus coagulans), used as probiotic in clinical practice, on acute watery diarrhoea in children.

OBJECTIVE: To assess the clinical efficacy of Lactobacillus sporogenes (Bacillus coagulans), as probiotic preparation, against dehydrating diarrhoea in children. METHODS: Double-blind, randomised, placebo-controlled, hospital-based clinical trial with children aged 6-24 months who had diarrhoea with some dehydration. Children received tablets of L. sporogenes (B. coagulans) or placebo (control group) and oral rehydration salt solution for correction of initial dehydration as well as maintenance therapy. Duration, frequency, volume of diarrhoea and intake of ORS of two groups were compared as outcome variables. RESULTS: One hundred and forty-eight children participated, of whom 78 (Study group) received L. sporogenes (B. coagulans) and 70 received placebo (Control group). Differences in recovery rate (P=0.2), duration (P=0.5), frequency (P=0.05), volume (P=0.1) of diarrhoea, intake of ORS (P=0.2) and other fluids (P=0.1) were not significant between both groups. Neither did a subgroup analysis of children who had rotavirus as sole enteropathogens show any significant differences in duration (P=0.5), frequency (P=0.6), volume (P=0.8) of diarrhoea, intake of ORS (P=0.8) and other fluids (P=0.8) among both groups. CONCLUSION: L. sporogenes (B. coagulans), as an adjunct to ORS, had no therapeutic impact on management of acute dehydrating diarrhoea of diverse etiology including rotavirus associated diarrhoea in children.

Pulmonary tuberculosis among HIV seropositives attending a counseling center in Kolkata.

The study was carried out to detect the prevalence of pulmonary tuberculosis among HIV-seropositive individuals (HIV/TB co-infection) who attended counseling center of National Institute of Cholera and Enteric Diseases, Kolkata. A total of 109 HIV-seropositive individuals were screened. Of them, 36 (33%) had HIV/TB co-infection diagnosed by chest X-ray and presence of acid fast bacillus (AFB) detected by repeated microscopic examination of sputum. Blood samples were examined for CD4 and CD8 counts and ratio. Findings of blood examination showed that low CD4 count (<50/µl) had statistically significant association (P = 0.007) with HIV/TB co-infection as compared to HIV infection only. However, no significant correlation with CD4:CD8 ratio in HIV/TB co-infection was observed.

Subtype prevalence, plasmid profiles and growing fluoroquinolone resistance in Shigella from Kolkata, India (2001-2007): a hospital-based study.

OBJECTIVES: Shigellosis is a major public health problem, and increasing antimicrobial resistance has complicated its treatment. We report isolation frequency, plasmid profiles and antimicrobial resistance of Shigella subtypes in Kolkata, India, from a prospective hospital-based study. METHODS: Fresh stool or rectal swabs were collected from children (<5 years) attending the Diarrhea Treatment Unit of a governmental paediatric referral hospital in Kolkata. Samples were processed following standard methods over a 7-year period from January 2001 to December 2007. RESULTS: Of 4478 samples collected, 516 (11.5%) were positive for Shigella spp. S. flexneri (312; 6.9%) was the most frequently isolated serogroup, ranking before S. sonnei (123, 2.7%), S. dysenteriae (48, 1.1%) and S. boydii (33, 0.7%). Although 10 subtypes of S. flexneri were identified, the common ones circulating locally were S. flexneri 2a (179), S. flexneri 6 (38) and S. flexneri 3a (36). Knowledge of Shigella subtypes is important for vaccine development. The majority of Shigella isolates (81.0%) were multidrug (two or more antimicrobial classes) resistant and showed high minimum inhibitory concentration (MIC) with commonly used drugs like ampicillin, tetracycline, co-trimoxazole and nalidixic acid. Emergence of fluoroquinolone (FQ)-resistant S. dysenteriae type 1 (100.0%) in 2002-2003 was followed by frequent isolation (>25.0%) of FQ-resistant S. flexneri 2a, and S. flexneri 3a in 2004, which restricted use of fluoroquinolones for treatment. A number of smaller plasmids (<20 kb) with distinct patterns have been observed for several years in predominant subtypes. CONCLUSION: Long-term surveillance of Shigellae and their antimicrobial resistance are mandatory in endemic areas to formulate treatment policy until any suitable candidate vaccine is available to control the disease.

Full genomic analysis of a human group A rotavirus G9P[6] strain from Eastern India provides evidence for porcine-to-human interspecies transmission.

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing countries.

Immunogenicity, reactogenicity and safety of human rotavirus vaccine (RIX4414) in Indian infants.

AIM: This study was undertaken to assess the immunogenicity, reactogenicity and safety of two doses of an oral live-attenuated human rotavirus vaccine, strain RIX4414 (Rotarix()) in an Indian setting. RESULTS: The seroconversion rate observed one month post-dose 2 in the RIX4414 group 58.3% [95% CI: 48.7; 67.4] was significantly higher when compared to the placebo group 6.3%; [95% CI: 2.5; 12.5]. The reactogenicity and safety profile was similar for both groups. PATIENTS AND METHODS: Healthy infants (N = 363), approximately eight weeks of age were enrolled to receive two doses of RIX4414 vaccine (n = 182) or placebo (n = 181) separated by one month. To assess the immune response, blood samples were taken before vaccination and one month post-dose 2 of RIX4414/placebo. Solicited symptoms were collected for eight-days post each dose and safety data was collected throughout the study. CONCLUSIONS: Two doses of RIX4414 (Rotarix()) were immunogenic, had a good safety profile and were well-tolerated when administered to healthy Indian infants. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov; NCT00289172; eTrack 103792.

Increase in prevalence of human group A rotavirus G9 strains as an important VP7 genotype among children in eastern India.

BACKGROUND: In addition to four globally important group A rotavirus (GARV) VP7 genotypes (G1-G4), recent surveillance studies have revealed importance of G9 strains as an aetiological agent of infantile diarrhoea. OBJECTIVE: Detection and genotyping of GARVs from children, admitted with gastroenteritis to Dr. B.C. Roy Memorial Hospital for Children, Kolkata, India. STUDY DESIGN: GARVs were detected in stool specimens by RNA electrophoresis in polyacrylamide gels. G- and P-genotyping were performed by seminested multiplex PCR assays. VP7 gene of rotavirus G9 and G12 strains were sequenced for further analysis. RESULTS: Of 249 GARV strains (n=668, May 2005-December 2006), G- and P-genotyping were successfully accomplished for 197 and 204 samples, respectively. G1 (41.6%) was most prevalent G-genotype followed by G2 (33%), G12 (14.2%), G9 (10.1%) and mixed genotype (1%). Prevalent P-genotypes were P[8] (54.4%), P[4] (31.4%), P[6] (7.3%) and mixed genotype (6.9%). Overall, G1P[8], G2P[4], G9P[8], G12P[8] and G12P[6] were identified as important G-P combinations. Phylogenetic analysis of 13 G9 strains revealed clustering within G9 lineage III. Nine of 28 G12 strains were sequenced and exhibited phylogenetic clustering with previously reported G12 strains from Kolkata. CONCLUSION: In comparison to our previous data (2003 to April 2005), G9 and G2P[4] strains established themselves in a short time span as important genotypes in eastern India.

Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India.

Endemicity of cryptosporidiosis in India has been documented with little genetic characterization of the parasites. Fifty Cryptosporidium-positive specimens collected between 2001 and 2004 from pediatric patients in Kolkata, India were analyzed for parasite genetic structure using multilocus sequence typing (MLST). Genotype analyses showed the presence of Cryptosporidium hominis, Cryptosporidium meleagridis and Cryptosporidium felis in 49, 2 and 1 patients, respectively (two patients had mixed infections of C. hominis and C. meleagridis). To assess the extent of genetic heterogeneity of C. hominis, minisatellites, microsatellites and polymorphic markers in three different chromosomes were sequenced, including genes encoding the 60kDa glycoprotein (GP60), a 47kDa protein (CP47), a mucin-like protein (Mucin1), a serine repeat antigen (MSC6-7), and a 56kDa trans-membrane protein (CP56) in chromosome 6, the 70kDa heat shock protein (HSP70) in chromosome 2, and a T-rich gene fragment (Chrom3T) in chromosome 3. Population sub-structure of C. hominis based on multilocus gene sequences showed that there were 25 multilocus subtypes defined by combined sequence length and nucleotide polymorphism, which formed four distinct groups in this population. Significant intra- and inter-genic linkage disequilibria were observed with minimum recombination or expansion of limited subtypes, all indicative of a mostly clonal population structure. The results highlight the importance of high resolution MLST in studying Cryptosporidium population sub-structure especially when length polymorphism may be inadequate in identifying unique subtypes. The significance of the diverse MLST within C. hominis in relation to geographical and temporal factors and clinical manifestations of disease warrants further investigations.

Molecular characterization of Cryptosporidium spp. from children in Kolkata, India.

The intracellular parasite Cryptosporidium is responsible for severe diarrhea in immunocompromised persons in developing countries. Few studies on the characterization of the parasite in India are available. In this study, molecular characterization of the parasite from diarrheic children was carried out by PCR-restriction fragment length polymorphism analysis. At least three genotypes were identified. Out of 40 positive samples, 35 were positive for C. hominis, 4 were positive for C. parvum, and 1 was positive for C. felis. This study clearly suggests that cryptosporidiosis in this region is caused largely by anthroponotic transmission.

Genetic variability of human rotavirus strains isolated from Eastern and Northern India.

An epidemiological study was conducted in Eastern and Northern India to determine the genomic diversity of rotaviruses in these parts of the country. In 2001, a total of 126 Group A rotavirus positive samples were detected from children below 4 years of age with diarrhoea from Kolkata, Dibrugarh and Bhubaneswar in Eastern India, and Chandigarh, a city in Northern India. All the samples were genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers. The strains with G1P[8] (32.5%) was predominant as reported earlier [Das et al. (2002) J Clin Microbiol 40:146-149] followed by G2P[4](4.7%) and only one sample was of G4P[8] specificity. Along with these common types some rare strains like G1P[6], G2P[8], G2P[6], G4P[4], and G4P[6] were also detected in 14.3% of cases. Thirty percent of samples in this study were mixed infections and 21 (16.7%) specimens remained untypeable either for the VP7 or for the VP4 gene. After sequencing of the VP7 gene, two G9 strains (RMC321 and ISO-3) were identified with P[8] and P[19] specificities. Sequence analysis revealed that they have much lower homology to the G9 strains (116E, INL1, and G16) isolated earlier from Indian subcontinent, but have much higher homology to isolates from Argentina, Brazil, Malawi, Taiwan, and USA suggesting a separate progenitor for these strains.

Shigella dysenteriae serotype 1, Kolkata, India.

Since July 2002, bacteriologically confirmed shigellosis cases have increased, and multidrug-resistant Shigella dysenteriae serotype 1 strains have reemerged in patients hospitalized with diarrhea in Kolkata, India. The isolated strains of S. dysenteriae 1 showed resistance to chloramphenicol (80%), ampicillin (100%), tetracycline (100%), co-trimoxazole (100%), nalidixic acid (100%), norfloxacin (100%), and ciprofloxacin (100%). Emergence of fluoroquinolone resistance in S. dysenteriae 1 strains complicated treatment of shigellosis patients. Six strains belonging to provisional serovars of S. dysenteriae were also identified for the first time in patients hospitalized with diarrhea in Kolkata, India.

Distribution of rotavirus VP7 genotypes among children suffering from watery diarrhea in Kolkata, India.

A combined reverse transcriptase-polymerase chain reaction (RT/PCR) was used to produce cDNA of the VP7 gene of rotavirus present in the stool samples. A total of 150 rotavirus positive stool samples were used in this study. Multiplex PCR, using the type specific primers, revealed the presence of G1 (49/150, 32.7%), G2 (27/150, 18%) and G4 (30/150, 20%) genotypes among the samples collected during 1999-2000 from children suffering from acute watery diarrhea. Eighteen samples (12%) were of mixed genotype and the remaining 16 samples (10.6%) could not be typed. Comparative analysis of the full length genes of the representative strains with corresponding genotypes incorporated in the human-rhesus rotavirus tetravalent vaccine (RRV-TV) formulation demonstrates variations of the circulating G1, G2 and G4 strains with the corresponding G genotypes present in the vaccine strain.