Resistance Pattern and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains Isolated in Bangladesh.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children as well as in travellers to ETEC endemic countries. Ciprofloxacin is a broad-spectrum antimicrobial agent nowadays used for the treatment of diarrhea. This study aimed to characterize ciprofloxacin resistant ETEC strains isolated from diarrheal patients in Bangladesh. METHODS: A total of 8580 stool specimens from diarrheal patients attending the icddr,b Dhaka hospital was screened for ETEC between 2005 and 2009. PCR and Ganglioside GM1- Enzyme Linked Immuno sorbent Assay (ELISA) was used for detection of Heat labile (LT) and Heat stable (ST) toxins of ETEC. Antimicrobial susceptibilities for commonly used antibiotics and the minimum inhibitory concentration (MIC) of nalidixic acid, ciprofloxacin and azithromycin were examined. DNA sequencing of representative ciprofloxacin resistant strains was performed to analyze mutations of the quinolone resistance-determining region of gyrA, gyrB, parC and parE. PCR was used for the detection of qnr, a plasmid mediated ciprofloxacin resistance gene. Clonal variations among ciprofloxacin resistant (CipR) and ciprofloxacin susceptible (CipS) strains were determined by Pulsed-field gel electrophoresis (PFGE). RESULTS: Among 1067 (12%) ETEC isolates identified, 42% produced LT/ST, 28% ST and 30% LT alone. Forty nine percent (n = 523) of the ETEC strains expressed one or more of the 13 tested colonization factors (CFs) as determined by dot blot immunoassay. Antibiotic resistance of the ETEC strains was observed as follows: ampicillin 66%, azithromycin 27%, ciprofloxacin 27%, ceftriazone 13%, cotrimaxazole 46%, doxycycline 44%, erythromycin 96%, nalidixic acid 83%, norfloxacin 27%, streptomycin 48% and tetracycline 42%. Resistance to ciprofloxacin increased from 13% in 2005 to 34% in 2009. None of the strains was resistant to mecillinam. The MIC of the nalidixic acid and ciprofloxacin of representative CipR strains were 256 µg/ml and 32µg/ml respectively. A single mutation (Ser83-Leu) in gyrA was observed in the nalidixic acid resistant ETEC strains. In contrast, double mutation in gyrA (Ser83-Leu, Asp87-Asn) and a single mutation in parC (Glu84-Ly) were found in ciprofloxacin resistant strains. Mutation of gyrB was not found in either the nalidixic acid or ciprofloxacin resistant strains. None of the ciprofloxacin resistant strains was found to be positive for the qnr gene. Diverse clones were identified from all ciprofloxacin resistant strains by PFGE analysis in both CF positive and CF negative ETEC strains. CONCLUSION: Emergence of ciprofloxacin resistant ETEC strains results in a major challenge in current treatment strategies of ETEC diarrhea.

Isolation and Characterization of Enteropathogenic Escherichia coli (EPEC) in Paediatric Diarrhoeal Patients by Detection of bfpA gene by PCR and HeLa cell Adherence Assay.

This study has been undertaken to investigate the isolation and identification of EPEC strains from paediatric diarrhoeal patients. The study was carried out in the department of Microbiology, Sir Salimullah Medical College & Mitford Hospital, Bangladesh during January to December, 2011. Total 272 samples were studied. Samples from patients with diarrhoea were collected from two tertiary care hospital. At first Esch. coli were isolated from these specimens using standard microbiological techniques and then EPEC strains were identified on the basis of presence of bundle forming pilus (bfpA) gene. Virulence of EPEC strains were determined by detection of bfpA gene and observing localized adherence (LA) in HeLa cell adherence assay. Esch. coli was isolated and identified from all the 272 samples from patients using standard microbiological techniques. Among 272 samples 20(7.35%) isolates were identified as EPEC on the basis of presence of bfpA gene detected by polymerase chain reaction. EPEC strains were identified from those 240 samples, from which Esch. coli had been isolated only. Out of twenty EPEC strains, 17 strains (85%) showed a pattern of localized adherence in Hela cell adherence assay. EPEC strains can be identified by bfpA gene detection and by adherence assays. HeLa cell adherence assay is the most specific method for detection of EPEC strains which has bfpA gene, responsible for localized adherence (LA) in HeLa cell line. Rapid and reliable detection of EPEC is required for successful microbiological surveillance and for treatment of EPEC mediated diarrhoeal disease.

Comparative population structure analysis of Campylobacter jejuni from human and poultry origin in Bangladesh.

Campylobacter jejuni is the most important cause of antecedent infections leading to Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The objective of the present study was to define the genetic diversity, population structure, and potential role of poultry in the transmission of Campylobacter to humans in Bangladesh. We determined the population structure of C. jejuni isolated from poultry (n = 66) and patients with enteritis (n = 39) or GBS (n = 10). Lipooligosaccharide (LOS) typing showed that 50/66 (76 %) C. jejuni strains isolated from poultry could be assigned to one of five LOS locus classes (A-E). The distribution of neuropathy-associated LOS locus classes A, B, and C were 30/50 (60 %) among the typable strains isolated from poultry. The LOS locus classes A, B, and C were significantly associated with GBS and enteritis-related C. jejuni strains more than for the poultry strains [(31/38 (82 %) vs. 30/50 (60 %), p < 0.05]. Multilocus sequence typing (MLST) defined 15 sequence types (STs) and six clonal complexes (CCs) among poultry isolates, including one ST-3740 not previously documented. The most commonly identified type, ST-5 (13/66), in chicken was seen only once among human isolates (1/49) (p < 0.001). Amplified fragment length polymorphism (AFLP) revealed three major clusters (A, B, and C) among C. jejuni isolated from humans and poultry. There seems to be a lack of overlap between the major human and chicken clones, which suggests that there may be additional sources for campylobacteriosis other than poultry in Bangladesh.

Geographical diversity in seasonality of major diarrhoeal pathogens in Bangladesh observed between 2010 and 2012.

The study aimed to determine the geographical diversity in seasonality of major diarrhoeal pathogens among 21 138 patients enrolled between 2010 and 2012 in two urban and two rural sites in Bangladesh under the surveillance system of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). Distinct patterns in seasonality were found for rotavirus diarrhoea which peaked in winter across the sites (December and January) and dipped during the rainy season (May) in urban Dhaka, August in Mirpur and July in Matlab, equated by time-series analysis using quasi-Poisson regression model. Significant seasonality for shigellosis was observed in Dhaka and rural Mirzapur. Cholera had robust seasonality in Dhaka and Matlab in the hot and rainy seasons. For enterotoxogenic Escherichia coli (ETEC) diarrhoea, clearly defined seasonality was observed in Dhaka (summer). Understanding the seasonality of such pathogens can improve case management with appropriate therapy, allowing policy-makers to identify periods of high disease burden.

Controlling scabies in madrasahs (Islamic religious schools) in Bangladesh.

OBJECTIVES: To assess the effectiveness of a scabies control programme in reducing the prevalence of scabies in urban Bangladesh madrasahs, where the condition is extremely common. STUDY DESIGN: A controlled trial involving four intervention madrasahs (total students 2359) and four control madrasahs (total students 2465) in Dhaka Metropolitan Area. METHODS: A baseline scabies sample survey was carried out on 40 and 44 students of four intervention and four control madrasahs, respectively. Another 40 students of the intervention madrasahs were administered a pre-intervention test on scabies knowledge. This was followed by mass treatment of all students, teachers and staff of the eight madrasahs with topical 5% permethrin cream. The subsequent intervention involved daily monitoring of students for five key personal hygiene practices, weekly 10-min scabies health education classes, supply of simple and inexpensive products to students to prevent cross-infestation to/from peers (e.g. plastic bags, clothes hangers), and chemotherapy of new students detected with scabies. After 4 months of the intervention, the prevalence of scabies, personal hygiene practices and scabies knowledge were assessed in students of the intervention madrasahs. RESULTS: Before the intervention, the prevalence of scabies was 61% and 62% in intervention and control madrasahs, respectively (P = 1.00). After mass scabies treatment in all eight madrasahs and 4 months of intervention, the prevalence of scabies was reduced to 5% and 50% in intervention and control madrasahs, respectively (P < 0.001). There were significant improvements in all five personal hygiene practices at the intervention madrasahs. Mean test scores for scabies knowledge were 40% before the intervention and 99% after the intervention in the four intervention madrasahs. The cost of this programme was US$1.60 per student, and primarily included products such as plastic bags and clothes hangers, and health education material. CONCLUSIONS: This programme demonstrates a pragmatic and cost-effective way to control scabies in a residential institutional setting. It is recommended that this programme should be scaled up to all residential madrasahs in Bangladesh.

Emergence of multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh.

The main objective of this study was to investigate the prevalence of bla (NDM-1) in Gram-negative bacteria in Bangladesh. In October 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratories, 1,816 consecutive clinical samples were tested for imipenem-resistant Gram-negative organisms. Imipenem-resistant isolates were tested for the bla (NDM-1) gene. Among 403 isolates, 14 (3.5 %) were positive for bla (NDM-1), and the predominant species were Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. All bla (NDM-1)-positive isolates were resistant to multiple antibiotics. Among ß-lactamase genes, bla (CTX-M-1-group) was detected in ten isolates (eight bla (CTX-M-15)), bla (OXA-1-group) in six, bla (TEM) in nine, bla (SHV) in seven, and bla (VIM) and bla (CMY) in two isolates each. The 16S rRNA methylase gene, armA, was detected in five K. pneumoniae isolates and in one E. coli isolate. rmtB and rmtC were detected in a Citrobacter freundii and two K. pneumoniae isolates, respectively. qnr genes were detected in two K. pneumoniae isolates (one qnrB and one qnrS) and in an E. coli isolate (qnrA). Transferable plasmids (60-100 MDa) carrying bla (NDM-1) were detected in 7 of the 11 plasmid-containing isolates. Pulsed-field gel electrophoresis (PFGE) analysis grouped K. pneumoniae isolates into three clusters, while E. coli isolates differed significantly from each other. This study reports that approximately 3.5 % of Gram-negative clinical isolates in Bangladesh are NDM-1-producing.

Intervention to increase detection of childhood tuberculosis in Bangladesh.

BACKGROUND: Despite a well-functioning adult tuberculosis (TB) control programme, children with TB remain grossly under-detected in Bangladesh. It is conservatively estimated that annually around 21,000 children with TB go undetected, due to an almost exclusive focus on sputum smear-positive TB and the absence of training or guidelines in paediatric TB. OBJECTIVE: To double child TB detection by increasing general awareness and training of health care workers at microscopy centres supported by the Damien Foundation (DF) Bangladesh. METHODS: A cluster-randomised trial was carried out with provision of child TB guidelines, training and logistics support to staff of 18 microscopy centres, while 18 non-adjacent microscopy centres continued their usual practice and served as controls. Paediatric data on TB suspect referral and case detection were collected at baseline and during the intervention at both control and intervention sites. RESULTS: Child TB case detection increased in both intervention and control microscopy centres, but the increase was three times the baseline in the intervention centres (from 3.8% to 12%) in comparison to less than double the baseline in the control centres (from 4.3% to 7%, P = 0.001). CONCLUSION: Simple guidelines and training on child TB case detection, together with basic logistics support, can be integrated into the existing National TB Control Programme and improve service delivery to children in TB-endemic areas.

Axonal variant of Guillain-Barre syndrome associated with Campylobacter infection in Bangladesh.

BACKGROUND: Campylobacter jejuni enteritis is the predominant bacterial infection preceding Guillain-Barré syndrome (GBS), an acute postinfectious immune-mediated polyradiculoneuropathy. The purpose of this study was to define the clinical phenotype of GBS and the relation with preceding C jejuni infections in Bangladesh. METHODS: We performed a prospective matched case-control hospital surveillance including 100 patients fulfilling the National Institute of Neurological Disorders and Stroke criteria for GBS from 2006 to 2007 in the Dhaka area of Bangladesh. Detailed clinical, electrophysiologic, serologic, and microbiologic data were obtained with a follow-up of 6 months. RESULTS: GBS affected predominantly young adult males living in rural areas. Sixty-nine percent of the patients had clinical evidence of a preceding infection. The most frequent symptom was diarrhea (36%). The majority of patients had a pure motor variant of GBS (92%) with relatively infrequent cranial nerve involvement (30%). Twenty-five percent of patients required respiratory support. Electrophysiologic studies showed that 67% of patients had an axonal variant of GBS. Eleven patients (14%) died, and 23 (29%) remained severely disabled during the follow-up. Positive C jejuni serology was found in an unprecedented high frequency of 57% as compared with 8% in family controls and 3% in control patients with other neurologic diseases (p < 0.001). C jejuni infection was significantly associated with serum antibodies to the gangliosides GM1 and GD1a, axonal neuropathy, and greater disability. CONCLUSIONS: We report an unusually high frequency of the axonal variant of Guillain-Barré syndrome in Bangladesh, associated with preceding Campylobacter jejuni infection, severe residual disability, and high mortality.

Comparison of enterotoxigenic Escherichia coli isolated from surface water and diarrhoeal stool samples in Bangladesh.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children. Although the prevalence of ETEC is high in Bangladesh and infections can be spread through food and contaminated water, limited information is available about ETEC in the surface water. We carried out studies to isolate ETEC from surface water samples from ponds, rivers, and a lake from a site close to field areas known to have a high incidence of diarrhea in Dhaka, Bangladesh, and Matlab, Bangladesh. ETEC strains isolated from the water sources were compared with ETEC strains isolated from patients with diarrhea at two hospitals in these areas. ETEC were isolated from 30% (45 of 150) of the samples from the surface water sources and 19% (518 of 2700) of the clinical specimens. One hundred ETEC strains isolated from patients with similar phenotypes as the environmental strains were compared for phenotypic and genotypic properties. The most common O serogroups on ETEC were O6, O25, O78, O115, and O126 in both types of strains. Pulsed-field gel electrophoresis analyses of the ETEC strains showed that multiple clones of ETEC were present within each colonization factor type and that some clones detected in the environment were also isolated from the stools of patients. The strains showed multiple and similar antibiotic resistance patterns. This study shows that ETEC is prevalent in surface water sources in Bangladesh suggesting a possible reason for the endemicity of this pathogen in Bangladesh.

Shiga toxin-producing Escherichia coli isolated from patients with diarrhoea in Bangladesh.

The prevalence of Shiga toxin-producing Escherichia coli (STEC) and its characteristics were determined among hospitalized patients with diarrhoea and children with diarrhoea in an urban slum community of Dhaka city using sensitive culture and PCR methods. Stool samples were collected from 410 patients with diarrhoea enrolled in the 2% surveillance system (every 50th patient attending the hospital with diarrhoeal disease is included) at the ICDDR,B hospital and from 160 children of 2-5 years of age with diarrhoea living in an urban slum in Dhaka, between September 2004 and April 2005. Shiga toxin genes (stx) were detected by multiplex PCR in the enrichment broth of nine samples (2.2%) from hospitalized patients and 11 samples (6.9%) from the community patients. STEC was isolated from five stool samples with positive PCR results using a colony patch technique. All five isolates were positive in the Vero cell assay and PCR fragments of stx genes were confirmed by sequencing. Two isolates were positive for the E. coli attaching-and-effacing (eae) gene and four were positive for the enterohaemolysin (hlyEHEC) gene and enterohaemolysin production. The five isolates belonged to five different serotypes:O32:H25, O2:H45, O76:H19, ONT:H25 and ONT:H19. It can be concluded that STEC is not a common pathogen in Bangladesh among hospitalized patients with diarrhoea nor among mild cases of diarrhoea in the community.

The emerging strains of Shigella dysenteriae type 2 in Bangladesh are clonal.

A total of 113 strains of Shigella dysenteriae type 2 isolated from patients attending the Dhaka diarrhoea treatment centre of ICDDR,B: Centre for Health and Population Research during the period 1999-2004 were studied. Serotype of the isolates was confirmed using commercially available antisera. Except for arabinose fermentation, all the strains had similar biochemical reactions. More than 60% of the strains were sensitive to commonly used antibiotics; only 6% (n=7) of the strains were resistant to nalidixic acid, and none of the strains were resistant to mecillinam and ciprofloxacin. All strains were invasive as demonstrated by the presence of a 140 MDa plasmid, ial, sen and ipaH genes, Congo Red absorption ability and by the Sereny test performed on representative strains. Plasmid patterns were heterogeneous but more than 50% of strains were confined to a single pattern. All strains possessed a 1.6 MDa plasmid and 87% of the strains contained a 4 MDa plasmid. Middle-range plasmids (90 MDa to 30 MDa) present in 36% of the strains were not associated with antibiotic resistance. All the strains were clustered within a single type with four subtypes by pulsed-field gel electrophoresis while ribotyping patterns of all the strains were identical.

Detection of intra-familial transmission of shigella infection using conventional serotyping and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) is commonly used in molecular epidemiology. However, this technique has never been used in studying intra-family spread of enteric diseases in Bangladesh. Our objective was to evaluate the intra-familial transmission of shigella infection using PFGE. Children of either sex, less than 10 years old, who were family contacts of shigella-infected index cases were the study population. PFGE was applied if the same serotypes/sub-serotypes of shigella were isolated from both the index case and the family contact children. In total, 227 index cases were studied. Shigella was isolated from 61 (27%) contact children on day 1 of enrolment, among which Shigella flexneri (41%) and S. boydii (41%) were dominant, followed by S. dysenteriae (10%), S. sonnei (3%), and shigella-like organisms (5%). Seventeen (28%) of the asymptomatic infections in contact children were caused by the same serotype of shigella as that found in the index case. The intra-familial shigella transmission rate was 8% (17/227). Of the 227 contact children, eight (4%) developed diarrhoea during a 10-day follow-up and shigella was isolated from five (2%) of these children, and three of them (S. flexneri 3a, 1b, and 3a) were identical to the strains from their respective index cases. Compared to children without asymptomatic carriage of shigella (2/166), the risk (odds ratio) of developing diarrhoea for the children with asymptomatic carriage of shigella identical to their cases (3/17) was 9.0 (95% CI 1.5-49.0, P=0.01). The attributable risk for symptomatic shigella infection by intra-familial transmission was 50%. Results of this study demonstrated that intra-familial transmission of shigella carries a higher risk for diarrhoea.

Shigella serotypes among hospitalized patients in urban Bangladesh and their antimicrobial resistance.

We studied the isolation of Shigella spp., and their antimicrobial resistance. S. flexneri (54 %) was most frequently isolated, followed by S. dysenteriae (20 %), S. boydii (16 %) and S. sonnei (10 %). Among S. flexneri (n = 122), 29 (24 %) were 2a, and 23 (19 %) were 2b. None of the Shigella strains were resistant to mecillinam or ciprofloxacin. Resistance to nalidixic acid was most frequent among S. dysenteriae type 1 (100%) followed by S. flexneri 2a (69%), and S. flexneri 2b (52 %). Systematic monitoring is needed to identify most prevalent serotypes, and to detect changes in the prevalence and antimicrobial resistance pattern.

Rickets among children of a coastal area of Bangladesh.

Many children with rachitic deformities have been reported in southern coastal area of Bangladesh but the actual rate of prevalence was not known. A survey was conducted to determine the magnitude of rachitic problem among the children of Chakaria thana of Cox's Bazar district of Bangladesh. Nine hundred children between 1-15 years selected randomly from 30 villages of total 340 villages. Face to face interview of the parents was taken and the children were examined for evidences of rickets. Serum calcium, phosphorus, alkaline phosphatase (ALP) were estimated and radiology of limbs were done in all clinically suspected cases and in a control of every eighth child. Seventy eight children (8.7%) had physical features suggestive of rickets. Fifty eight (6.4%) children had 'clinical rickekts' (positive physical feature(s) but normal ALP and negative radiology), 12 (1.3%) children had 'biochemical rickets' (positive physical features and raised ALP but negative radiology) and 8 (0.9%) children had 'confirmed rickets' (positive physical features, raised ALP and positive radiology). Out of 78 children with rachitic feature(s), Pectus carinatum was found as the most common clinical feature in 26 (33.3%) children followed by genu valgum in 23 (29.4%) cases. Twenty two normal children (2.2%) had raised level of ALP (>300U/L). The prevalence of rickets is high in children of Chakaria and further study is needed to find out the exact aetiology of rickets in children there.

Altering trends in the dominance of Shigella flexneri serotypes and emergence of serologically atypical S. flexneri strains in Dhaka, Bangladesh.

Of 469 recently isolated Shigella flexneri strains, 452 agglutinated with Shigella flexneri-specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with S. flexneri 2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.

Three case definitions of malaria and their effect on diagnosis, treatment and surveillance in Cox's Bazar district, Bangladesh.

In countries where malaria is endemic, routine blood slide examinations remain the major source of data for the public health surveillance system. This approach has become inadequate, however, as the public health emphasis has changed from surveillance of laboratory-confirmed malaria infections to the early detection and treatment of the disease. As a result, it has been advocated that the information collected about malaria be changed radically and should include the monitoring of morbidity and mortality, clinical practice and quality of care. To improve the early diagnosis and prompt treatment (EDPT) of malaria patients, three malaria case definitions (MCDs) were developed, with treatment and reporting guidelines, and used in all static health facilities of Cox's Bazar district, Bangladesh (population 1.5 million). The three MCDs were: uncomplicated malaria (UM); treatment failure malaria (TFM); and severe malaria (SM). The number of malaria deaths was also reported. This paper reviews the rationale and need for MCDs in malaria control programmes and presents an analysis of the integrated surveillance information collected during the three-year period, 1995-97. The combined analysis of slide-based and clinical data and their related indicators shows that blood slide analysis is no longer used to document fever episodes but to support EDPT, with priority given to SM and TFM patients. Data indicate a decrease in the overall positive predictive value of the three MCDs as malaria prevalence decreases. Hence the data quantify the extent to which the mainly clinical diagnosis of UM leads to over-diagnosis and over-treatment in changing epidemiological conditions. Also the new surveillance data show: a halving in the case fatality rate among SM cases (from 6% to 3.1%) attributable to improved quality of care, and a stable proportion of TFM cases (around 7%) against a defined population denominator. Changes implemented in the EDPT of malaria patients and in the surveillance system were based on existing staff capacity and routine reporting structures.