Antimicrobial resistance of bacterial enteropathogens isolated from stools in Madagascar.

BACKGROUND: Diarrheal diseases are a major public health problem in developing countries, and are one of the main causes of hospital admissions in Madagascar. The Pasteur Institute of Madagascar undertook a study to determine the prevalence and the pathogenicity of bacterial, viral and protozoal enteropathogens in diarrheal and non-diarrheal stools of children aged less than 5 years in Madagascar. We present here the results of the analysis of antimicrobial susceptibility of the bacteria isolated during this study. METHODS: The study was conducted in the community setting in 14 districts of Madagascar from October 2008 to May 2009. Conventional methods and PCR were used to identify the bacteria; antimicrobial susceptibility was determined using an agar diffusion method for enterobacteriaceae and MICs were measured by an agar dilution method for Campylobacter sp. In addition to the strains isolated during this study, Salmonella sp and Shigella sp isolated at the Pasteur Institute of Madagascar from 2005 to 2009 were included in the analysis to increase the power of the study. RESULTS: Twenty-nine strains of Salmonella sp, 35 strains of Shigella sp, 195 strains of diarrheagenic E. coli, 203 strains of C. jejuni and 71 strains of C. coli isolated in the community setting were tested for antibiotic resistance. Fifty-five strains of Salmonella sp and 129 strains of Shigella sp isolated from patients referred to the Pasteur Institute of Madagascar were also included in the study. Many E. coli and Shigella isolates (around 80%) but fewer Salmonella isolates were resistant to ampicillin and trimethoprim/sulfamethoxazole. A small proportion of strains of each species were resistant to ciprofloxacin and only 3% of E. coli strains presented a resistance to third generation cephalosporins due to the production of extended-spectrum beta-lactamases. The resistance of Campylobacter sp to ampicillin was the most prevalent, whereas less than 5% of isolates were resistant to each of the other antibiotics. CONCLUSION: The highest prevalence of antimicrobial resistance was to ampicillin and trimethoprim/sulfamethoxazole. Antibiotic treatment is not recommended for children with diarrhea in Madagascar and the emphasis should be placed on oral rehydration.

Campylobacter infection in a cohort of rural children in Moramanga, Madagascar.

BACKGROUND: Campylobacter infection is the most common cause of bacterial gastroenteritis in developing countries, including Madagascar. Reports of pathogenicity have not been consistent and repeated exposures over time seem to lead to the development of protective immunity in developing areas. We conducted this study to support evidence for these hypotheses by exploring the association between infection and age, the reoccurrence of infection and the pathogenicity of Campylobacter. METHODS: We carried out a community-based longitudinal study of children under the age of 24 months in two rural villages in Moramanga, Madagascar. Children were visited twice a week and a stool specimen was collected in cases of diarrhoea. Stools specimens were collected bimonthly from all children enrolled, regardless of symptoms. Children were followed-up until the age of 36 months. RESULTS: Between January 2010 and May 31st 2012, 508 children were included in the cohort. We detected 319 episodes of Campylobacter infection in total, and 43.3% (n = 220) of the children had at least one episode of intestinal Campylobacter infection. The rate of Campylobacter isolation from stool specimens was 9.3%. The annual incidence rate for symptomatic Campylobacter infection was 0.05 episodes/child. The probability of Campylobacter infection was highest between the ages of six and 23 months. Taking children under six months of age as the reference group, the age-specific odds ratio for the association was 5.0 (95% CI: 2.9-8.6) for children aged six to 11 months, 5.7 (95% CI: 3.3-10.0) for children aged 12 to 17 months and 3.3 (95% CI: 1.8-5.8) for children aged 18 to 23 months. A second episode of infection occurred 63 days after the first episode in children with primary infections, and after 137 days in children with multiple infections (p < 0.01). First episodes of Campylobacter infection were associated with diarrhoea (odds ratio = 16.1; 95% CI: 1.8-140.8). CONCLUSION: Our findings suggest that protective immunity to Campylobacter may be acquired over time, following repeated exposures. However, Campylobacter infection prevention measures should be reinforced in the first year of life, as this age seems to be associated with the highest risk of diarrhoea during Campylobacter infection.

Molecular characterization of multidrug-resistant extended-spectrum ß-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar.

BACKGROUND: We investigated the molecular characteristics of multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar. RESULTS: Forty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(6')-Ib,aac(6')-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods.Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone. CONCLUSIONS: Our results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo.

Dissemination of multidrug resistant Acinetobacter baumannii in various hospitals of Antananarivo Madagascar.

This study reports the dissemination of multidrug-resistant (MDR) OXA-23-producing Acinetobacter baumannii clones in hospitals in Antananarivo, Madagascar. A total of 53 carbapenem-resistant A. baumannii isolates were obtained from September 2006 to March 2009 in five hospitals. These resistant strains represent 44% of all A. baumannii isolates. The double disk synergy test was performed to screen for production of metallo-beta-lactamases. Polymerase chain reaction (PCR) and DNA sequencing were performed for the detection of bla(AmpC), bla(OXA-51),bla(OXA-23), bla(OXA-24), bla(IMP), bla(VIM). The presence of the insertion sequence ISAba1 relative to blaOXA-23 and blaOXA-51 was assessed by PCR. Isolates were typed by Rep-PCR. All the isolates were MDR and produced the OXA-23 carbapenemase, which was confirmed by sequencing. PCR analysis for AmpC and OXA-51 gave positive results for all strains studied. No isolates produced metallo-beta-lactamases. In all isolates ISAba1 laid upstream of blaOXA-23. The A. baumannii isolates were separated into two genotypes; genotype A had a higher prevalence (41 strains) than genotype B (12 strains). Genotype A was present in four hospitals, whilst genotype B had spread in two hospitals. The high frequency of MDR OXA-23-producing A. baumannii in various hospitals in Antananarivo is curious since carbapenems are not available in Madagascar, but it emphasises the need for infection control procedures and strict adherence to them to prevent the spread of these resistant organisms in Antananarivo and also the need to control the use of carbapenems in the future.

Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries.

The main purpose of this study was to assess the accuracy of various techniques available for diagnosis of malaria. Blood samples were collected from 313 patients with clinical suspicion of uncomplicated malaria in 2 primary health centers in Madagascar. The presence of Plasmodium parasites was assessed by conventional microscopy, 2 rapid diagnostic tests (one HRP2-based test, PALUTOP(+4), and one pLDH-based test, OptiMAL-IT), and real-time polymerase chain reaction (PCR), which is used as the "gold standard" method. The degree of agreement observed was very high for microscopy (0.99) and the HRP2-based test (0.93) and high for the pLDH-based test (0.82). Public-health implications are also discussed in this paper.

Case-control study of the etiology of infant diarrheal disease in 14 districts in Madagascar.

BACKGROUND: Acute diarrhea is a major cause of childhood morbidity and mortality worldwide. Its microbiological causes and clinico-epidemiological aspects were examined during the rainy seasons from 2008 to 2009 in 14 districts in Madagascar. METHODS: Stool specimens of 2196 children with acute diarrhea and 496 healthy children were collected in a community setting. Intestinal parasites were diagnosed by microscopy and bacteria by culturing methods. Rota-, astro and adenoviruses were identified using commercially available ELISA kits and rotaviruses were confirmed using reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Intestinal microorganisms were isolated from 54.6% of diarrheal patients and 45.9% of healthy subjects (p = <0.01). The most common pathogens in diarrheic patients were intestinal parasites (36.5%). Campylobacter spp. and Rotavirus were detected in 9.7% and 6.7% of diarrheic patients. The detection rates of Entamoeba histolytica, Trichomonas intestinalis and Giardia lamblia were much greater in diarrheal patients than in non diarrheal subjects (odds ratios of 5.1, 3.2, 1.7 respectively). The abundance of other enteropathogens among the non diarrheal group may indicate prolonged excretion or limited pathogenicity. CONCLUSION: In developing countries, where the lack of laboratory capacities is great, cross sectional studies of enteropathogens and their spatial distribution, including diarrheal and non diarrheal subjects, are interesting tools in order to advise regional policies on treatment and diarrheic patient management.