Trafficking of methicillin-resistant staphylococci and co-colonization with vancomycin-resistant enterococci.

OBJECTIVES: To determine the trafficking of methicillin-resistant staphylococci between the hospital and community as well as the occurrence of co-colonization with vancomycin-resistant enterococci (VRE). SUBJECTS AND METHODS: From November 2005 to April 2006, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MRCoNS)-positive patients at the Salmaniya Medical Complex, Bahrain were assessed for VRE co-colonization. Characterization of vancomycin resistance genotype by PCR was carried out. Close family contacts were screened for MRSA and pulsed-field gel electrophoresis (PFGE) analysis of MRSA isolates from patient-family member pairs was conducted. RESULTS: One hundred and eighty-two patients (93 MRSA; 89 MRCoNS) and 356 family members were enrolled. Seven MRSA and 41 MRCoNS strains were isolated from the family members. PFGE analysis revealed the presence of variants of a single MRSA clone among patients and their relatives. A total of 112 patients (62 MRSA; 50 MRCoNS) provided stool for VRE screening. Of these 13 stool specimens (11.6%) were VRE-positive. All the VRE isolates were from MRSA-positive patients, thus positivity rate among MRSA patients was 20.9% (n/N = 13/62). These were predominantly Enterococcus gallinarum with vanC1 genotype and one strain was Enterococcus faecium (vanB genotype). Two E. gallinarum isolates harbored an additional vanB gene. The majority of VRE isolates were from patients in medical and surgical units (n/N = 10/13; 77%). Male gender, prolonged hospitalization and presence of co-morbidities were significantly associated with MRSA/VRE co-colonization (p < 0.05). CONCLUSION: MRSA/VRE co-colonization with MRSA trafficking between the hospital and community environment is a public health concern occurring in our setting.

Predominance of CTX-M genotype among extended spectrum beta lactamase isolates in a tertiary hospital in Saudi Arabia.

OBJECTIVE: To determine the molecular characterization of extended-spectrum beta-lactamases (ESBL) isolates from a tertiary center in Saudi Arabia using multiplex polymerase chain reaction (PCR) technique and assess their antibiotic susceptibility pattern. METHODS: Prospective study conducted at the Saudi Aramco Dhahran Health Center, Dhahran, Saudi Arabia between April-December 2006. Extended-spectrum beta-lactamases phenotype of isolates identified by automated methods was confirmed using E-test. Multiplex PCR for the detection of blaTEM, blaSHV and blaCTX-M was performed. Susceptibility to a panel of antibiotics was determined. RESULTS: One hundred isolates (Escherichia coli [E.coli] n=84; Klebsiella pneumoniae [K. pneumoniae] n=16) were studied and 71% harbored the blaCTX-M gene. For E.coli isolates 43 (51%) harbored CTX-M+TEM combination and 21 (25%) had CTX-M alone. In contrast, only one K. pneumoniae isolate (6.2%) harbored the CTX-M+TEM combination and 3 (18.8%) isolates had CTX-M only. One E.coli and 7 K. pneumoniae isolates were blaSHV positive. The blaCTX-M gene was found predominantly in urinary isolates (n=63/71; 88.7%). The presence of blaCTX-M was significantly higher in isolates from outpatients compared to inpatient (p<0.05). Sensitivity to imipenem was 100% and 78% to nitrofurantoin. Resistance to amoxicillin-sulbactam was significantly higher in blaCTX-M positive isolates (p<0.05). CONCLUSION: The findings indicate a high-level of blaCTX-M positive ESBL isolates circulating in our setting with the dissemination of these in the community. The trend of multidrug resistance profile associated with carriage of blaCTX-M gene is cause for concern.

Probiotics for the treatment of bacterial vaginosis.

BACKGROUND: The dominance of lactobacilli in healthy vaginal microbiota and its depletion in bacterial vaginosis (BV) has given rise to the concept of oral or vaginal instillation of probiotic Lactobacillus strains for the management of this condition. OBJECTIVES: To ascertain the efficacy of probiotics in the treatment of BV. SEARCH STRATEGY: We searched electronic databases irrespective of publication status or language. These included: Cochrane Central Register of Controlled Trials (CENTRAL), the HIV/AIDS and STD Cochrane Review Groups' specialized registers, the Cochrane Complementary Medicine Field's Register of Controlled Trials, MEDLINE (1966 to 2008), EMBASE (1980 to 2007), ISI science citation index (1955 to 2007), CINAHL (Cumulative Index to Nursing & Allied Health Literature (1982 to 2007).We handsearched of specialty journals, conference proceedings and publications list on the website of the International Scientific Association of Probiotics and Prebiotics (http://www.isapp.net/default.asp).For unpublished studies or ongoing trials, we contacted authors from relevant publications, nutraceutical companies and probiotic-related scientific associations. We searched electronic databases on ongoing clinical trials. SELECTION CRITERIA: Randomized controlled trials using probiotics for the treatment of women of any age diagnosed with bacterial vaginosis, regardless of diagnostic method used. The probiotic preparation could be single or "cocktail" of strains, any preparation type/dosage/route of administration. Studies comparing probiotics with placebo, probiotics used in conjunction with conventional antibiotics compared with placebo or probiotics alone compared with conventional antibiotics were eligible for inclusion. DATA COLLECTION AND ANALYSIS: We screened titles and abstracts , obtained full reports of relevant trialsand independently appraised them for eligibility. A data extraction form was used to extract data from the four included studies. For dichotomous outcomes, odds ratios (OR) and 95% confidence intervals (CI) were derived for each study using RevMan (versions 4.2 and 5). We did not perform meta-analysis due to significant differences in the probiotic preparations and trial methodologies. MAIN RESULTS: Analysis suggests beneficial outcome of microbiological cure with the oral metronidazole/probiotic regimen (OR 0.09 (95% CI 0.03 to 0.26)) and the probiotic/estriol preparation (OR 0.02, (95% CI 0.00 to 0.47)). For the probiotic/estriol preparation, the OR and 95% CI for physician-reported resolution of symptoms was OR 0.04 (95% CI 0.00 to 0.56). AUTHORS' CONCLUSIONS: The results do not provide sufficient evidence for or against recommending probiotics for the treatment of BV. The metronidazole/probiotic regimen and probiotic/estriol perparation appear promising but well-designed randomized controlled trials with standardized methodologies and larger patient size are needed.

Campylobacter enteritis in the Arabian Gulf.

Diarrhoea illnesses constitute a common cause of morbidity and mortality worldwide. In recent years, Campylobacter spp. has been recognized as the leading cause of bacterial enteritis in both developed and developing countries. The biology of Campylobacters as well as the mechanism by which they cause disease is yet to be fully explained. In addition, non-availability of fast and reliable diagnostic methodology and the growing trend of antibiotic resistance continue to pose significant challenges. The absence of national surveillance programs for campylobacteriosis, particularly in developing countries, makes it difficult to give an accurate picture of the true infection prevalence and the molecular epidemiology of isolates circulating in the populations, a situation which had hitherto existed in the Arabian Gulf region. However, in recent years, emerging data from studies in the Arabian Gulf region has not only enhanced our understanding of the epidemiology of Campylobacter both in humans and poultry in the region, but has also contributed to the overall understanding of the mechanism of Campylobacter enteritis, antibiotic resistance, and improved diagnostic approaches. In this review, we examine these emerging data from the Arabian Gulf region.

Extended spectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae: trends in the hospital and community settings.

BACKGROUND: To assess the prevalence of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and Klebsiella strains in nosocomial and community-acquired infections. METHODOLOGY: The study was conducted at a centralized microbiology laboratory in the Eastern Province of Saudi Arabia. Laboratory records (January 2004 - December 2005) were assessed. Associated resistance to a panel of antibiotics was determined. RESULTS: A total of 6,750 Gram-negative organisms were assessed for ESBL-phenotype. ESBL was detected in 6% (409/6,750) of isolates, the majority of which were E. coli (83%). ESBL producers were significantly higher among isolates from in-patients 15.4% (143/927) versus out-patients (4.5%; 266/5,823); p < 0.05. Old age (older than 60 years) represented a significant risk for having an ESBL-producing pathogen. Urine was the major source of ESBL isolates in in-patients (46.1%) and out-patients (74.4%). The proportion of urinary E. coli isolates which were ESBL producers was significantly higher among in-patients (53/506; 10.4%) compared to out-patients (182/4,074; 4.4%); p<0.05. Old age (older than 60 years) represented a significant risk for having an ESBL-producing pathogen. Urine was the major source of ESBL isolates in in-patients (46.1%) and out-patients (74.4%). The proportion of urinary E. coli isolates which were ESBL producers was significantly higher among in-patients (53/506; 10.4%) compared to out-patients (182/4,074; 4.4%); p<0.05. Among in-patients, 60% of the ESBL associated infections were nosocomial. All were sensitive to imipenem but high levels of resistance to gentamicin, amikacin, amoxicillin-clavulanic acid and ciprofloxacin was shown. CONCLUSION: The findings document evidence of the spread of multiresistant ESBL-producers into the community. This has significant implications for patient management, and indicates the need for increased surveillance and molecular characterization of these isolates.

Campylobacter jejuni induces diverse kinetics and profiles of cytokine genes in INT-407 cells.

OBJECTIVE: To examine the kinetic ability of embryonic human epithelial INT-407 cells to express messenger ribonucleic acid mRNA for various cytokines and chemokines in response to Campylobacter jejuni C. jejuni stimulation. METHODS: In an experimental single-blind study, cultured embryonic human epithelial INT-407 cells were treated with different concentrations of viable C. jejuni, its sonicated, and filtered supernatant. A modified non-radioactive in situ hybridization using probe cocktails was used to measure mRNA levels for the pro-inflammatory cytokines interleukin IL-1beta, IL-6, interferon-gamma IFN-gamma, tumour necrosis factor TNF-alpha, transforming growth factor TGF-beta1, and IL-8, and the anti-inflammatory cytokines, IL-4 and IL-10. The study was carried out from September 2005 to March 2007 at the Department of Microbiology, Immunology, and Infectious Diseases, College of Medicine, Arabian Gulf University, Bahrain. RESULTS: Viable C. jejuni, sonicated bacteria and filtered supernatant induced high mRNA expression for the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, TNF-alpha, TGF-beta1, and IL-8, which peaked at the 12 hours post stimulation. Anti-inflammatory cytokines IL-4 and IL-10 mRNA expression were induced maximally at 3 hours post stimulation mainly by sonicated bacteria and filtrated supernatant, however, not with living bacteria. Untreated embryonic human epithelial INT-407 cells expressed low amount of mRNA for the various cytokines and chemokines at all time points. For each cytokine, 4 samples were used per time hour. CONCLUSION: This study demonstrated that embryonic human epithelial INT-407 cells in response to viable C. jejuni or its cytotoxins can alter cytokine and chemokine mRNA expression patterns and kinetics suggesting a potential role for theses mediators in the immunopathogenesis of the infection caused by this pathogen, which might be relevant for future immunotherapeutic interventions during severe bacterial infections.

Management of acute diarrhoea in primary care in Bahrain: self-reported practices of doctors.

This nationwide study was conducted to assess the extent of adherence of primary-care physicians to the World Health Organization (WHO)-recommended guidelines on the use of oral rehydration therapy (ORT), antimicrobials, and prescribing of other drugs used in treating symptoms of acute diarrhoea in Bahrain. A questionnaire-based, cross-sectional survey was carried out in primary-care health centres. During a six-week survey period (15 August-30 September 2003), 328 (25.2%) completed questionnaires were returned from 17 of 20 health centres. In a sample of 300 patients, oral rehydration salts (ORS) solution was prescribed to 89.3% (n=268) patients; 12.3% received ORS alone, whereas 77% received ORS in combination with symptomatic drugs. Antimicrobials were prescribed to 2% of the patients. In 11.4% of the cases, rehydration fluids and other drugs were given parenterally The mean number of drugs was 2.2+0.87 per prescription. In approximately one-third of the patients, three or more drugs were used. Primary-care physicians almost always adhered to the WHO guidelines with respect to ORT and antimicrobials. However, in several instances, ORT was prescribed along with polypharmacy, including irrational use of drugs for symptomatic relief. Effective health policies are needed to reduce the unnecessary burden on the healthcare system.

Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools.

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015-0.016 ng C. jejuni and C. coli DNA mul(-1) in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

Clinical relevance of virulence genes in Campylobacter jejuni isolates in Bahrain.

There are no data describing the genetic make-up of Campylobacter strains (an important aetiological agent of diarrhoea) circulating in the Arabian Gulf region. Here, the molecular characterization of two virulence genes in Campylobacter jejuni from Bahrain and the relationship with clinical infection are reported. Molecular screening for cytolethal distending toxin (cdtB) and invasion-associated marker (iam) genes was carried out on C. jejuni stool isolates collected from January 2002 to January 2004 in Bahrain. The molecular characterization was correlated with the patients' socio-demographic and clinical parameters. Of the 96 C. jejuni strains tested, 50 (52 %) were cdtB+/iam+, 30 (31 %) were cdtB+/iam- and 16 (17 %) were cdtB-/iam-. Sixty-nine per cent (66/96) of patients were less than 3 years old, with significantly higher detection of cdtB+/iam+ and cdtB+/iam- strains (P < 0.001 and P < 0.01, respectively) in this age group. Seventy patients (73 %) were symptomatic. In the group that were less than 3 years old, 62 and 85 % of those with cdtB+/iam+ and cdtB+/iam- strains, respectively, were symptomatic compared with 100 % for those over 3 years of age. However, the presence of cdtB-/iam- strains still resulted in clinical infection in the children under 3 years but not in the older patients. This is the first report describing the molecular characterization of virulence genes in Campylobacter isolates from this region. The findings indicate that strains of different virulence genetic make-up are circulating in the population, with children under the age of 3 years being most vulnerable. Further work on the molecular characterization, gene expression and determination of the invasive phenotypes of C. jejuni strains circulating in different regions is needed.

Human papillomavirus infection among women attending health facilities in the Kingdom of Bahrain.

OBJECTIVE: To investigate the occurrence of human papillomavirus (HPV) infection and the associated risk factors in Bahrain's female population. METHODS: This study was carried out between March to December 2004, which includes cervical scrapings for Pap smear and HPV-DNA testing using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis, obtained from 100 women attending the Gynecology Clinic at Salmaniya Medical Center and Sheikh Sabah Health Center in the Kingdom of Bahrain. We distributed questionnaires that include the sociodemographic data as well as information on risk factors such as smoking, parity, and the contraceptive used. RESULTS: Eleven women (11%) with normal cytology were HPV-positive. The RFLP analysis detected HPV-types 16, 18, 45, 62 and 53. Positive women were significantly older (43.3 +/- 10.1 years) than negatives (36.5 +/- 9.9 years; p=0.04), however, there was no difference in age of first sexual contact (positive: 18.1 +/- 5.7 years versus negative: 20.6 +/- 4.4 years). Polygamy, smoking and hormonal contraception was not identified as risk factors, but positive women showed higher parity. CONCLUSION: In this study on HPV infection in Bahrain, the 11% positivity with high risk HPV types, in the presence of normal cytology suggests that in addition to the cervical cancer screening program, offer of HPV testing deserves consideration.

Neonatal sepsis 1991-2001: prevalent bacterial agents and antimicrobial susceptibilities in Bahrain.

OBJECTIVES: To investigate the organisms causing neonatal sepsis and their modifications over an extended period, to assess their changing sensitivities to antibiotics and to verify whether the policy for screening pregnant women for group B streptococci (GBS) carriage is desirable in our settings. SUBJECTS AND METHODS: Medical records of all infants with positive blood culture from the Neonatal Intensive Care Unit at Salmaniya Medical Complex between 1991 and 2001 and Bahrain Defense Force Hospital between 1999 and 2001 were reviewed. RESULTS: Of the 7,978 neonates in both hospitals 335 (4.19%) had culture-proven bacteremia. Gram-positive bacteria were isolated at constant rate over the 11-year period. The main agents isolated were coagulase-negative Staphylococcus (CoNS) in 138 cases (41%), Staphylococcus aureus in 28 newborns (8%) and GBS in 26 patients (7.8%, 0.2/1,000 live births). All of them were sensitive to penicillin G, erythromycin and clindamycin. Gram-negative bacteria were declining but Escherichia coli was isolated in 35 cases (10%). Of special concern is the increasing percentage (5.7%) of Candida isolation. No clear trend toward increasing resistance was observed, although a major difference among the two institutions was evident. Klebsiella and Enterobacter spp. showed resistance to many of the antibiotics tested, thereby posing difficult therapeutic choices. CONCLUSION: Good quality specimens are essential to evaluate the role of CoNS. The increasing threat of fungal infection must be carefully tackled. Specifically tailored policies for GBS prevention must be defined according to the local epidemiology.

Effect of antibiotic sub inhibitory concentration on cytolethal distending toxin production by Campylobacter jejuni.

OBJECTIVES: To investigate the effect of pre-exposure antibiotics on cytolethal distending toxin (CDT) production and toxigenic effect of C. jejuni. METHODS: Sonicates and filtrates were prepared from known cdt+ and cdt- isolates of C. jejuni which had been pre-exposed to varying concentrations (MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC) of erythromycin and ciprofloxacin. The CDT toxigenic effect was examined using INT 407 and HeLa cells. RESULTS: A trend of increased toxigenic effect was observed with pre-exposure to antibiotics. This was more pronounced with erythromycin pre-exposure compared to ciprofloxacin. Although a trend of increasing toxigenic effect with decreasing antibiotic concentration was demonstrable, some differences were observed between isolates. In one isolate the increased toxigenic effect was statistically significant (P<0.05) at 1/4 MIC in INT 407 cells and at 1/8 MIC in HeLa cells. CONCLUSIONS: This study provides evidence of an association between CDT production by C. jejuni and pre-exposure to antibiotics. Pre-exposure to ciprofloxacin and erythromycin at concentrations below MICs could potentiate CDT activity. Further work is needed to elucidate the mechanism involved. We recommend that these antibiotics be used in the treatment of C. jejuni enteritis only when strongly indicated and with careful monitoring of patients.

Status of malaria in the Kingdom of Bahrain: a 10-year review.

BACKGROUND: Malaria was eradicated in Bahrain about 20 years ago. However, because of a large immigrant population, cases of imported malaria continued to be seen. This paper presents an evaluation of the data from 1992 to 2001 to assess the levels of indigenous and imported malaria cases and the potential for reemergence of malaria transmission in the country. METHODS: Epidemiologic and parasitologic data on confirmed malaria cases during the review period were analyzed. Data on vector breeding activity were also analyzed to determine the potential for reemergence of local transmission. RESULTS: From 1992 to 2001, 1,572 cases of malaria were reported. All were imported malaria cases. There was a consistent decline in the number of cases, from 282 (the peak level) in 1992 to 54 in 2001. Eighty-four percent (1,318/1,572) of infections were contracted as a result of travel to or previous domicile in five countries, namely India, Pakistan, Sri Lanka, Bangladesh and Sudan. Plasmodium vivax was the etiologic agent in 85.6% (1,346/1,572) of cases, with Plasmodium falciparum accounting for 14% (220/1,572). During this period, the percentage of examined breeding sites where Anopheles mosquito larvae were identified remained fairly constant, ranging between 0.05% and 0.1%. CONCLUSIONS: From 1992 to 2001, there was a consistent decline in the number of imported malaria cases in Bahrain. There were no cases of locally acquired malaria during this period. The low level of vector breeding spots with Anopheles mosquito larvae suggests that the potential for reemergence of local transmission remains low.

Induction of alpha and beta chemokines by intestinal epithelial cells stimulated with Campylobacter jejuni.

OBJECTIVES: To investigate the production of dynamic alpha and beta chemokines represented by interleukin-8 (IL-8) as alpha chemokine and CCL2 (monocyte-chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1beta, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1alpha, CCR1/5 ligand), (CCL5, regulated upon activation, normal T-cell expressed and secreted (RANTES, CCR5 ligand) as beta chemokines by the human intestinal cell line INT407 stimulated with factors produced by living Campylobacter jejuni (C. jejuni) and those present within sonicated and filtrated bacteria. METHODS: We used immunohistochemical technique modified to detect intracellular production of cytokines protein and RT-PCR to read RNA messages for evaluation of de novo cytokine synthesis. RESULTS: Living bacteria induced increased numbers of IL-8, CCL4 and CCL2 but not CCL3 or CCL5 producing cells. Low numbers of IL-8, CCL4 and CCL2 producing cells were detected with filtrated supernatant compared to living and sonicated bacteria. A non-significant low number of chemokine producing cells was noted when comparing numbers of chemokine producing cells stimulated with living C. jejuni to those stimulated with sonicated bacteria, indicating that the triggering factors involved in stimulation with living bacteria were still active after sonication, but they were largely lost upon filtration. The mRNA signals for IL-8 were noted in conformity with its protein levels as increased IL-8 mRNA signals were registered after stimulation with living and sonicated bacteria but not with filtrated supernatant. CONCLUSIONS: Preferential production of chemokines probably induced by membrane associate factors of C. jejuni acting on intestinal epithelial cells is presented. These chemokines are suggested to be part of an inflammatory network affecting cell types that contribute to initiation and/or resolution of the infection.

Campylobacter-stimulated INT407 cells produce dissociated cytokine profiles.

OBJECTIVES: To study the action of factors produced by living Campylobacter jejuni (C. jejuni) against those present within sonicated and filtrated bacteria on induction of potential cytokines by the human intestinal cell line INT407. METHODS: We used immunohistochemical technique modified to detect intracellular production of cytokines protein and RT-PCR to read RNA messages for evaluation of de novo cytokine synthesis. RESULTS: The data herein display dissociation of cytokine profiles induced on by living C. jejuni. Exposure of INT407 cells to 10(6) live bacteria showed the highest numbers of cytokine producing cells of all examined cytokines. IFN-gamma was the highest induced cytokine followed by IL-10, TNF-alpha and lastly IL-4. Also, abrogation of induction of the proinflammatory cytokines IFN-gamma and TNF-alpha but not the antiinflammatory cytokines IL-4 and IL-10 by sonicated and filtrated bacteria was depicted. At the mRNA level, TNF-alpha signals were noted in accordance with its protein levels since increased TNF-alpha mRNA signals were registered only after stimulation with living bacteria. Very low or no induction of TNF-alpha was registered with non-stimulated cells. CONCLUSIONS: These results illustrate for the first time a role for factors from living bacteria in directing the immune response towards Th1 type. Characterization of such factors may be essential for future immunotherapeutic interventions during severe bacterial infections.

Causative pathogens of severe diarrhea in children.

OBJECTIVES: To investigate the enteropathogens in children with diarrhea attending Salmaniya Medical Complex, Bahrain. METHODS: Fecal samples from 805 children up to 15 years were examined for parasites, ova and cysts by direct wet preparation, formol-ether concentration and modified Ziehl-Neelsen stain, during the period November 1998 through to June 2000. Samples were cultured for Salmonella, Shigella, Campylobacter and Enteropathogenic Escherichia coli. Antibiotic sensitivity tests were performed on the relevant clinical isolates by agar disk diffusion method. All stools from children below 3 years of age (653 samples) were processed for adenovirus and rotavirus using a commercially available latex agglutination test (Diarlex). In addition, reverse transcription-polymerase chain reaction was performed on 200 randomly selected samples using oligonucleotide primers for Rotavirus A, B and C. RESULTS: Four subjects were found positive for parasites. Eighty-three (10.3%) samples were found positive for Salmonella (46 isolates), Shigella (26 isolates), Campylobacter jejuni (7 isolates), and Enteropathogenic Escherichia coli (4 isolates). Rotavirus was found in 91 (13.9%) samples and 4 samples (0.6%) were found positive for adenovirus. Out of 200 samples examined by reverse transcription-polymerase chain reaction, 73 (36.5%) were positive for group A rotavirus. CONCLUSIONS: Rotavirus type A appeared to be the most common single agent in our pediatric population, followed by the classical bacterial pathogens. Adenovirus and parasites appeared to play a very minor role in diarrhea. Thus, we suggest the introduction of rotavirus diagnostic tests in microbiological examination of diarrheic stools of children below 3 years of age.