Real-time PCR identification of agents causing diarrhea in Rwandan children less than 5 years of age.

BACKGROUND: Knowledge about causes of acute diarrhea among children in developing countries is insufficient. Molecular methods might improve diagnostics of infectious gastroenteritis, but due to the high sensitivity, findings may be difficult to interpret. METHODS: Feces samples from Rwandan children 0.5-5.0 years of age, with diarrhea for <96 hours (patients, n = 544) or without diarrhea for 14 days (controls, n = 162), were analyzed by real-time polymerase chain reaction targeting 17 pathogens. RESULTS: At least 1 agent was detected in 94% of patients and in 79% of controls, with higher rates in sick children for rotavirus (42% vs. 2%, P < 0.0001) and enterotoxigenic Escherichia coli (ETEC)-estA (21% vs. 9%, P = 0.0006). Detection rates did not differ significantly for adenovirus (39% vs. 36%), ETEC-eltB (29% vs. 30%), Campylobacter (14% vs. 17%) or Shigella (13% vs. 10%), but for Shigella the threshold cycle (Ct) values were lower (pathogen loads were higher) in sick children than in controls. By multivariate analysis, including gender and age, detection of rotavirus (P < 0.0001), ETEC-estA (P = 0.001), Shigella (P = 0.004) and norovirus genogroup II (P = 0.009) was associated with symptomatic infection, and a Ct value below a cutoff (in the range 28-29) improved identification of ETEC-estA, Shigella and norovirus genogroup II. CONCLUSION: Real-time polymerase chain reaction can detect essentially all diarrheagenic agents, and provides Ct values that improve identification of clinically relevant infections.

Comparison of rectal swabs and faeces for real-time PCR detection of enteric agents in Rwandan children with gastroenteritis.

BACKGROUND: Molecular diagnostics have emerged as an efficient and feasible alternative for broad detection of pathogens in faeces. However, collection of stool samples is often impractical in both clinical work and in epidemiology studies. The aim of this study was to investigate the diagnostic performance of rectal swabs as compared with traditional faeces samples for detection of enteric agents by PCR. METHOD: Three hundred twenty-six pairs of rectal swab and stool samples, obtained from Rwandan children aged 0.5-4.99 years, with or without diarrhoea, were analysed by multiple real-time PCR amplifying 3 viral, 6 bacterial and one protozoan target. RESULTS: For all agents there was a significant correlation (R2 0.31-0.85) between Ct values in faeces and rectal swabs. For most agents the Ct values, a marker for target concentration, were significantly lower (by 1-3 cycles) in faeces, indicating pathogen content up to ten times higher than in rectal swabs. Despite this, there was no significant difference in detection rate between faeces and rectal swabs for any agent, reflecting that pathogen concentration was far above the limit of detection in the majority of cases. CONCLUSION: The similar detection rates and the Ct value correlations as compared with traditional faeces samples indicate that rectal swabs are accurate for real-time PCR-based identification of enteric agents and may be used also for quantitative estimation of pathogen load.

Decreased susceptibility to commonly used antimicrobial agents in bacterial pathogens isolated from urinary tract infections in Rwanda: need for new antimicrobial guidelines.

The aim of this study was to obtain data on susceptibility patterns of pathogens responsible for both community and hospital urinary tract infections (UTIs); and analyzed risk factors for infection caused by ciprofloxacin-resistant Escherichia coli and extended-spectrum ß-lactamase (ESBL)-producing strains in Rwanda. Of 1,012 urine cultures prospectively studied, a total of 196 (19.3%) yielded significant growth of a single organism. The most common isolate (60.7%) was Escherichia coli. The antibiotics commonly used in UTIs are less effective except Fosfomycin-trometamol and imipinem. The use of ciprofloxacin in the previous 6 months (odds ratio [OR] = 7.59 [1.75-32.74]), use of other antibiotics in the previous 6 months (OR = 1.02 [1.02-2.34]), and production of ESBL (OR = 19.32 [2.62-142.16]) were found to be associated with ciprofloxacin resistance among the E. coli isolates. Risk factors for ESBL positivity were the use of ciprofloxacin and third-generation cephalosporin in the preceding 6 months (OR = 3.05 [1.42-6.58] and OR = 9.78 [2.71-35.25], respectively); and being an inpatient (OR = 2.27 [1.79-2.89]). Fosfomycin-trometamol could be included as a reasonable alternative for the therapy of uncomplicated UTI in Rwanda.