Study of Spectrum of Diarrheal Agents and the Contributing Factors

Abstract

Diarrhea can be defined as “passing of a liquid stool more than three times within 24 hours”, it is consistency rather than frequency which is more important and major cause infantile deaths in underdeveloped world in young children<5 years. In Pakistan five million children are born every year, 10% of these die before reaching their first birthday and 14% die before the age of five years which accounts to nearly 70,000 - 150,000 children under the age of five years annually i.e. nearly 2000 children die every day. More than one third of the total hospital admissions are due to diarrheal disorder and ultimately leading to 5-6 episodes of infection per child annually leading towards malnutrition and stunted growth. Comprehensive local studies to determine full spectrum of types and subtypes of bacterial, viral, or protozoal pathogens as causative agents of diarrhea and the contributing factors for high prevalence of gastrointestinal infection in young children < five years of age have not been conducted in different provinces of Pakistan. The role of hand hygiene, food handlers, quality of water, vender food and its safety data is missing and information regarding the storage and transport of food materials, fast food items sold in local market as important contributing factors needs to be determined. Serious health consequences due to multiple episodes of avoidable diarrhea are not highlighted due to lack of availability of reliable data. In view of the above observations, we designed this study to determine the spectrum of diarrheal pathogens in children reporting to the diarrheal unit of local government hospital catering to the needs of urban and rural low socioeconomic areas of Karachi and other cities of Sindh province. This study also aimed to conduct a detailed survey of sanitary, phytosanitary and environmental conditions as contributing factors for high incidence of diarrhea and to determine the quality of vender food, water as well as role of food processing techniques for consumer safety. This study was carried out during Jan 2013 to June2015. In this duration a total of 1465 patients were recruited, demographic data for qualitative data sets like basic information of included patients gender, age group, socio- economic status, personal hygiene, clinical information regarding vomiting diarrhea and fever and diarrheal episode per twenty-four hours, BMI body mass index etc. was recorded. Stool samples of diarrheal patients who reported with acute watery diarrhea at NICH and CIVIL hospitals which are tertiary care public hospital, who cater to the needs of Karachi city and areas xiv of interior Sindh province were processed for the presence of protozoal, bacterial and viral pathogens, which were identified by conventional microscopy, culture, serology and isolated genotypes of importance were confirmed by advanced molecular techniques like PCR and RTPCR. All lactose and non-lactose fermenting bacterial colonies were processed for the identification of bacterial pathogens and verified biochemically. All bacterial isolates including E. coli, Salmonella, Shigella, and V.cholera were serotyped by commercially available strain specific antiserum, and determination of antibiotic susceptibility screening against all bacterial pathogens isolated from patients stool samples, food and water pathogens, vended food handlers from near and around diarrheal patient’s residential areas, enviornrnental contaminants from patients and their attendants hands was done by Kirby Bauer disk diffusion method against commonly prescribed antibiotics. Fecal coli forms from water samples were isolated by processing samples by MPN technique and food sample were processed by pour plate method. Pathogenic E. coli cannot be distinguished from non-pathogenic E. coli by culture and biochemical characteristics only. So it is pre confirmed that weather it is pathogenic or non-pathogenic by using strain specific anti sera. The characterization of the genes that code for virulence factors is only possible by the molecular identification. In Pakistan the molecular typing of Pathogens from diarrheal stool samples for E. coli and Rotavirus genotypes is not done routinely in diagnostic settings and we never knew that which type of pathogenic strains are prevalent in our country. That is why all E. coli strains were characterized by PCR method using specific primers for genes: E.coli; uidA, st / It for ETEC; eae/bfpA for EPEC; stx1/inv for EIEC; stx2 for EHEC and amplified products were visualized by gel electrophoreses. Similarly, All Rotavirus were characterized for VP4, P[8], P[4], P[6], P[9], P[10], P[11] and RotaVirus VP7, (G8), (G1), (G2), (G4), (G3), (G9) capsid protean coding genes using specific primer sets by RT-PCR method by converting genomic RNA to CDNA. Analysis of recorded demographic data showed that out of 1465 samples 44.8% were female and 55.2% were males, who presented with the complain of acute watery diarrhea. It was also noted that predominant age groups suffering from diarrhea were twelve months old (65.3%), followed by12-14 months (20.1%) and > 36 months was 6%. According to BMI recorded results, we found that 10% children were overweight and 5% were obese. Using the number of family members living per square area indicated that 73% patients had 7-8 family members on average and 73.3% study population had poor personal hygiene standards at the time of interview. Clinical sign and xv symptoms of fever, vomiting and diarrheal episodes showed that patients present with combination of clinical indications with history of fever (22.4%), vomiting (48.07%). We recorded that 70% of patients reported 1-2 episodes of vomiting per 24 hours, whereas 62.3% has 3-4 episodes of persistent diarrhea. According to records majority of sick children (90.5%) reported that they did not receive compulsory routine childhood vaccination. An analysis of spectrum of diarrheal pathogens indicated that majority of patients were suffering from protozoal infection (24.02%) including Giardia lamblia (6.2%), Entamoebae histolytica (4.8%), Ascaries eggs (4.1%). In some cases, we found multiple pathogen i.e parasites, bacteria, as well viruses (1.7%), Entamoebae coli (1.5%), live worms (1.4%), H. nana (0.6%), Blastocyst hominis (0.2%) were also present. Next category of infectious agents included bacterial pathogens (15.35%). Among E. coli, isolates, EPEC (48.6%) was found to be most dominant diarrheal pathogen followed by ETEC (24.3 %), EAEC (21.6 %), EIEC (5.4%) and EHEC (0.0%). In addition to E. coli we also found other pathogens like Salmonellae (3.0%), V. cholera (2.4%), Shigellae (1.8%), C. jejuni (1.5%) as well as combination of more than one pathogen i.e. polyclonal infection (E. coli + Protozoa). Antibiotic susceptibility showed high level of resistance among diarrheal isolates. Most isolates were resistant to Cotrimaxazole (Sxt), Ciprofloxacin (CIP), Amoxicillin (AML), Imipenum (IMP), Amikacin (AK), Augmentin (AMC), Tazocin and piperacillin (TZP), Cefxime (CXM), Cefotaxime (CTX) and Chloramphenicol (C). Most probable reason for this high level of resistance (60% -75%) is misuse or over use under situations where they are not needed like their use in Protozoal and Rotavirus infection. Findings indicated that antibiotic resistance among the environmental isolates from hand print of patient’s attendant as well as from food and water was comparatively low (20- 55%) against Ceftriaxone, Ciprofloxacin, Gentamicin and Imipenum. Perhaps environmental contaminants go in gut flora acquire resistance due to selective pressure of antibiotics used during blind therapy. A total of 120 hands and finger tip prints as well as environmental samples from patients and their attendants were collected and analyzed. Similarly, 150 water samples along with 120 vended food and surrounding environmental, samples from residential areas which frequently reported diarrheal cases were collected and analyzed. Variety of pathogens like E. coli, Salmonella, Pseudomonas, Proteus spp, Klebsiella pneumonia, S. aureus, S. epidermidis including fungal infection were xvi identified from these samples. Fecal contamination was found in different drinking water samples (16%-32%) in addition to above mentioned enteric and other serious disease causing pathogens. Seasonal variation for the presence of pathogens was noted as they were more frequently isolated in June to August during summer season. While isolation rate for Rota virus was more in December to January, however E coli was detected throughout the year. One of the common viral pathogen in infectious diarrhea is Rotavirus and more than 6% patients were found to be positive. Common Rotavirus genotypes which were detected in our patients were G10P4, G2P4, G2P6, G3P4, G8P4, G8P6, and G9P4 as determined by RT-PCR. In our studied population we found protozoal pathogens as the most dominant diarrheal etiological agents followed by EPEC and Rota virus along with other diarrheal pathogens in children under five years of age. Majority of diarrheal pathogens exhibited high level of resistance to major antibiotics used to treat different infectious diseases, which suggested irrational use of antibiotics for diarrheal infection which needs to be controlled. High level of antimicrobial resistance detected in this study among diarrheal pathogens is a serious public health concern, which is probably due to inappropriate use of antibiotic for simple manageable diarrheal, viral and protozoal infections where they are not required. It is therefore suggested that in addition to using conventional culture techniques, PCR and advanced molecular techniques should also be included in the diagnostic protocol for diarrheagenic pathogens to develop a better understanding of infectious agents and management of young patients. In our study food, water and environmental contaminants were recorded as contributing risk factors in the transmission of pathogens in infectious diarrheal episodes. Therefore, it is suggested that boiled drinking water and high sanitation standards as well as clean environmental interventions and hand washing be adopted to reduce the high incidence of avoidable diarrheal episodes in young infants and hospitalization and treatment cost in socioeconomically deprived and marginalized suffering population. All these interventions and proper management of diarrheal patients may reduce emergence of antimicrobial resistance among diarrheal pathogens and repeated episodes of diarrhea which deprive infants from essential nutrients leading to malnutrition, making them susceptible to diarrhea and other serious diseases as well as high morbidity and mortality rate in children less than five years of age.