Case-Control Study of Enteropathogens Associated with Childhood Diarrhea in Dhaka, Bangladesh

The International Centre for Diarrhoeal Disease Research, Bangladesh, is a major center for research into diarrheal diseases. The center treats more than 100,000 patients a year. To obtain useful information representative of all patients, a surveillance system in which a 4% systematic sample of all patients is studied in detail, including etiological agents of diarrhea, was installed in October 1979. The first paper on etiology for the surveillance patients was published in 1982, which identified a potential enteric pathogen in 66% of patients. In subsequent years, several new agents of diarrhea have been identified. To assess the importance of a broader spectrum of diarrheal agents including the ones identified relatively recently, we studied 814 children with diarrhea. The children were up to 5 years of age and were part of the surveillance system. They were matched with an equal number of community controls without diarrhea. The study was conducted from February 1993 to June 1994. A potential enteric pathogen was isolated from 74.8% of diarrheal children and 43.9% of control children (P = 0.0001). Even though the first study was not a case-control study, it identified rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp. , and Vibrio cholerae O1 as major pathogens. The present study identified these pathogens as being significantly associated with diarrhea. In addition, the study also identified six additional agents, including enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile, and Cryptosporidium parvum, as being significantly associated with diarrhea. Plesiomonas shigelloides, Salmonella spp., diffusely adherent E. coli, enteroaggregative E. coli, Entamoeba histolytica, and Giardia lamblia were not significantly associated with diarrhea. Enteroinvasive E. coli, enterohemorrhagic E. coli, and Cyclospora cayetanensis were not detected in any of the children. The major burden of diseases due to most pathogens occurred in the first year of life. As in the previous study, seasonal patterns were seen for diarrhea associated with rotavirus, V. cholerae, and enterotoxigenic E. coli, and infections with multiple pathogens were common. With a few exceptions, these findings are in agreement with those from other developing countries. This knowledge of a broader spectrum of etiological agents of diarrhea in the surveillance patients will help us plan studies into various aspects of diarrheal diseases in this population.     

Three-year prospective study of intestinal pathogens in Madrid, Spain.

During the period July 1980 through June 1983, in a General Hospital in Madrid, the following organisms were detected from 6,970 patients with gastroenteritis: 710 Salmonella spp. 506 Campylobacter jejuni, 379 Shigella spp., 12 Yersinia enterocolitica, 1,466 rotavirus, 134 Giardia lamblia, and 4 Entamoeba histolytica. Chloramphenicol showed good activity against most tested strains of Salmonella spp., Shigella spp., and C. jejuni. The incidence of Salmonella spp. and Shigella spp. was very marked in the hot dry months of the year, rotavirus predominated during the cold months, and no seasonal variations of importance were seen for C. jejuni and G. lamblia.     

Etiology of childhood diarrhea in Korea.

To assess the role of recently recognized enteropathogens in childhood diarrhea in Korea, 231 children with diarrhea admitted to and 104 children without diarrhea seen at the well-baby clinic or the outpatient department of Hanyang University Hospital in Seoul, Korea, were evaluated during a 14-month period. Stools were cultured for bacterial pathogens, including enterotoxigenic (heat-labile and heat-stable enterotoxin-producing) and enteroadherent organisms. Only those stools obtained from patients with diarrhea were examined for rotavirus. All Escherichia coli isolates were screened for Shiga-like toxin (SLT) I, SLT-II, enterohemorrhagic E. coli fimbriae, and enteroinvasiveness by colony hybridization. One or more pathogens were identified in 75.8% of the children with diarrhea. Rotavirus was the most frequently identified pathogen, accounting for 47% of the cases. Other major enteropathogens were enterotoxigenic E. coli (22%), Clostridium difficile (16%), enteroadherent E. coli (15%), and enteropathogenic E. coli (6%). Shigella spp., Campylobacter jejuni, Salmonella spp., SLT-I-and enterohemorrhagic-E. coli-fimbria-probe-positive E. coli serotype O26:H11 and enteroinvasive E. coli were isolated from only a few patients. Aeromonas hydrophila and E. coli O157 were not isolated. Compared with those of the controls, the isolation rates of heat-stable-enterotoxin-producing E. coli (P less than 0.05), C. difficile (P less than 0.025), and enteroadherent E. coli (P less than 0.05) were significantly higher in the patients with diarrhea. The greatest number of rotavirus, enterotoxigenic E. coli, and C. difficile cases were identified during the cool, dry months of October and November.     

Significance of Cryptosporidium in acute diarrhoea in North-Eastern India.

In a hospital-based study, stool samples from 2095 patients of all ages were examined for different fungal, protozoal and bacterial enteropathogens over a period of 2 years (July 1994-June 1996). Cryptosporidium was detected in 151 specimens (7.2%) and was the third commonest pathogen found. The highest prevalence of this organism was in the group aged 16-45 years and during the rainy months (July-Oct.). Diarrhoea caused by the protozoon was of mild to moderate severity and features of dysentery were absent. Amongst other enteropathogens, Candida albicans was the most frequently isolated, followed by enteropathogenic and enterotoxigenic Escherichia coli, Salmonella spp., Campylobacter jejuni, Entamoeba histolytica, Giardia duodenalis (lamblia), Shigella spp., Vibrio cholerae and Aeromonas spp.     

Etiology of childhood diarrhea in Beijing, China.

To determine the role of recently recognized enteropathogens in childhood diarrhea in China, 221 children with diarrhea and 108 controls seen at the Beijing Children's Hospital were studied during April and May 1989. Stools were examined for ova, parasites, and rotavirus, cultured for bacterial pathogens, and probed for enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteropathogenic adherence factor-positive (EAF+) E. coli. Pathogens were identified in 56.5% of children with diarrhea and 43.5% of controls (P = 0.04). Detection of enteropathogens was significantly greater in patients examined within 1 week of symptom onset (65%) than in patients examined later (39%; P = 0.01). ETEC was the most frequently detected pathogen in children with diarrhea, accounting for 20% of the cases. Other agents identified in patients included the following: salmonellae, 12%; rotavirus, 7%; EIEC, 7%; EHEC, 7%; members of the Aeromonas hydrophila group, 6%; EAF+ E. coli, 5%; Ascaris lumbricoides, 3%; shigellae, 3%; campylobacters, 2%; and Vibrio spp., 0.5%. The isolation rates of salmonellae (P = 0.02), EAF+ E. coli (P = 0.04), and mixed pathogens (P = 0.05) were significantly greater for diarrhea patients than for controls. Resistance to multiple antimicrobial agents occurred in 39% of the Salmonella isolates, 22% of the Aeromonas isolates, and 17% of the Shigella isolates. Multiresistant salmonellae (P = 0.05) and shigellae were recovered from diarrheal stools only. Ciprofloxacin, cefotaxime, and imipenem were the only agents tested to which all bacterial isolates were susceptible in vitro. These results suggest that both traditional and newly recognized agents are important causes of childhood diarrhea in Beijing and that therapy may be complicated by indigenous antimicrobial resistance.     

Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies. Escherichia coli enteric virotypes, especially enteropathogenic E. coli (EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter (mainly Campylobacter jejuni), and Shigella flexneri were also often identified. Enterotoxigenic E. coli, Salmonella, and Giardia lamblia were sporadically recognized. Unusual findings included two enteroinvasive E. coli strains, one Shigella dysenteriae 2 isolate, and a non-O:1 Vibrio cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.     

High frequency of coinfecting enteropathogens in Aeromonas-associated diarrhea of hospitalized Peruvian infants.

Rectal swabs from 391 infants less than 18 months of age who were hospitalized with acute diarrhea and from 138 similarly aged healthy infants were examined for the etiologic agents of diarrhea. Aeromonas spp. were recovered from 205 of 391 (52.4%) diarrheic patients, whereas they were recovered from 12 of 138 (8.7%) controls (P less than 10(-11). Among the 205 Aeromonas-positive diarrheic patients, 118 (57.6%) were found to be coinfected with other common enteropathogens. Of the 164 Aeromonas-positive initial diarrheic specimens, 82 (50.0%) had one or more other enteropathogens present; 30 patients were coinfected with rotavirus, 20 with enterotoxigenic Escherichia coli, 16 with Campylobacter spp., 14 with Shigella spp., 13 with enteropathogenic E. coli, 4 with Vibrio spp., 1 with Salmonella spp., and 1 with Plesiomonas spp. of Aeromonas strains from cases compared with that from controls supports an etiologic role for this organism. However, frequent concomitant infections with other well-recognized enteropathogens and a lack of disease correlation with common Aeromonas phenotypes suggest that only a subset of Aeromonas strains may be diarrhea causing and that such strains may be common to several of the existing species.     

Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T(m)) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10(5) food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10(4) food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10(5) food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.     

Role of the microbiology laboratory in the diagnosis of nosocomial diarrhea

Diarrhea that occurs in hospitalized patients is frequent and may be due to infectious or noninfectious causes. In adults with nosocomial diarrhea, the most commonly detected agent is Clostridium difficile; in children, rotaviruses are predominant. Various studies have shown that bacterial enteric pathogens (e.g. Salmonella spp., Shigella spp., Campylobacter spp...) or parasites are common causes of community-acquired diarrhea but rarely cause nosocomial enteritis. Stool cultures for these pathogens and ova and parasite examination should not be performed in patients hospitalized for more than three days unless there are plausible clinical or epidemiological reasons to do so. In contrast, C. difficile toxins assay (and rotavirus screening in children) should be primarily requested. The detection of C. difficile toxin B by stool cytotoxicity assay remains the 'gold standard'. Identification of toxin A (or A + B) can also be performed by immuno-enzymatic (ELISA) tests: results may be obtained in three hours. Electronic microscopy is the standard method for rotavirus diagnosis but tests using latex agglutination or immuno-enzymatic assay are now available. Various typing methods have been developed and may be routinely used in epidemiological investigations.     

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.     

Epidemiology of bacterial pathogens associated with infectious diarrhea in Djibouti.

During a survey examining the causes of diarrhea in the East African country of Djibouti, 140 bacterial pathogens were recovered from 209 diarrheal and 100 control stools. The following pathogens were isolated at comparable frequencies from both diarrheal and control stools: enteroadherent Escherichia coli (EAEC) (10.6 versus 13%), enterotoxigenic E. coli (ETEC) (11 versus 10%), enteropathogenic E. coli (EPEC) (7.7 versus 12%), Salmonella spp. (2.9 versus 3%), and Campylobacter jejuni-C. coli (3.3 versus 5%). Surprisingly, the EAEC strains isolated did not correspond to well-recognized EPEC serogroups. No Yersinia spp., enteroinvasive E. coli, or enterohemorrhagic E. coli were isolated during the course of this study. Only the following two genera were recovered from diarrheal stools exclusively: Shigella spp. (7.7%) and Aeromonas hydrophila group organisms (3.3%). Shigella flexneri was the most common Shigella species isolated. Patients with Shigella species were of a higher average age than were controls (27 versus 13 years), while subjects with Campylobacter or Salmonella species belonged to younger age groups (2.6 and 1.6 years, respectively). Salmonella cases were more often in females. Shigella diarrhea was associated with fecal blood or mucus and leukocytes. ETEC was not associated with nausea or vomiting. Anorexia, weight loss, and fever were associated with the isolation of Salmonella and Aeromonas species. EAEC, ETEC, EPEC, and Shigella species were resistant to most drugs used for treating diarrhea in Africa, while the antibiotic most active against all bacteria tested was norfloxacin. We conclude that in Djibouti in 1989, Shigella and Aeromonas species must be considered as potential pathogens whenever they are isolated from diarrheal stools and that norfloxacin should be considered the drug of choice in adults for treating severe shigellosis and for diarrhea prophylaxis in travelers.     

Evaluation of the Vitek EPS enteric pathogen screen card for detecting Salmonella, Shigella, and Yersinia spp.

We evaluated the Vitek EPS card as a screen for the enteric pathogens Salmonella spp., Shigella spp., and Yersinia enterocolitica. Salmonella spp., Shigella spp., and Y. enterocolitica (125, 54, and 5 isolates, respectively) and 81 nonenteric pathogens that might be selected for screening from primary plates (non-lactose fermenters) were tested. The EPS card correctly identified 183 of 184 pathogens tested (sensitivity, 99.5%). Of 81 nonenteric pathogens screened with the EPS card, 8 were identified as possible enteric pathogens (specificity, 90.1%). We reviewed our stool culture records over the past 1.5 years and analyzed the specificities of TSI-urea screens for 300 stool cultures that had suspicious colonies. From 55 of 300 stool cultures, either Salmonella spp. or Shigella spp. were isolated, and from 245 stool cultures, no pathogen was isolated. Of the 245 negative cultures, 166 gave false-positive screening-test results that resulted in further biochemical identification procedures (Analytab Products or Vitek identification). Thus, the specificity of the TSI-urea screen in our experience was 32.2%. The Vitek EPS card was shown to be a more cost-effective screening procedure than the TSI-urea screen.     

The role of Proteae in diarrhea

A survey was undertaken on the occurrence of Protease in the human fecal flora and its coincidence with other well-documented enteropathogens such as Campylobacter, Salmonella, Shigella, Staphylococcus aureus, Yersinia, protozoa and rotavirus. A total of 2000 fecal specimens was investigated, 1000 from patients suffering from diarrhea and 1000 from healthy persons which served as controls. Proteus mirabilis was isolated more frequently from diarrhea cases than from healthy people. The difference was statistically significant (P less than 0.001). There was no correlation between its occurrence and isolation of Campylobacter, Salmonella, Shigella, Staphylococcus aureus, and protozoa. However, a questionable coincidence was found with rotavirus (P less than 0.2) and a more certain correlation with Yersinia enterocolitica (P less than 0.025). Proteus mirabilis in patients suffering from infection by other known enteropathogens was largely absent, suggesting that the organisms were independent causative agents of intestinal disorders. Notwithstanding this, they may additionally play a role as opportunists in enteric diseases due to some other pathogens.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

Selective criteria for the microbiological examination of faecal specimens.

To assess the effectiveness of predetermined investigation criteria for the examination of faecal samples from inpatients, cultured stool specimens were prospectively examined for Salmonella spp, Shigella spp, Campylobacter spp and Clostridium difficile, and screened microscopically for intestinal parasites. Out of a total of 505 specimens, 421 (83%) fulfilled the criteria for examination for C difficile, 254 (50%) for Salmonella spp, Shigella spp, and Campylobacter spp, and 87 (17%) for intestinal parasites. Isolation rates for these organisms in those groups of patients where examination was indicated were 22.5% for C difficile and 9.1% for Salmonella spp, Shigella spp, and Campylobacter spp; the detection rate for parasites was 3.5%. In those patients where the criteria did not suggest investigation, the isolation or detection rates were 3.6% for C difficile, 0% for Salmonella spp, Shigella spp, and Campylobacter spp, and 1.7% for intestinal parasites, suggesting that the use of predetermined investigation criteria was effective.     

Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens

The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.     

In vitro activity of norfloxacin and other antibacterial agents against gastro-intestinal pathogens isolated in Sweden

The in vitro activity of norfloxacin was compared to that of ampicillin, doxycycline, chloramphenicol, trimethoprim in combination with sulfamethoxazole (1/20), and erythromycin, against 272 clinical isolates of gastro-intestinal pathogens. Norfloxacin was the most active compound of those tested with MICs in the range 0.004-2 mg/l. Concentrations inhibiting 90% of the strains (MIC 90) were 0.008 mg/l for Vibrio cholerae, 0.016 mg/l for Aeromonas hydrophila, 0.032 mg/l for Vibrio cholerae non 01, 0.064 mg/l for Vibrio parahaemolyticus, Yersinia enterocolitica 03, enterotoxigenic (ETEC) and enteropathogenic (EPEC) Escherichia coli and Shigella species, 0.125 mg/l for Salmonella species, and 0.5 mg/l for Campylobacter species. Resistance to one or several of the other drugs was seen with higher or lower frequency in all the bacterial species tested. No cross-resistance between any of the other agents and norfloxacin was recorded.     

Demonstration of a lack of synergistic effect of rotavirus with other diarrheal pathogens on severity of diarrhea in children.

The severity of group A rotavirus (RV) diarrhea was compared with that of mixed infections of RV with diarrheagenic Escherichia coli, Vibrio cholerae O1, and Shigella species by a scoring system. The severity of mixed infections of RV and E. coli was the same as that of infections with RV alone. RV infections mixed with V. cholerae and Shigella species mimicked cholera and shigellosis, respectively.     

Neuraminidase production by Vibrio cholerae O1 and other diarrheagenic bacteria

Vibrio cholerae O1 strains belonging to both biotypes (classical and El Tor) and both serotypes (Ogawa and Inaba) produced neuraminidase which was released rather than cell bound. Classical strains made more neuraminidase than did El Tor strains. About one-third of V. cholerae non-O1 strains and one-fourth of Aeromonas hydrophila strains were neuraminidase positive. Strains of enterotoxigenic Escherichia coli, Vibrio parahaemolyticus, and Shigella spp. did not produce detectable neuraminidase.     

Detection of Campylobacter jejuni in healthy monkeys and monkeys with enteric infections by PCR

Campylobacter were detected by PCR in feces of monkeys of different species (clinically healthy, with diarrhea, and dead from acute enteric infections). High prevalence of these bacteria in monkeys was revealed. The incidence of C. jejuni DNA in monkeys with acute enteric infections was higher than in healthy animals (69.6 and 51.3%, respectively). The highest percentage (92.3) of positive results was observed in Macaca mulatta with enteric diseases and in macaque dead of these diseases. The presence of C. jejuni in monkeys with diarrhea and the absence of pathogenic enterobacteria (Shigella, Salmonella, Yersinia) in feces probably attest to etiological relationship of acute enteric infections with Campylobacter.