Indian J Pediatr (2011) 78:165–170 DOI 10.1007/s12098-010-0249-0

ORIGINAL ARTICLE

Bacterial Enteropathogens and Risk Factors Associated with Childhood Diarrhea Abdel Moati Kh. Al Jarousha & Mahmoud A. El Jarou & Iyad A. El Qouqa

Received: 2 June 2010 / Accepted: 16 September 2010 / Published online: 6 October 2010 # Dr. K C Chaudhuri Foundation 2010

Abstract Objective To investigate the etiology of bacterial enteropathogens causing diarrhea among children; to elucidate the risk factors, sign and symptoms involved in developing of infection; and to identify the antimicrobial susceptibility pattern. Methods Stool samples from diarrheal cases with clinical diagnosis of gastroenteritis compared to matched controls, were examined for detection of bacterial enteropathogens by conventional cultural method from February 2006 through January 2007. Results Ninety three of diarrheal cases comprising enteropathogen isolates. A recognized bacterial enteric pathogen was identified in 31% of the cases and 8% of the control group. The frequency of isolated enteropathogens from faeces of diarrheal cases was recorded in the following order, Enterohemorrhagic E. coli 8.3%, Shigella spp. 6.7%, Campylobacter jejuni 5%, Salmonella spp. 4%, Yersinia enterocolitica 2.7%, Aeromonas spp. 4.7%, and Plesiomonas spp. 1.3%. E.H.E coli, Shigella and Salmonella had shown a significant statistical association with diarrhea (p<0.05). Compared to matched uninfected control, multivariate analysis revealed that malnutrition status (OR adjusted A. M. K. Al Jarousha (*) Laboratory Medicine Department, Al Azhar University, P.O. Box 1277, Gaza, Palestine e-mail: [email protected] M. A. El Jarou Medical Microbiology Department, Shifa Hospital, MOH, Gaza, Palestine e-mail: [email protected] I. A. El Qouqa Medical Technology Department, Hejazi Medical Center, Medical Services, Gaza, Palestine e-mail: [email protected]

8.37; p<0.001) was the only independent factor associated with infection. Antibiotic susceptibility profile indicated that enteropathogens were generally susceptible to meropenem, ceftriaxone, followed by amikacin and ciprofloxacin. Almost all enterpathogens were resistant to ampicillin and amoxicillin. Conclusions The results of this study emphasize the importance of bacterial enteropathogens in causing severe diarrhea in children. E.H.E. coli was the most predominant pathogen. Malnutrition was identified as the main independent risk factor associated with developing of infection. Ampicillin and amoxicillin should not be used as empirical treatment in acute diarrhea. Keywords Enteropathogens . Risk factors . Diarrhea . Children

Introduction Infectious diarrheal diseases are of great concern throughout the world, as they are responsible for considerable morbidity and mortality, especially in developing countries. It has been reported that diarrheal diseases cause approximately three million deaths worldwide per year [1]. More than 20 viral, bacterial, and parasitic enteropathogens are currently associated with acute diarrhea. Infections caused by Shigella, Salmonella, Campylobacter jejuni/coli, Vibrio cholerae, Aeromonas and Plesiomonas spp., protozoa and helminthes occur more commonly among low-income groups in poorer areas [2, 3]. Several factors can contribute to intestinal infections like duration of maternal breastfeeding, food and environmental contaminations, maternal education and employment, home location, family income, infant’s birth weight, access to

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treated water and basic sanitary conditions, seasonal variations and hosts’ underlying conditions [4, 5]. Antimicrobial resistance in enteric pathogens is of great importance in the developing world, where the rate of diarrheal diseases is highest. The progressive increase in antimicrobial resistance among enteric pathogens in developing countries is becoming a critical area of concern [6]. This study was undertaken, to investigate the bacterial etiology of diarrhea in children admitted in hospitals or outpatient clinics, to elucidate the risk factors, sign and symptoms of infection, and to identify the antimicrobial susceptibility pattern of the most important pathogens involved in the epidemiology of the acute diarrhea.

Indian J Pediatr (2011) 78:165–170

Sample Collection The samples of study population were collected according to standard microbiology laboratory techniques. Stool collection was achieved via sterile plastic containers provided with screw capes with spoon. The rectal swabs were collected by using sterile cotton swabs in cases of children less than 1 year old. Cary Blair transport media was used for preservation of samples. The samples were processed within two hrs of collection. Microbiological Analysis

Material and Methods

Stools were processed and analyzed for enteric bacteria on the day of sample collection. Standard culture and identification methods were used to identify enteric pathogens [8].

Study Design

Demographical Characteristics

The present study was designed as a case-control study and was conducted on children aged up to 12 yrs from February 2006 through January 2007. Stool samples were collected consecutively for the diagnosis of infectious diarrhea, from children attending Gaza Strip hospitals, particularly El-Dorra pediatric, Shohdaa El-Aqsa, European Gaza and El-Naser pediatric hospitals. The total number of children enrolled in the study were 465. Three hundred children were considered as gastroenteritis patients and 165 were considered as a matched control group.

The cases were initially seen by physician. Clinical and demographical data subsequently collected through wellprepared questionnaire, where the guardians of the cases were questioned about clinical symptoms, treatment, previous or concurrent disease and other relevant information including malntritional status (that was assessed by low hemoglobin level{anemia}) house crowdness index (per room) maternal education, and family income.

Study Population

The antibiotic susceptibility of enteropathogens isolates were determined by Kirby Bauer disk diffusion method, with the following antibiotics: ampicillin (AM), amoxicillin (AC), chloramphenicol (C), nalidixic acid (NA), tetracycline (T), cephalexin (CN), trimethoprime-sulfamethoxazole (SXT), ceftazidime (CAZ), ceftriaxone, (CRO) gentamicin (GN), amikacin (AK), cefuroxime (CXM), ciprofloxacin (CIP), and meropenem (MER) [9].

The study population comprised of children admitted to pediatrics department or who presented in the outpatient clinics with diarrhea (diarrheal group). Control patients without diarrhea were matched for age and sex, geography and selected from patients with complaint other than diarrhea like chest infection, skin, orthopedic or presented in the clinic for vaccination. The control children who received antibiotics during the preceding 2 weeks were excluded from the study. The selection of the cases was carried out according to symptoms or signs of gastroenteritis. The suspected gastroenteritis was defined as a case that had acute diarrhea with three or more stools a day [7]. Outpatients were first examined by a physician and those requiring further care were admitted to the hospital ward. A physician performed a physical examination and assessed the patient’s dehydration status, according to clinical signs and symptoms. The study was conducted and approved officially by Helsinki Committee and Palestinian MOH. In compliance with Helsinki declaration, a consent form was signed by guardians of the children, after explaining the importance and objectives of the study.

Antimicrobial Susceptibility Test

Statistical Analysis The SPSS PC 15.0 software package (SPSS Inc., Chicago, Ill., USA) was used for the data analysis. For continuous data, mean, standard deviation, as well as median with interquartile range were computed. Categorical variables were analyzed using χ2 and Fisher’s exact tests. Potential risk factors were assessed by calculating the odds ratio. Variables found to be significant by univariate analysis at p values < 0.1 were selected for inclusion in multivariate modeling. Backward stepwise logistic regression was used to model the association between infection positivity and selected correlates adjusted for confounding factors identified in univariate analysis. p<0.05 was considered statistically significant.

Indian J Pediatr (2011) 78:165–170

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Results Study Population In the current study, cases suffering from gastroenteritis (diarrheal group) were three hundred children aged between 1 month and 12 years (mean age 5.01±3.06 years and median age 4 years with interquartile range of [3–8] years). The control group comprised of one hundred sixty five children who were free from symptoms of gastroenteritis and were attending the clinics for other complains, like chest infection and vaccination. The mean age group was 5.01±3.14 yrs and median age of 5 yrs with interquartile range of 2–8 yrs. Table 1 illustrates the enteropathogens isolated from stool samples of gastroenteritis patients. The most frequent enteropathogens isolated from the stool of the gastroenteritis patients were E.H.E. coli 25 (8.3%), followed by Shigella spp. 20 (6.7%). The serotypes of Shigella spp. were, S. flexinari 12/20 (60%), S. sonnei 6/20 (30%) and S. boydii 2/20 (10%), C. jejuni had frequency of 15(5%) in diarrheal group and serogroups A, C, D, F and Y were identified; each had one strain, whereas serogroup B, G and O had two strains, meanwhile 4 strains of serogroup of C. jejuni were unknown. Aeromonas spp 14 (4.7%) Salmonella spp. 12 (4%) and Y. enterocolitica 8 (2.7%) Y. enterocolitica serotype were (O3 4/8, O8 2/8 and O9 2/8) and the lowest frequency was of Plesiomonas spp. 4 (1.3%). In control group: E.H.E.coli 5 (3%) were the most frequent isolate, followed by C. jejuni 3 (1.8%), Shigella spp. 2 (1.2%), Aeromonas spp. 2 (1.2%) and Salmonella spp. 1 (0.6%). Y. enterocolitica as well as Plesiomonas spp. were not detected in control group. The Gastroenteritis infection may be caused by a single or multiple microorganisms. In the present study, 88 (94.6%) cases were positive for single microorganisms and 5 (5.4%) cases were positive for multiple microorganism’s (E.H.E.coli and Shigella, E. H.E.coli and Aeromonas, Salmonellla and Shigella, Campylobacter and Aeromonas , Aeromonas and Plesiomonas). The differences in the isolation rates between patients and control subjects particularly for, E.H.E.coli, Shigella , and Salmonella, had shown a significant Table 1 Enteropathogens isolated from stool specimens

a Fisher exact test two tailed; p<0.05 statistically significant

Enteropathogens

E.H.E. coli Shigella spp. C. jejuni. Aermonas spp. Salmonella spp. Y. enterocolitica Plesiomonas spp.

statistical association (p<0.05). Meanwhile the isolation rates for Campylobacter, Yersinia, Aeromonas and Plesiomonas did not reach statistical significance. As shown in Table 2, E.H.E. coli was the only frequently isolated enteropathogen in age group younger than 1 year. Generally, the enteropathogens are mostly encountered in both age group 1–3 and 4–6 yrs. Therefore, the age groups were categorized into two merged groups (groups 1–6 yrs and 7–12 yrs excluding age group <1 yrs as the majority of children were exclusively breast fed. Statistical analysis showed a significant difference in proportions of acquisition of infection for E.H.E. coli (p<0.001). Total enteropathogens isolated also revealed also a significant association with age group 1–6 yr, p=0.002). The major symptoms recorded among infected cases with enteropathogens were vomiting, abdominal pain, fever and dehydration with proportion of 38% (53/139), 42% (67/160), 42% (46/110), 26.4% (16/65), respectively. The statistical relationship between the infected cases and the development of the symptoms was analyzed and showed a significant association (p<0.05) for both abdominal pain and fever. Demographical Characteristics and Risk Factors of Diarrheal Cases and Control Groups Risk factors of infection in relation to matched uninfected control patients by univariate analysis are displayed (Table 3). Infected cases were significantly more likely to have malnutrition (OR=8.57; p<0.001), crowdness index≥3 per room supply (OR=1.9; p=0.015). Severe variables tended to be more associated with developing infection but did not reach statistical significance. However, in multivariate logistic regression, malnutrition was the only independent risk factor (OR=8.37; P<0.001) for developing infection. Antibiotic Susceptibility Profile The antibiotic susceptibility profile of isolated enteropathogens is depicted in (Fig. 1), it shows that 97% of enteropathogen isolates were susceptible for meropenem; 90.8% ceftriaxone; 86.7% amikacin; 77.6% ciprofloxacin;

Diarrheal cases n=300

Control group n=165

Frequency

Frequency

25 20 15 14 12 8 4

% 8.3 6.7 5.0 4.7 4.0 2.7 1.3

5 2 3 2 1 0 0

p-value % 3.0 1.2 1.8 1.2 0.6 0.0 0.0

0.026 0.008 0.089 0.051 0.039a 0.055 0.30

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Indian J Pediatr (2011) 78:165–170

Table 2 Frequency of isolated enteropathogens by age group from diarrheal cases Enteropathogens

<1y n=27

EH.E.coli. Shigella spp. C. jejuni Aeromonas spp. Salmonella spp. Y. enterocolitica Plesiomonas spp. Total %

1–3y n=70

2 0 0 0 0 0 0 2

4–6y n=114

16 2 5 2 1 6 1 33

(7.4%)

7 10 6 8 7 2 3 43

(47.1%)

Discussion This is the first case control study focusing on detection of different bacterial enteropathogens in diarrheal children from

Table 3 Characteristics and risk factors associated with enteropathogens

a Fisher exact two tailed; URTI, upper respiratory tract infection; UTI, urinary tract infection; P<0.05 statistically significant; CI, confidence interval

Variables

10–12y n=29

0 6 3 2 3 0 0 14

(37.7%)

76.5% ceftazidime; 74.5% gentamicin71.4%; cefuroxime 62% chloramphenicol and 40% nalidix acid. Almost all strains were resistant to ampicillin.

7–9y n=60

(23.3%)

0 2 1 2 1 0 0 6

25 20 15 14 12 8 4 98

(20.7%)

(32.7%)

Gaza Strip. In this investigation, seven types of bacterial enteropathogens were isolated from diarrheal cases, which constitute a prevalence of 31% (93/300). Diarrhea in children in developing countries other than Gaza Strip has been reported to have a known etiology in 50–60% of the diagnosed cases [2]. The main difference between these reports is the inclusion of diarrheagenic E. coli which increases the frequency of causes of diarrhea with known etiology. In the present study, E.H.E coli was responsible for

Cases N=93 (%)

Uninfected Control N=152 (%)

Malnutrition Family income low high Mother education low high Crowdness <3 Per Room ≥3 Per room Residence North Gaza

35 (37.6)

10 (6.6)

Gaza Mid-Zone South Gaza Gender Male Female Age groups (Yr) 0–6 7–12 Other disease URTI UTI Skin

Total n=300

OR (95% CI)

p-value

8.57 (3.98–18.44)

<0.001

70(46.1) 82(53.9)

1.30(0.78–2.19)

0.31

30(19.7) 122(80.3)

1.66(0.91–3.03)

0.095

88(57.9) 64(42.1)

1.9 (1.13–3.21)

0.015

30(32.3)

40(26.3)

1.33 (0.76–2.35)

0.32

39(41.9) 10(10.8) 14(15.1)

60(39.5) 22(14.5) 30(19.7)

1.11(0.65–1.87) 0.71(0.32–1.58) 0.72(0.36–1.44)

0.70 0.40 0.35

48(51.6) 45(84.4)

84(55.3) 68(44.7)

0.86 (0.51–1.45)

0.58

74 (79.6) 19 (20.4)

105 (69.1) 47( 30.9)

1.74 (0.95–3.21)

0.072

2(2.2)

7(4.6)

0.45(0.093–2.24)

0.49a

2(2.2) 1(1.1)

5(3.3) 4(2.6)

0.65(0.12–3.4) 0.40(0.044–3.65)

0.71a 0.65a

49(52.7) 44(47.3) 27(29) 66(71) 39(41.9) 54(58.1)

Indian J Pediatr (2011) 78:165–170 97

100

90.8

90

Frequency (%) 86.7 77.6 76.5 74.5

80

Frequency (%)

169

71.4

70

62

60 50 40

40

34.6

30 18

20

9

10

9 2

AC

C N SX T AM

A

T

C

N

N

XM C

AZ

IP

G

C

C

O AK

R C

M

ER

0

Antibiotics

Fig. 1 Antimicrobial susceptibility pattern of isolated enteropathogens against different antibiotics

8.3% of diarrheal cases. It has surpassed several points of interest related to diarrhagenic E. Coli, the positive strains belonging to classical E.H.E coli serotypes were associated with diarrhea. However, E.H.E. coli was isolated from diarrheal and non-diarrheal (control) cases simultaneously. The classical E.H.E coli was isolated in high proportion in children below 3 yrs of age with no isolation from children above 7 yrs and was the only isolate from children below 1 yr of age indicating that this pathogen is an important cause of sporadic diarrheal illness in infants. In the present study, Shigella spp. was observed at a lower frequency than E.H.E coli and was the most frequent agent in children more than age of 4–6 yrs of age and no infection in less than 1 yr of age. Shigella spp was linked to severe diarrhea particularly in age group 4–6 yrs. This was by statistical significant association when compared with age group 7–12 yrs. Shigella spp in certain studies has a wide spectrum and is more severe in infants who are not breast fed and malnourished [10]. In Thailand., infants particularly when breastfed were generally spared from shigellosis [11]. However, the results are consistent to the preceding data in Thiland. Campylobacter was the third identified pathogen in the present study with proportion of 5%. It was noted that this organism is commonly encountered in stool of children aged 1–6 yrs and absent in children less than 1 yr of age. It is known that Campylobacter infection is usually a mild self-limiting diarrheal disease, however, severe and prolonged infection causes enteritis and bacteremia [12, 13]. Campylobacter is considered as zoonotic diasease with raw or undercooked poultry being an important source of campylobacter infection. Contaminated milk water, beef, lamb, and sea food also contribute to human infections. Thus, the main source of infection in the present study is difficult to determine. Aeromonas spp. was detected in proportion higher than expected (4.67%) and more than Salmonella spp. This

pathogen was detected for the first time and no previous information dealing with this pathogen was available in our area. Aeromonas spp. was detected in both diarrheal cases and control group in considerable proportions, which means that a considerable number of cases have a silent infection with no signs of diarrhea. However, the role of Aeromonas spp as significant diarrheal disease agent is controversial, which can be linked to acute diarrhea in some controlled studies but not others [14, 15]. Salmonella spp was isolated in this study in lower proportion 4% and was against the expected rate since Salmonella spp is endemic in our area and survival of salmonella infected patients in different conditions with adverse effect recorded in several studies. The low rate of isolation for Salmonella may be due to more attention by the physician and the lab work for isolation, identification and treatment. Therefore, there is a decline in the proportion of Salmonellosis in our area. Y. enterocolitica was isolated only from age group 1–6 yrs with no successful isolation achieved in the age group of less than 1 yr and more than 6 yrs. The low proportion of Y. enterocolitica may be due to difficulty in isolation since it need specific enrichment conditions [15]. Moreover, Y. enterocolitica recorded in the present study showed a high percent susceptibility for the oral antibiotics that are regularly used in the treatment especially sulfamethaxazole-trimethoprime which is used as empirical treatment in several infections. Plesiomonas spp. was isolated as the lowest frequent pathogen in the present study (1.33%). The causative role of Plesiomonas spp. in diarrheal diseases is not firmly established [16]. The isolation of different enteropathogens indicated that those children up to 6 yrs were more susceptible to infection. This could be attributed to lesser skills and not fully compliant with self-hygiene instructions. Univariate analysis showed that two risk factors had association with diarrhea, malnutrition, and crowdness, however, in multivariate analysis, malnutrition has been shown to be the only independent risk factor for diarrheal infection. Several studies have been conducted in the world and have shown similar risk factors to the present findings [11, 17]. The common symptoms recorded in the current study were abdominal pain, followed by vomiting, fever, and dehydrations. Statistical analysis showed that abdominal pain and fever were associated significantly with diarrheal disease. The antibiotic susceptibility tests showed that the new generation antibiotics and the newly introduced meropenem had lowest resistant rate, while the older generations like ampicillin, amoxicillin had highest resistant rate. This indicates that multiple uses of these antibiotics for different

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infection led to emergence of resistant mutant strains. Consequently, they are not effectively used as empirical antibiotics in eradication of diarrheal diseases and therefore, there is a need for other antibiotics to be used in that field. The large percentage of cases with no recognized enteropathogens raises a number of issues; first, technique used for identifying known pathogens may have not been optimal during the course of this study. The other different organisms that may cause diarrhea are Rotavirus and Adenovirus. Secondly even when specifically sought some pathogens may not detected because of excretion in insufficient number, in particular, intermitten excretion or collection of faces only during the recovery phase may result in failure to isolate the causative pathogens. Thirdly and of most importance, there may be other (some as yet unknown) pathogens causing severe diarrhea in children.

Conclusions The results of this study emphasize the importance of bacterial enteropathogens in causing severe diarrhea among children. Hence, it is advisable to be diagnosed routinely in all public and private hospitals labs. Measures to control childhood diarrhea diseases in Gaza Strip should be directed at prevention of infection. The old ampicillin and amoxicillin should not be used as empirical treatment and should be replaced by new ones. Manutrition factor has shown to be main independent risk factor for developing infection. Acknowledgements We acknowledge the support and assistance of all pediatricians, nurses and laboratory staff of El-Dorra pediatric, Shohdaa El-Aqsa, European Gaza and El-Naser hospitals, for their assistance and cooperation.

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