Enteric pathogens associated with diarrhea in children in Fayoum, Egypt

Diagnostic Microbiology and Infectious Disease 56 (2006) 1 – 5 www.elsevier.com/locate/diagmicrobio

Bacteriology

Enteric pathogens associated with diarrhea in children in Fayoum, EgyptB,BB Hanon El-Mohamadya,*, Ibrahim A. Abdel-Messiha, Fouad G. Youssef a, Mohamad Saidc, Hossaini Faragc, Hind I. Shaheena, David M. Rockabranda, Stephen B. Lubyb, Rana Hajjeha, John W. Sandersa, Marshall R. Montevillea, John D. Klenaa, Robert W. Frencka a

Enteric Diseases Research Program, U.S. Naval Medical Research Unit N — 3, Box 5000, FPO AE 09835-0007 Cairo, Egypt b Centers for Disease Control and Prevention (CDC), Atlanta, GA c Ministry of Health and Population, Cairo, Egypt Received 21 September 2005; accepted 9 February 2006

Abstract In a cross-sectional study of children b 60 months old from Fayoum, Egypt, presenting with diarrhea, 46% (162/356) had detectable enteric pathogens. Bacterial pathogens were identified in 25% (89/356), whereas rotavirus and Cryptosporidium were detected in 21% (54/253) and 15% (39/253), respectively. Cryptosporidium is an important pathogen in this region. D 2006 Elsevier Inc. All rights reserved. Keywords: Egypt; Enteric pathogens; Cryptosporidium

Diarrheal diseases continue to be a major cause of morbidity and a leading cause of mortality in children less than 5 years of age living in developing countries (Kosek et al., 2003). Implementation of effective control measures for diarrheal illness requires an awareness of the most common pathogens within a region. For the past decade, numerous studies evaluating diarrheal diseases among children living in the Nile River Delta, northern Egypt, have been conducted (Abu-Elyazeed et al., 1999; Naficy et al., 1999; Rao et al., 2003; Wierzba et al., 2006). Enterotoxigenic Escherichia coli (ETEC) has been shown to be the most commonly identified cause of bacterial-induced diarrhea in these studies (Abu-Elyazeed B

This research was supported by the United States Department of Defense Global Emerging Infectious Disease Surveillance Program (GEIS) no. 323987M906. BB The opinions and assertions contained herein are the private opinions of the authors and are not to be construed as official or as reflecting the views of the Department of Navy, Department of Defense, the United States Government, or the Egyptian Ministry of Health and Population. 4 Corresponding author. Tel.: + 20-202-348-0341; fax: + 20-202-3843847. E-mail address: [email protected] (H. El-Mohamady). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.02.007

et al., 1999; Naficy et al., 1999; Rao et al., 2003; Wierzba et al., 2006). Rotavirus, although responsible for fewer episodes of diarrhea relative to ETEC, is the most commonly identified cause of diarrhea among children seeking medical care for severe illness (Wierzba et al., 2006). In addition, Cryptosporidium parvum has also been identified as a prevalent and virulent agent of childhood diarrhea in the Nile River Delta (Abdel-Messih et al., 2002; Antonios et al., 2001). Although numerous studies concerning childhood diarrhea have been conducted in Egypt, only one study has been published involving children from southern Egypt (Mikhail et al., 1989). Therefore, a pilot study was initiated to determine the prevalence of selected enteric pathogens associated with diarrheal disease in children living in the Tamiya District of the Fayoum governorate located in southern Egypt. Between August and September 2003, 356 children, aged V 60 months median age, 12 (interquartile range [IQR] 8–20 months) with acute diarrhea, defined as the occurrence of 3 or more unformed (or z 1, if bloody) stools in a 24-h period (Abu-Elyazeed et al., 1999), were enrolled after informed consent was obtained from a parent or legal guardian. Each child was asked to provide 2 rectal swabs and a stool sample for analysis. Methods detailing specimen

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Table 1 Enteric pathogens isolated from children with diarrhea, Fayoum Hospital, August to September 2003 Pathogens (number of samples) Tested

Number of positive cases (n)

Bacteria (N = 356)a ETEC Campylobacter spp. C. jejuni C. coli Shigella spp. S. flexneri S. dysenteriae S. boydii Salmonella spp. Aeromonas hydrophila Vibrio fluvialis Parasitic (n = 253)a Cryptosporidium Viral (n = 253) Rotavirus Mixed infectionb No pathogen identifiedc

%

37 19 16 3 7 4 2 1 2 4 2

10.8 5.6

27

10.7

43 21 194

17 6.1 54

2.0

0.6 1.1 0.6

a

Rectal swabs were collected from 100% (n = 356) of subjects and a stool sample collected from 71% (n = 253). b Mixed infections: 2 ETEC and Campylobacter; 1 Campylobacter and Shigella; 5 Cryptosporidium and ETEC; 2 Cryptosporidium and Campylobacter; 2 rotavirus and Campylobacter; 4 rotavirus and ETEC; 3 Cryptosporidium and rotavirus; 1 rotavirus and Cryptosporidium and ETEC; and 1 rotavirus and Cryptosporidium and Salmonella. c This group includes diarrheal cases where no pathogen was detected.

collection, handling, and storage have been previously reported (Wierzba et al., 2006). Testing for enteric bacteria (E. coli, Campylobacter spp., Shigella spp., and Salmonella spp.), as well as E. coli heat-stable and heat-labile enterotoxins, and colonization factor antigens (CFAs) were performed as previously described (Wierzba et al., 2006). Commercially available ELISA diagnostic kits were used according to the manufacturer’s instructions to test for the presence of C. parvum (Techlab, Blacksburg, VA) and rotavirus (Premier RotacloneR, Meridian Bioscience, Cincinnati, OH) antigens in stool samples. Statistical comparisons were made using the v 2 test for comparison among proportions, Fisher’s exact test or the Kruskal-Wallis test. All data were double-entered into Epi-info V6 (CDC, Atlanta, GA) and analyzed using SAS V8 (SAS Institute, Cary, NC). Statistical significance was 2-tailed and set at P b 0.05 for each analysis. Overall, an enteric pathogen was identified in 162 children (46%) (Table 1). Bacterial pathogens were isolated from 25% (89/356) of the children, and ETEC was the most commonly isolated bacterial pathogen followed by Campylobacter spp. and Shigella spp. Rotavirus and C. parvum were identified in 21% (54/253) and in 15% (39/253), respectively. A single enteric pathogen was identified in 91% of the cases with a detectable pathogen (141/162) (Table 1). In the remaining 21 cases, 18 samples had a bacterial pathogen

Table 2 Clinical characteristics of diarrhea in Egyptian children less than 60 months old seeking treatment in Fayoum Hospital, August–September, 2003 Fayoum hospital

Characteristics Age (months) 0–11 12–23 24–60 Diarrhea type Acute watery Dysentery Persistent Hospitalized Fever present Blood in stool Vomiting present Dehydration present Rectal temperature N 38.2 Median (Q1–Q3) Max no. of stool (n = 351) No. of stool in last 24 (n = 355) Days to evaluation (n = 353)

Mixed, n (%)

Campylobacter, n (%)

Cryptosporidium, n (%)

ETEC, n (%)

Rotavirus, n (%)

Shigella, n (%)

21 (6.1)

19 (5.5)

27 (10.6)

37 (10.8)

43 (16.9)

7

15 (71.4) 3 (14.3) 3 (14.3)

13 (68.4) 3 (15.8) 3 (15.8)

21 (77.8) 6 (22.2) 0 (0.0)

7 (18.9) 18 (48.7) 12 (32.4)

21 (48.8) 19 (44.2) 3 (7.0)

3 (42.9) 3 (42.9) 1 (14.2)

19 2 0 2 18 2 14 9 1

17 1 1 0 16 1 7 6 2

24 (88.9) 2 (7.4) 1 (3.7) 0 (0.0) 22 (88.0) 3 (11.5) 16 (59.3) 11 (42.3) 1 (3.7)

34 1 2 4 32 1 16 15 3

38 3 1 6 39 3 36 21 4

4 3 0 0 6 3 4 2 0

(90.5) (9.5) (0.0) (9.5) (94.7) (10.0) (66.7) (42.9) (4.8)

(89.5) (5.3) (5.3) (0.0) (84.2) (5.3) (36.8) (31.6) (10.5)

(91.9) (2.7) (5.4) (11.1) (88.9) (2.7) (44.4) (42.9) (8.3)

(90.5) (7.1) (2.4) (14.3) (97.5) (7.3) (83.7) (51.2) (9.3)

No pathogen identified (n = 200)

(57.1) (42.9) (0.0) (0.0) (85.7) (42.9) (57.1) (28.6) (0.0)

93 (46.5) 47 (23.5) 60 (30.0) 170 20 6 4 167 20 100 73 9

(86.7) (10.2) (3.1) (2.0) (85.6) (10.1) (50.0) (36.7) (4.5)

5 (3.5,8.5)

6 (4,7)

5 (3,9)

7 (4,10)

6 (4,10)

7 (5,10)

5 (4,7)

5 (4,7)

5 (4,6)

6 (4,8)

7 (5,10)

6 (4,10)

7 (5,12)

6 (4,7)

3 (2,5)

3 (2,4)

3 (3,5)

3 (2,5)

3 (2,4)

2 (2,4)

4 (3,7)

P value from Fisher’s exact test for comparisons among proportions, or for continuous data, Kruskal-Wallis test. Significant P values are reported as P = 0.061 blood in stool, P = 0.0013 vomiting present, and P = 0.062 median days to evaluation. Mixed pathogens were excluded from the analysis.

H. El-Mohamady et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 1 – 5

parasitic pathogens could be identified. If all volunteers had submitted a stool specimen rather than just a rectal swab, more pathogens would most likely have been identified. The frequencies of C. parvum, and rotavirus infection inversely correlated with age, and infections were more prevalent in infants aged 0 –11 months (P = 0.02 for both infections) (Table 2). However, children aged 12– 24 months were more likely to experience diarrhea due to ETEC infection ( P = 0.0001) supporting the observation that passive immunity transferred during breast-feeding from mother to children less than 1 year of age is protective against ETEC infection (Hanson et al., 1994). Consistent with previous observations, patients presenting with rotavirus had the highest reported history of vomiting (84%, n = 36), dehydration (51%, n = 21), and hospitalization (14%, n = 6) (Hanson et al., 1994; Wierzba et al., 2006). Of the 52 ETEC isolates, 23 (44%) were phenotypically positive for the E. coli heat-stable toxin (ST), 21 (40%) for the E. coli heat-labile toxin (LT), and 8 (15%) for both LTST (Table 3). Fifty-six percent of ETEC cases (n =29) expressed a known colonization factor (Table 3). Toxin expression in ETEC from the Fayoum district is in agreement with a similar clinic-based study of Egyptian children in northern Egypt that showed 45% and 40% of the ETEC isolates were ST and LT, respectively (Wierzba et al., 2006). However, results differ from community-based studies of diarrhea in Egyptian children aged under 3 years in northern Egypt where the frequency of ST-expressing ETEC isolates was higher than LT-expressing ETEC isolates by at least 2-fold (Peruski et al., 1999; Shaheen et al., 2004). Of interest, the percentage of isolates expressing a detectable CFA was similar to that previously reported from ETEC isolates from northern Egypt (Abu-Elyazeed et al., 1999). Our data concur with a previous study conducted in northern Egypt in which isolates expressing CFA IV, especially CS6, were the most commonly recovered, but differs from a hospital-based study that found strains expressing CFA/I were more common (Peruski et al., 1999; Wierzba et al., 2006).

Table 3 Phenotypic characterization of ETEC isolates in children with diarrhea from the Fayoum hospital Colonization factors CFA/I CFAII CS1CS3 CFAIV CS4CS6 CS5CS6 CS6 CS7 CS19 O159 None detectedb

LT (n = 21)

LTST (n = 8)

ST (n = 23)

0

0

2

2

0

0

1

1

0 0 2 4 0 0 15

0 4 0 0 3 1 0

1 3 6 0 1 0 9

1 7 8 4 4 1 24

3

Total (n = 52)a

a

Three cases had multiple phenotypes detected. Using a panel pf 13 monoclonal antibodies, CFA/I, CS1-CS8, CS12, CS14, CS17 and CS19. b

present as well as either rotavirus or C. parvum. In 3 cases, 2 bacterial pathogens were detected. The most common mixed infections observed were a bacterial pathogen with Cryptosporidium (n = 9) or with rotavirus (n = 8). Children infected with multiple potential pathogens had no significantly different clinical symptoms compared to children infected with a single enteric pathogen. No pathogen was found in 194 (54%) (Table 1) of the cases. The percentage of identified pathogens is less than has been found in other studies performed in the northeast of Egypt (69%) (Zaki et al., 1986), but is consistent with a previous report characterizing diarrheal agents in children less than 18 month of age done in Aswan, in southern Egypt (Mikhail et al., 1989). Several possible explanations could account for the difference in pathogen isolation rate. It is possible that there is a higher rate of infection with enteric pathogens for which we did not specifically test, such as other diarrheagenic E. coli, caliciviruses, or other parasites. It might also be due to the collection technique. All 356 volunteers submitted rectal swabs from which bacterial pathogens could be cultured, but only 253 submitted a stool specimen from which viral and

Table 4 Antimicrobial susceptibility patterns for Campylobacter spp., Shigella spp., and Salmonella spp. isolates from children with diarrhea from Fayoum, Egypt, 2003 Percentage of isolates susceptible to Enteric bacterial pathogen Campylobacter spp. C. coli (n = 3) C. jejuni (n = 23) Shigella spp. S. boydii (n = 1) S. dysentery (n = 2) S. flexneri (n = 5) Salmonella spp. (n = 3)

AMP

CHL

ERY

NAL

CIP

TET

SXT

STR

AZT

LEV

67 52

NA NA

100 100

0 13

0 13

33 17

ND ND

33 22

100 100

33 26

0 0 20 0

100 0 60 33

100 100 20 0

100 100 100 0

100 100 100 67

100 0 20 0

0 100 20 33

NA NA NA NA

100 100 80 ND

ND ND ND ND

CHL was not tested with Campylobacter spp. ERY and STR were not applied for Shigella spp and Salmonella spp. AZT and LE were not applied to Salmonella spp. AMP = ampicillin; CEP = cephalothin; CHL = chloramphenicol; ERY = erythromycin; NAL = nalidixic acid; CIP = ciprofloxacin; TET = tetracycline; SXT = sulfamethoxazole/trimethoprim; STR = streptomycin; AZT = azthromycin; LEV = levofloxacin; NA = not applicable; ND = not determined.

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Antimicrobial susceptibility testing (AST) was performed on Campylobacter isolates using E-test, according to the manufacturer’s instructions (AB-BIODISK, Solna, Sweden). AST was also performed on Shigella and Salmonella isolates using the Kirby–Bauer disk diffusion method and interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI, 2005) guidelines (Bauer et al., 1966). Most Campylobacter isolates were found to be resistant ciprofloxacin, nalidixic acid, and tetracycline (Table 4), although all samples were found to be sensitive to the macrolides (erythromycin and azithromycin). The majority of Shigella isolates were determined to be resistant to 2 or more antimicrobial agents including ampicillin, tetracycline, and sulfamethoxazole/trimethoprim. Notably, one Shigella isolate was resistant to azithromycin. Antibiotic resistance testing was not performed for ETEC isolates. Similar to findings from an active surveillance study conducted in northern Egypt, our results indicate that Egyptian Campylobacter isolates are rapidly approaching universal resistance to the fluoroquinolones (Putnam et al., 2003). The availability and misuse of antimicrobial agents in developing countries like Egypt are likely to contribute to this problem (Engberg et al., 2001). Nevertheless, Campylobacter isolates remain sensitive to macrolide therapy. Thus, it is important that disease surveillance continues to monitor for a wide range of pathogens and antimicrobial agents (Engberg et al., 2001). ETEC was the most commonly isolated bacterial pathogen in this study. This finding is similar to that observed in several community-based studies and to a clinical-based study conducted in northern Egypt and to a hospital-based study conducted in southern Egypt (Mikhail et al., 1989; Rao et al., 2003; Wierzba et al., 2006; Zaki et al., 1986). Similar rates of Campylobacter were detected when compared to a previous study conducted in southern Egypt; however, isolation of Shigella spp. and Salmonella spp. was not as frequent. A possible explanation for this could be the limited duration and timing (i.e., at the end of the diarrheal season) of our study. Rotavirus was found to be the single most common cause of diarrhea, consistent with other studies conducted in Egypt (Wierzba et al., 2006). Significantly, this study determined that there was a higher percentage of C. parvum-positive cases than studies conducted elsewhere in Egypt (Abdel-Messih et al., 2002; Mikhail et al., 1989). Thus, although this study suggests that regional variations in pathogen distribution exist within Egypt, they are fairly minor. Furthermore, this study highlights the fact that Cryptosporidium is an underappreciated cause of pediatric diarrhea in Egypt, and the Fayoum district may be a suitable region for further study of Cryptosporidium pathogenesis and related host-immune responses. Acknowledgments The authors thank the Enteric Diseases Research Program Laboratory, NAMRU-3 members for their tireless

efforts in the laboratory and the social workers in the health care units. Special thanks to Cary Schlett for the management and analysis of data and valuable editorial contributions. The authors also thank Professor A.M. Svennerholm (University of Gfteborg, Sweden) for providing the monoclonal antibodies reagents for the ETEC ST/LT and CFA characterization. This research was conducted in compliance with all applicable federal regulations governing the Protection of Human Subjects in Research CPH no. 174 and DOD no. NAMRU3.2005.0005.

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