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Pathogens associated with persistent diarrhoea in children in developing countries Systematic review of observational studies FINAL DRAFT1
Katharine Abba1, Rebecca Sinfield1, Tony Hart2, Paul Garner1
1 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA 2 Department of Microbiology, University of Liverpool
Correspondence: [email protected] Date: 11 June 2007
Abstract Background: Persistent diarrhoea in children is common in children in developing countries, but best treatment is not known. We sought all relevant studies that reported pathogens in the stool to help describe the commonest causes and whether there were geographical patterns. Methods: Systematic review of descriptive studies in children in middle and low income countries with persistent diarrhoea. Quality of the laboratory tests was assessed, data on pathogens were extracted and summarised, including data from control groups when these were presented. With E coli, often several types were reported, and we took the commonest type as a conservative estimate of prevalence. Results across studies were compared for geographical patterns. If there were no large differences in pathogen prevalence, we aggregated data between studies to give a guide of overall prevalence. Results: Fourteen studies included. Some used episodes of diarrhoea as the unit of analysis, others used children; quality of reporting the laboratory procedures was variable, and classification (particularly for E coli types) changed over time. Studies suggested overall enteropathic E coli was commonly associated with diarrhoea. Other organisms were detected but across all studies their prevalence was under 10%. For studies with control groups, no obvious pattern emerged. Conclusions: A number of common pathogens are associated with persistent diarrhoea, but there is insufficient information to provide indirect evidence of potentially effective presumptive treatment of this condition. No geographic patterns are apparent.
1 This review has not been refereed.
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Introduction Background Diarrhoea causes an estimated 13.2% of child deaths worldwide1, most in children under the age of five in developing countries (2002 estimate2). In these children, around 3 to 19% of acute diarrhoea episodes become persistent3, and 50% of diarrhoea deaths are due to persistent diarrhoea. The World Health Organization (WHO) defines diarrhoea as the passing of three or more loose stools (which take the shape of a container) within a 24 hour period. A new episode of diarrhoea can occur after two full days without diarrhoea. Episodes of diarrhoea lasting for less than 14 days are defined as acute, episodes starting as an acute episode but lasting for 14 or more days are defined as persistent. As the number of deaths from acute diarrhoea reduces following widespread use of oral rehydration therapy, the contribution of persistent diarrhoea to overall diarrhoea mortality is increasing. In additional, persistent diarrhoea may adversely affect nutritional status; in one study, three months after a persistent diarrhoea episode, children had significantly lower weight for age and weight for height Z scores than three months before the episode4. Children living in poor areas with poor hygiene and sanitation conditions and children with poor nutritional status are most at risk of developing persistent diarrhoea3. Children with HIV/ AIDS are at particular risk; at initial presentation to hospital with HIV/AIDS, around 36-50%5,6,7 of children have persistent diarrhoea. Dysentery and more severe diarrhoeal illness are more likely to become persistent than milder episodes3. Previous antibiotic use and irrational use of antibiotics for acute diarrhoea are also risk factors3 for persistent diarrhoea. Causes The causes of persistent diarrhoea in populations are complex and poorly understood, and in individuals are often unknown. Pathogens associated with persistent diarrhoea are also found in healthy children without diarrhoea4. Some, such as Cryptosporidium, Giardia lamblia and enteroaggregative Escherichia coli (EAggEC) are thought to be particularly associated with persistent diarrhoea3 in some locations. Children with persistent diarrhoea and HIV infection may have different patterns of enteric pathogens than those without HIV8. Pathogens detected in persistent diarrhoea are often not the same as those detected in the original acute episode, suggesting that secondary infections may be important. In addition, children may be infected with more than one enteric pathogen, making it difficult to identify which, if any, is causing the illness. Some may have no detectable pathogens. When and how to treat As health facilities in developing countries tend to have limited diagnostic facilities, treatment often needs to be presumptive, based on symptoms and the mostly likely cause of the symptoms. The current recommendations of Integrated Management of Childhood Illness programme9 for treating persistent diarrhoea is that children with bloody diarrhoea are treated with antibiotics for Shigella, and children with watery diarrhoea not treated with antibimicrobials; except where Shigella, Giardia or Entamoeba histolytica are found. When persistent diarrhoea is caused by certain specific pathogens, the illness is likely to respond to appropriate treatment. However, the use of antimicrobials presumptively should be approached with caution. They can cause drug resistance and there are potential reactions of some micro-organisms: entero-haemorrhagic E coli (EHEC) may release toxins more readily when a person is treated with certain types of antibiotic, causing potentially severe illness10. Given the limited knowledge about the causes of persistent diarrhoea, we carried out a systematic review of studies that have attempted to identify aetiological agents in patients with this condition in developing countries. We hoped in particular to identify whether there were particular organisms that were commonly associated with this condition, and if there were any geographical patterns. Either could help guide treatment of this condition in the absence of diagnostic facilities. The review aimed to address the following questions about children with persistent diarrhoea:
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• Which enteric pathogens are identified and what are the frequencies of each? • How do the frequencies compare with control groups? • Are there any important regional differences? Objectives 1. To summarise studies that describe pathogens found in children with persistent diarrhoea, and comparisons with control children where this is available. 2. To explore possible influences on the variation between studies, including possible regional differences. Criteria for considering studies for this review Types of studies Descriptive studies, for example those analysing routinely collected data, data collected as part of a surveillance project, or point survey data, which provided data on faecal pathogens in at least 30 children with persistent diarrhoea. Types of participants Children under the age of six years living in low or middle income countries with any form of diarrhoea which has lasted more than 14 days. Where the age range of participants was not specifically stated but it was apparent that all or almost all were below the age of six, these studies were included. Participants could be recruited from any setting including the general community, primary health care services, and hospital outpatient and inpatient services. Studies could also include an appropriate comparison group of children without diarrhoea. Observations Presence of different faecal pathogens, identified by laboratory diagnosis of fresh faecal samples or rectal swabs, in participants with persistent diarrhoea and, where available, a suitable comparison group without diarrhoea. Studies also including children or adults of other ages, or diarrhoea lasting for less than 14 days, were included, provided that data from observations for children under the age of six with persistent diarrhoea could be extracted. Studies presenting data on only one particular species of pathogen were excluded. Methods of the review The search strategy, data extraction methods, quality assessment and data analysis methods are contained in annex 1. For E coli types, we extracted the data in two ways: assuming each E coli type was found in different individuals (upper estimate); and assuming that all had mixed infections, choosing the most frequent type (lower estimate).
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Results Selection of studies We included a total of 17 reports. Two of these reported on the same surveillance project, including a slightly different age range of children; both reports were included because they presented data on different pathogens. The initial search returned 1,789 references, from which we selected 102 for full text retrieval; of these we retrieved 98 and four were not available. Table 1 lists the reasons for exclusions of reports not included, and the number in each category. We found reports of a further five relevant studies through the references lists of the other studies and through another search for a related review. We excluded one of these because it may have included some of the same participants as another study already included. Characteristics of included studies Details of the characteristics of the individual studies included in the review are presented in Tables 2A and 2B. Table 2A shows the characteristics of the eight studies with a comparison group without diarrhoea, Table 2B shows the nine studies without a comparison group. Two papers based on the same data set are included separately as they presented data on a slightly different age group. Within each table, the studies are arranged by WHO region of where they carried out. A summary of the information presented in the table is presented below. Geographical Locations Nine studies (described ten reports) were carried out in the South-East Asia region; three (four reports) in Bangladesh and six in India. Five were carried out in the Americas, including three in Brazil (all undertaken in poor urban areas of a particular city), one in Guatemala and one in Mexico. Two were carried out in the Western Pacific region, one in a refugee camp on the border between Thailand and Cambodia, and one in Vietnam. There were no studies from the African Region. Participants Fourteen studies included children from birth to a specific age; in one study it was two years, in six it was three years, in one it was four years, in five it was five years and in one it was six years. One included children aged one to 36 months, one included children aged one to five years, and one did not give an age range, but stated a mean participant age of 16.6 months with a standard deviation of 30.1 months. There was no apparent pattern by region, for each region there were studies using different age ranges. Three studies (two in Southeast Asia, one in the Americas) excluded children with bloody diarrhoea, or included only those with watery diarrhoea. Three included children with all types of persistent diarrhoea, while the other studies did not describe the type of diarrhoea included. Seven studies had additional inclusion or exclusion criteria. Four included only children with additional signs and symptoms; two (in Southeast Asia) stated that the children must have lost weight or failed to gain weight during the episode, another (again in Southeast Asia) that the children should have a weight for length of less than or equal to 90% of the standard, one (in the Western Pacific) that children should also have either a rectal temperature of over 38oC, colic or vomiting. One study (in the Americas) excluded children with specific diagnoses such as caeliacs disease or short bowel syndrome, another (in the Americas) excluded those with diarrhoea that had persisted for longer than 18 days. One study in the Americas excluded children who had received antibiotics in the previous 72 hours. None of the included studies looked specifically at children with HIV infection, and none presented data on children with HIV infection as a subgroup. Source of participants Six of the included studies, including three from in South-East Asia and three from the Americas, employed active surveillance to identify all cases of persistent diarrhoea within a specific cohort of children in a defined community. Two, one in South-East Asia (two reports) and one in the Western Pacific, sampled children attending hospital outpatient departments,
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including children who were also subsequently admitted to the wards. Seven, four in South-East Asia, two in the Americas and one in the Western Pacific, sampled only children who were admitted to hospital, and one study in South-East Asia included both inpatients and outpatients at a hospital. Sampling strategies All six of the community cohort studies included in this review included all the children identified with persistent diarrhoea over the duration of the project. Two of the studies recruiting patients at healthcare facilities also sampled all consecutive eligible patients. One study included a random sample (method of randomisation not stated) one (two reports) used a systematic sampling method (every 25th patient). Reports of the further six studies did not describe their sampling methods. Date of studies The majority of the included studies were carried out over ten years ago, and none presented any data collected after 2000. Taking the mid-year of the data collection period as the reference point, three (two in Southeast Asia and one in the Americas), collected data in 1985 or before, seven (three in Southeast Asia, two in the Americas and two in the Western pacific), between 1986 and 1990, one (in the Americas) between 1991 and 1995, and two (one in Southeast Asia and one in the Americas) and between 1996 and 1999. Three papers, all from Southeast Asia, did not state the data of the data collection, two of these papers were published in 1995 and one in 1990. Duration of study All the included studies, except one which collected data for six months (in the Western Pacific), collected data for a period of one year or more. Five collected data for periods of whole years; three (two in Southeast Asian, one in the Western Pacific) for one year, one for two years (in the Americas), and one (in South-East Asia) for three years. Six collected data for other lengths of time: two (one in Southeast Asia, one in the Americas) for more than one but less than two years, three (one in South-East Asia, two in the Americas) for more than two but less than three years, and one (in the Americas) between three and four years. Three study reports did not give any information about dates or durations of data collection. Unit of analysis Five of the community cohort type studies used episodes as the unit of analysis, with children in both the persistent diarrhoea and comparison group (where used) often included in the analysis more than once. One of the studies recruiting children from a community surveillance project was actually reporting baseline data for a clinical trial, and therefore included individual children only once. The remaining studies presented data as for individual children, although occasionally children may have been included more than once, as none reported procedures to exclude children who presented to the facility more than once during the study period. Comparison groups Eight study reports (five in Southeast Asia, three in the Americas) presented pathogen frequency data on a comparison group of children without diarrhoea. A further two described a comparison group but did not present any relevant data. In six studies, the comparison participants were matched to participants with persistent diarrhoea using specific criteria, in two the diarrhoea and comparison groups were not matched. Five of the studies with comparison groups were community cohort studies which recruited the comparison group from the same community. The other three were based at health facilities and chose children attending or admitted to the hospital with illnesses other than diarrhoea.
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Quality of reporting for laboratory procedures Table 3 gives a summary of the quality of reporting of the laboratory procedures used in the study, plus an indication of whether there was any mention of antimicrobial susceptibility testing of bacteria being carried out, and whether quality control procedures were described. A tick indicates that the procedure was described or referenced (see methods section for definitions), a cross that it was not described or referenced, and a dash that it was not relevant to that study, for example, because it did not present data on a particular class of pathogens. The quality of reporting varied widely across studies in all WHO regions, with one study describing none of the procedures, while two reported six out of seven. Only two studies reported testing bacterial isolates for antimicrobial susceptibility, and one reported using a quality control procedure. Findings Individual study results Table 4 shows, for each study, the percentage of cases tested that tested positive (estimated prevalence), and the 95% confidence limits around the estimates, for different pathogens included in the review, in children with persistent diarrhoea, and, where available, children without diarrhoea (in italics). The estimated prevalence of each pathogen varied considerably between studies, and tended to be very imprecise, due to the relatively small sample sizes used in the studies. For studies with control groups, there was no significant difference in prevalence between the persistent diarrhoea and the comparator. As prevalence for individual organisms did not show extreme variations, we combined the data across studies to provide an approximate indicator of prevalence across studies. Combined study results In children with persistent diarrhoea Table 5 shows, for each WHO Region and for all the studies combined, the number of studies testing for each pathogen, the combined number of persistent diarrhoea cases tested, the weighted mean percentage across studies of cases tested testing positive, and the range between studies. There were no apparent differences by region. Under Table 5 we list the E coli types tested for in each study, using current category conventions. In two studies (Bangladesh 1992a, India 1992), it was uncertain whether one category of E coli referred to EPEC or EHEC; in these cases it was assumed to be EPEC. Figure 1 show the weighted mean percentage across all studies of cases tested testing positive. No studies tested for Astrovirus. One study tested for Norovirus (Brazil 2000) but was excluded from the analysis because the sample size was less than 30. Two studies tested specifically for EHEC and found no EHEC infections. Two different sets of E coli estimates are presented: those based on the upper estimate of the range, and those based on the lower estimates. Studies with only one estimate are included in both estimates. For each pathogen in each region, a median percentage of cases testing positive across the studies was also calculated. These are not presented, as in most cases they were within 5% (absolute values) of the mean. The only exception was the lower estimate for E coli in the Americas, where the mean was 35% and the median was 25%.
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Figure 1: Percentage of cases tested that tested positive for different pathogens in children with persistent diarrhoea
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Comparison between children with persistent diarrhoea and no diarrhoea Table 6 shows, for each pathogen, the number of studies testing for it in both children with persistent diarrhoea and a comparison group of children with no diarrhoea, total sample size, and weighted mean percentage across studies of cases and comparisons testing positive for different pathogens in studies undertaken in South-East Asia, the Americas, and all studies. There were no studies with a comparison group from the Western Pacific region. Figure 2 shows the weighted mean percentages of cases tested that tested positive for different pathogens within the persistent diarrhoea groups and comparison groups without diarrhoea in all eight studies with a comparison group. As described in the methods section, the data from the comparison group in each study was weighted according to the sample size of the group with persistent diarrhoea, to make the combined mean directly comparable with the combined mean for the persistent diarrhoea group. While children with persistent diarrhoea had a higher prevalence than those without diarrhoea of any kind of detectable enteric pathogen, for individual pathogens any differences were very small.
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Figure 2: Weighted mean percentage of cases tested that tested positive for different pathogens in children with persistent diarrhoea and comparison children with no diarrhoea
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No Diarrhoea
Discussion For this review, we were able to identify, retrieve and assess 16 studies, of which eight had a comparison group without diarrhoea. The studies varied widely in their methods, methodological quality, and the pathogens that they tested for. Most had quite small sample sizes, so estimates of prevalence from individual studies are imprecise. Studies were undertaken in a range of locations, yet none were carried out within the Africa region. The majority of were more than ten years old. In the assessed studies, children with persistent diarrhoea were infected with a wide range of enteric pathogens, the rates of each varying greatly between studies, although there were no apparent differences between WHO regions. The estimated prevalence of each pathogen was low (below 10%), with the exception of E coli, which had an estimates prevalence of around 30%. In every study, a proportion of children did not test positive for any enteric pathogen, although no study included tests for all relevant micro-organisms. In studies comparing children with persistent diarrhoea with a comparison group of children without diarrhoea, there were few differences between the two groups in the prevalence of any particular pathogen. When data from all eight studies was combined using a mean weighted according to the number of children with persistent diarrhoea who were tested, there were still no apparent differences for individual organisms, although a higher percentage of children with persistent diarrhoea tested positive for any enteric pathogen. The results of the review suggest that the routine use of an antimicrobial treatment for children with persistent diarrhoea without specific symptoms is unlikely to be recommended, unless a causative organism is identified, or there is an outbreak of a particular diarrhoea disease in the area.
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References Included studies India 1995a Ananthan S, and Anantha Subramanian. Microbial etiology and
assessment of immunological status of children with persistent diarrhoea. J Com Dis. 27(3):193-195
Cambodia 1992 Arthur, J. D.; Bodhidatta, L.; Echeverria, P.; Phuphaisan, S., and Paul, S. Diarrheal disease in Cambodian children at a camp in Thailand. Am J Epidemiol. 1992 Mar 1; 135(5):541-51.
Bangladesh 1992a Baqui, A. H.; Sack, R. B.; Black, R. E.; Haider, K.; Hossain, A.; Alim, A. R.; Yunus, M.; Chowdhury, H. R., and Siddique, A. K. Enteropathogens associated with acute and persistent diarrhea in Bangladeshi children less than 5 years of age. J Infect Dis. 1992 Oct; 166(4):792-6.
Guatemala 1992 Bartlett, A. V.; Torun, B.; Morales, C.; Cano, F., and Cruz, J. R. Oral gentamicin is not effective treatment for persistent diarrhea. Acta Paediatr Suppl. 1992 Sep; 381:149-54.
India 1989 Bhan, M. K.; Khoshoo, V.; Sommerfelt, H.; Raj, P.; Sazawal, S., and Srivastava, R. Enteroaggregative Escherichia coli and Salmonella associated with nondysenteric persistent diarrhea. Pediatr Infect Dis J. 1989 Aug; 8(8):499-502.
India 1999 Bhandari, N.; Bahl, R.; Dua, T.; Kumar, R., and Srivastava, R. Role of protozoa as risk factors for persistent diarrhea. Indian J Pediatr. 1999 Jan-1999 Feb 28; 66(1):21-6.
India 1992 Bhatnagar, S.; Bhan, M. K.; George, C.; Gupta, U.; Kumar, R.; Bright, D., and Saini, S. Is small bowel bacterial overgrowth of pathogenic significance in persistent diarrhea? Acta Paediatr Suppl. 1992 Sep; 381:108-13.
Brazil 1995 Fang, G. D.; Lima, A. A.; Martins, C. V.; Nataro, J. P., and Guerrant, R. L. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J Pediatr Gastroenterol Nutr. 1995 Aug; 21(2):137-44.
Mexico 2003 Guerra-Godinez, J. C.; Larrosa-Haro, A.; Coello-Ramirez, P.; Tostado, H. R.; Rivera-Chavez, E.; Castillo de Leon, Y. A.; Bojorquez-Ramos Mdel, C., and Aguilar-Benavides, S. Changing trends in prevalence, morbidity, and lethality in persistent diarrhea of infancy during the last decade in Mexico. Arch Med Res. 2003 May-2003 Jun 30; 34(3):209-13.
Bangladesh 1992b Henry, F. J.; Udoy, A. S.; Wanke, C. A., and Aziz, K. M. Epidemiology of persistent diarrhea and etiologic agents in Mirzapur, Bangladesh. Acta Paediatr Suppl. 1992 Sep; 381:27-31.
India 1995b Jindal et al. A study of infective aetiology of chronic diarrhoea in children in Amritsar. J Indian Med Assoc. 93 (5): 169-171
Brazil 2000 Lima, A. A.; Moore, S. R.; Barboza, M. S. Jr; Soares, A. M.; Schleupner, M. A.; Newman, R. D.; Sears, C. L.; Nataro, J. P.; Fedorko, D. P.; Wuhib, T.; Schorling, J. B., and Guerrant, R. L. Persistent diarrhea signals a critical period of increased diarrhea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil. J Infect Dis. 2000 May; 181(5):1643-51.
Bangladesh 1991 Mahalanabis, D.; Alam, A. N.; Rahman, N., and Hasnat, A. Prognostic indicators and risk factors for increased duration of acute diarrhoea and for persistent diarrhoea in children. Int J Epidemiol. 1991 Dec; 20(4):1064-72.
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India 2001 Mishra, O. P.; Dhawan, T.; Singla, P. N.; Dixit, V. K.; Arya, N. C., and Nath, G. Endoscopic and Histopathological Evaluation of Preschool Children With Chronic Diarrhoea. Journal of Tropical Pediatrics. 2001 Apr; 47(2):77-80.
Vietnam 1992 Ngan, P.K.; Khanh, N.G; Tuong, C.V.; Quy, P.P; Anh, D.N; Thuy, H.T.. Persistent diarrhoea in Vietnamese children: a preliminary report. Acta Paediatric Supplment 381:124-6, 1992
Brazil 1990 Schorling, J. B.; Wanke, C. A.; Schorling, S. K.; McAuliffe, J. F.; de Souza, M. A., and Guerrant, R. L. A prospective study of persistent diarrhea among children in an urban Brazilian slum. Patterns of occurrence and etiologic agents. Am J Epidemiol. 1990 Jul; 132(1):144-56.
Bangladesh 1988 Shahid, N. S.; Sack, D. A.; Rahman, M.; Alam, A. N., and Rahman, N. Risk factors for persistent diarrhoea. BMJ. 1988 Oct 22; 297(6655):1036-8.
Others 1. Mathers CD, Bernard C, Moesgaard Iburg K, Inoue M, Ma Fat D, Shibuya S, Stein C, Tomijima N, Xu H. Global Burden of Disease in 2002: data sources, methods and results. Global Programme on Evidence for Health Policy Discussion Paper No. 54 World Health Organization. 2003 (revised 2004) 2. Global burden of disease and injury. In: Murray C, Lopez A, eds. Global health statistics. Cambridge, MA, Harvard School of Public Health on behalf of the World Health Organization and the World Bank, 1996 (Series Vol. 2). 3. Lima AAM and Guerrant RL. Persistent diarrhoea in children: epidemiology, risk factors, pathophysiology, nutritional impact, and management. Epidemiologic Reviews 1992, 14:222-242 4. Lima AAM, Moore SR, Barboza MS, Soares AM, Schleupner MA, Newman RD, Sears AM, Nataro JP,Fedorko DP, Wuhib T, Schorling JB and Guerrant RL. Persistent diarrhoea signals a critical period of increased diarrhoea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil. Journal of Infectious Diseases. 2000, 181:1643-51. 5. Lumbiganon P, Kosalaraksa P, Loapaiboon M. Survival of children with AIDS; experience in a university hospital in northeast Thailand. J Med Assoc Thai. 2000; 83(6):652-6 6. Emodi IJ, Okafor GO. Clinical manifestations of HIV infection in children at Enugu, Nigeria. J Trop Pediatr. 1998. 44(2):73-6 7. Lodha R, Upadhyay A, Vishal K, Kabal SK. Clinical profile and natural history of children with HIV infection. The Indian Journal of Pediatrics 2006. 73(3):201-204 8. Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, Ndeezi G, Downing R, Feng X, Akiyoshi DE and Tzipori S. Cryptosporidiosis and microsporidiosis in Ugandan children with persistent diarrhoea with and without concurrent infection with human immunodeficiency virus. Am J Trop Med Hyg 2005, 73 (5): 921-925 9. Handbook IMCI: Integrated Management of childhood illnesses. WHO/ UNICEF 2006. 10. Kimmitt PT, Harwood CR and Barer MR. Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.
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Annex 1: Methods of the review Search strategy for identification of studies The search for suitable studies was be performed by Vittoria Lutje, using the following electronic databases: MEDLINE (1966 to date) via the OVID interface. EMBASE (1980 to date) via the OVID interface. LILACS database - Latin American and Caribbean Health Sciences Literature (1982 to date) - via Virtual Health Library interface. WEB OF SCIENCE (Science Citation Index Expanded – 1945 to present). The following search strategy was used in MEDLINE: 1. Persistent (diarrhea OR diarrhea) ti, ab. 2. Chronic (diarrhea OR diarrhea) ti, ab. 3. Watery (diarrhea OR diarrhea) ti, ab 4. (Diarrheal disease*) OR (diarrhoeal disease*) ti, ab 6. 1 OR 2 OR 3 OR 4 ) NOT cancer NOT (inflammatory bowel disease*) NOT (ulcerative colitis) 7. Diarrhea, Infantile/classification OR diarrhea, infantile/etiology OR diarrhea, infantile/microbiology OR diarrhea, infantile/parasitology OR diarrhea, infantile/pathology [MeSH] 8. 6 OR 7 9. Child* OR infant* OR pediatr* ti, ab 10. 8 AND 9 11. Case-control studies [MeSH] OR Epidemiological studies {MeSH] 12. Descript* OR surve* OR monitor* ti, ab 13. 11 OR 12 14. 10 AND 13, Limits:humans The strategy was amended where necessary to search the other databases listed. No language restrictions were applied. The reference lists of included studies were also scrutinised for additional relevant studies. Methods of the review Study selection Two authors independently inspected titles and abstracts identified by the literature search in order to identify potentially relevant publications. All potentially relevant publications identified by at least one reviewer were obtained in full text format, with the exception, due to time constraints, of reports not available within the United Kingdom. One author then applied the inclusion criteria to select which studies to include in the review, consulting with another in cases of uncertainty. All publications were scrutinised for duplication of study results. Data extraction For each study report, two authors worked together to extract information describing the characteristics of the study and assess its quality, using a proforma as a guide. The data collected was as follows:
• Location: Geographical location of the study, including any relevant background socio-economic or environmental information, and WHO region
• Study dates: Start and end dates of the study • Participant characteristics: Age range, any additional inclusion criteria or local factors
such as HIV infection or malnutrition. • Sampling: Where the participants were recruited from, for example, outpatient clinic,
hospital ward, community, and how they were sampled • Comparison group: Whether the study also had a comparison group without diarrhoea,
and how they were selected and whether and how they were matched to the diarrhoea group
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• Sample sizes: Of the persistent diarrhoea group and comparison group with no diarrhoea
• Unit of analysis: Data for the diarrhoea group presented by children or episodes One author extracted results data for each study report. This included:
• Types of enteric pathogen tested for in each group (persistent diarrhoea and comparison group without diarrhoea)
• Numbers of children (or episodes) tested for each pathogen • Number or percentage of children, episodes or samples where each pathogen was
detected. • Number or percentage of children with any detected enteric pathogen.
We extracted data on selected pathogens only, identified as important potential causes of diarrhoea, in terms of both prevalence and affect, by an expert microbiologist (TH). For E coli, we combined all enteropathic types (any which have the capacity to cause enteric illnesses), with the exception of the entero-heamorrhagic type (EHEC), into one category, because different studies classified E coli very diversely, making it impossible to compare types across studies. E coli of an unspecified type, or classed as ‘untypable’ was excluded from the analysis because it was not possible to assess whether it was an enteropathic type. E. coli described by the authors as diff. E coli, EAEC-D, EAEC (difuse) or E coli showing diffuse Hep-2 adherence were also excluded because it is uncertain whether they are pathogenic. EHEC was kept separate where possible due to its different behaviour. For each study a note was made of the E coli types tested for, as described in the study. The list of pathogens for which we extracted data, where available, is shown below:
Viruses Rotavirus Enteric adenovirus Astrovirus Norovirus Bacteria Campllobacter Shigella Salmonella Vibrio cholerae Enteropathic E coli (excluding EHEC) Entero heamorrhagic E Coli (EHEC) Parasites Giardia lamblia Crytosporidium Entamoeba histolytica
Where it was not clear in the report whether some individual patients may have tested positive more than one type of E coli, we used all the available information (including for example, number of patients with any pathogens detected, number with more than one pathogen detected) to calculate the highest and lowest number or percentage of children, episodes or samples that may have tested positive for enteropathic E coli. Where any organisms presented in a study paper were excluded from the analysis, we did not use any data presented on ‘any pathogen’ from that study. We planned to also extract data for any subgroups presented in the reports (for example by age or HIV status), but no reports presented data for subgroups. Some additional data was also collected to describe the quality of reporting of the laboratory procedures used for the study. These included:
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• Laboratory processes: o Were the any methods or timings described for collected and testing faecal
samples for pathogens? o Were any methods described or referenced for the detection of parasites, other
then the word ‘microscopy’? (e.g. stain, magnification) o Were any methods described or referenced for the culture of bacteria, other
than the word ‘culture’? (e.g. media, temperature) o Were any methods described or references for the identification of cultured
bacteria? o Were any methods described for the identification of viruses? (the name of the
test was sufficient) • Quality control: Were methods for testing and controlling the quality of the laboratory
tests used in the study described? • Antimicrobial susceptibility testing: Which of the pathogens detected in the study, if any,
were tested for antimicrobial susceptibility Data analysis For each relevant reported pathogen in each study, we calculated, if both were not provided in the report, the number or percentage of children, in both the persistent diarrhoea and comparison groups, with the pathogen detected. We also calculated the standard deviation and the 95% confidence intervals around the percentage. The equation we used to calculate confidence intervals was:
95% CI = mean +/- 1.96 SD Studies were grouped by the WHO Region where they were undertaken. For each region, and for all the studies together, we noted or calculated the following for each pathogen and for any pathogen:
For the groups with persistent diarrhoea:
• Number of studies where the pathogen was tested for and presented. • Total number of participants or episodes included in these studies. • The maximum and minimum percentage across studies of children or episodes where
the pathogen was detected. • The median percentage across studies of children or episodes where the pathogen was
detected. • Mean percentage across studies (weighted by study sample size) of children or
episodes where each pathogen was detected.
We also calculated each of these summary statistics for the subset of studies that had a comparison group without diarrhoea. For the groups without persistent diarrhoea we calculated similar summary statistics. However, in order to make the mean percentage across trials comparable with that for the persistent diarrhoea group, we weighted the contribution of each study according to the size of group with persistent diarrhoea, rather than the group with no diarrhoea. To avoid presenting data that is potentially misleading due to very small sample sizes we do not present any data based on a combined sample size of less than 30.
14
Appendix: Abbreviations used for different types of enteropathic Escherichia .coli
ETEC Enterotoxigenic E coli
EIEC Enteroinvasive E coli
EPEC Enteropathogenic E coli
EAggEC Enteroaggregative E coli
EHEC Enteroheamorrhagic E coli
Table 1: Reasons for exclusion of papers identified in the initial screening process as potentially relevant Reason for exclusion Papers
Diarrhoea status of participants Included children with acute diarrhoea only 9 Wrong definition of persistent diarrhoea 2 Asymptomatic children only 1 Included all children in a community, whether or not they had diarrhoea 1 No separate analysis for cases of acute and persistent diarrhoea 38
Other participant characteristics
Participants were of the wrong age range 8 Participants resided in a high income country 2
Study design factors
Less than 30 participants 5 Faecal samples taken in the acute stage (before day 14) of the persistent episode 5 Tested for one organism only 4 Sampled only children who had been in close contact with Shigella dysentery 1 Reviewed hospital records only for children found positive for enteric pathogens 1
Not relevant
No data on frequency of pathogens presented 5
Table 2A: Characteristics of included studies: those with a comparison group without diarrhoea Study report
Location Start date Duration
Participants (cases)
Source of participants (cases)
Sampling method
Number of cases
Unit of Analysis
Source of comparison group
Matching of comparison group
Number of comparisons
Bangladesh 1992a
Households in Matlab (rural)
1988 1 year
< 5 years Active household surveillance
All cases identified
varied 68 - 184
Episodes Same study cohort
Age and proximity of residence
varied 67 - 164
Bangladesh 1992b
Households in Mirzapur (rural)
1987 2 years
< 6 years Active household surveillance
All cases identified
varied 53 - 153
Episodes Same study cohort
Age and proximity of residence
varied 46 - 213
India 1989 Hospital in New Delhi 1984 1 year
< 2 years, no blood in stools, weight loss during episode
Hospital admissions
Consecutive cases
92 Children Other wards with non-gastro-intestinal illnesses
Age and nutritional status
92
India 1992a Hospital in New Delhi 1988 28 months
< 3 years, no blood in stools, weight for height ≤ 90% of standard
Hospital admissions
Not described 81 Children Nutrition clinic with failure to thrive and weight for height ≤ 90% of standard
Not matched 32
India 1999 Households in a slum area of New Delhi
Not stated < 3 years Active household surveillance
All cases identified
115 Episodes Same study cohort
Age, nutritional status and proximity of residence
115
Brazil 1990 Households in an urban slum, Fortaleza, northeast Brazil
1984 28 months
< 5 years Active household surveillance
All cases identified
40 Episodes Same community study cohort.
Not matched 38
Brazil 1995 Children’s hospital in Fortaleza, northeast Brazil
1988 32 months
< 3 years, not had antibiotics within previous 72 hours
Hospital admissions
Not described 56 Children Children on other wards.
By age 42
Brazil 2000 Households in a shantytown, Fortaleza, northeast Brazil
1989 45 months
< 4 years Active household surveillance
All cases identified
88 Episodes Same community study cohort
By age and sex 443
Table 2B: Characteristics of included studies: those without a comparison group Study report
Location Start date Duration
Participants (cases)
Source of participants (cases)
Sampling method
Number of cases
Unit of analysis
Source of comparison group
Matching of comparison group
Number of comparisons
Bangladesh 1988
Diarrhoea treatment centre, Dhaka
1983 3 years
< 5 years Outpatient attendances
Systematic every 25th case
410 Children N/A N/A N/A
Bangladesh 1991
Diarrhoea treatment centre, Dhaka
1983 3 years
1 month to 3 years
Outpatient attendances
Systematic every 25th case
varied 391 - 445
Children N/A N/A N/A
India 1995a
Children’s hospital in Madras
Not stated < 5 years Hospital admissions
Not described 100 Children N/A N/A N/A
India 1995a
Hospital in Amritsar
Not stated < 3 years Hospital admissions
Not described 150 Children N/A N/A N/A
India 2001 Hospital in Varanasi 1998 15 months
1 to 5 years, loss of weight or failure to gain weight
Hospital admissions & Outpatient attendences
Randomly 57 Children N/A N/A N/A
Guatemala 1992
Households of a rural indigenous community
1988 20 months
< 3 years with watery diarrhoea for 14 to 18 days
Active household surveillance
All cases identified
49 Children N/A N/A N/A
Mexico 2003 Children’s hospital 1997 Unknown between 24 and 36 months
mean age 16.6 months, SD 30.1 months, without specific diagnosis
Hospital admissions
Not described 89 Children N/A N/A N/A
Cambodia 1992
Hospital serving a resettlement camp on border of Thailand and Cambodia
1989 6 months
< 5 years and: rectal temperature over 38oC, colic or vomiting
Hospital admissions
Consecutive cases
79 Children N/A N/A N/A
Vietnam 1992
Children’s hospital in Hanoi
1988 12 months
< 3 years Hospital admissions
Not described.
83 Children N/A N/A N/A
Table 3: Studies describing or referencing different laboratory procedures
Bang
lades
h 198
8
Bang
lades
h 199
1
Bang
lades
h 199
2a
Bang
lades
h 199
2b
India
1989
India
1992
India
1995
a
India
1995
b
India
1999
India
2001
Camb
odia
1992
Vietn
am 19
92
Braz
il 199
0
Braz
il 199
5
Braz
il 200
0
Guate
mala
1992
Mexic
o 200
3
Sample Storage and transport x √ √ √ √ √ x √ √ x √ x √ √ √ √ x
Parasites Stain, magnification or mount x - √ √ √ x x √ √ x √ x √ √ √ √ √
Media, temperature, etc x √ √ √ √ √ √ √ - x √ x √ √ √ √ √ Bacteria Identification methods x √ √ √ √ √ √ √ - x √ x √ √ x √ √
Viruses Test used √ √ - √ √ x - - - - √ √ √ √ √ √ -
Susceptibility testing of bacteria x x x √ x x x x - x √ x x x x x x General
Quality control methods x x x x x x x x √ x x x x x x x x
Score 1/7 4/6 4/6 6/7 5/7 3/7 2/6 4/6 3/3 0/6 6/7 1/7 5/7 5/7 4/7 5/7 3/6
Table 4A: Percentage of sampled cases and comparisons testing positive for different pathogens in studies carried out in the South-East Asia region, with calculated 95% confidence intervals
Bangladesh 1988
Bangladesh 1991
Bangladesh 1992a
Bangladesh 1992b India 1989 India 1992 India 1995a India 1995b India 1999 India 2001
Virus
Rotavirus - 2 (0 to 3) -
1 (0-2) 0
6 (2 to 10) 3 (1 to 5)
2 (0 to 5) 1 (0 to 3)
0 0 - - - -
Enteric adenoviris - - - - 1(0 to 3) 2 (0 to 5) - - - - -
Bacteria
Camplylobacter - 10 (7 to 13) -
11 (6 to 16) 16 (10 to 21)
8 (4 to 13) 8 (2 to 14)
4 (0 to 9) 3 (0 to 7)
4 (2 to 8) 3 (0 to 9)
0 -
1 (0 to 2) - - -
Shigella 14 (11 to 17) - -
6 (2 to 9) 2 (0 to 4)
5 (2 to 9) 7 (3 to10)
3 (2 to 7) 0
2 (0 to 6) 0
0 -
2 (1 to 7) - -
2 (0 to 5) -
Salmonella - - - 1 (0 to 2) 0
16 (9 to 24) 0
5 (2 to 10) 0
0 -
9 (4 to 13) - - -
Vibrio cholerae - 3 (1 to 4) -
1 (0 to 3) 2 (0 to 4)
0 0 - -
0 - - - -
*All Enteropathic E coli - upper estimate -
6 (4 to 9) -
33 (26 to 31) 43 (35 to 50)
25 (13 to 36) 20 (8 to 31)
32 (22 to 41) 10 (14 to 21)
26 (6 to 76) 19 (5 to 33)
76 (68 to 84 -
23 (16 to 29) - - -
*All Enteropathic E coli - lower estimate - -
21 (14 to 27) 34 (27 to 41) - -
44 (34 to 55) 13 (1 to 24)
-
21 (15 to 28) -
-
EHEC - - - - 0 0 - - - - -
Parasites
Giardia lamblia 2 (1 to 4) - -
4 (1 to 8) 2 (0 to 4)
4 (2 to 8) 4 (1 to 8)
8 (2 to 13) 5 (1 to 10)
2 (0 to 6) 22 (8 to 36)
8 (3 to 13) -
2 (1 to 7) -
20 (13 to 27) 4 (1 to 8) -
Crytosporidium - - 0 2 (0 to 4) - - -
0 - -
1 (0 to 4) 5 (1 to 9) -
Entomoeba histolytica - - 0 0
1 (0 to 3) 5 (1 to 9)
1 (0 to 3) 0
0 1 (0 to 3)
4 (0 to 8) -
1 (0 to 4) -
8 (3 to 13) 5 (1 to 9)
9 (1 to 16) -
At least one pathogen - - - - 65 (55 to 75) 20 (15 to 25) -
88 (82 to 94) -
- -
-
95% confidence intervals in parentheses, comparison group without diarrhoea in italics
Table 4B: Percentage of sampled cases and comparisons testing positive for different pathogens in studies carried out in the Western Pacific and Americas regions, with calculated 95% confidence intervals
Western Pacific The Americas
Cambodia 1992
Vietnam 1992 Brazil 1990 Brazil 1995 Brazil 2000
Guatemala 1992 Mexico 2003
Virus
Rotavirus 19 (10 to 29) -
5 (0 to 9) -
20 (8 to 32) 21 (8 to 34)
9 (4 to 16) 2 (0 to 7)
9 (4 to 16) 2 (0 to 6)
9 (0 to 19) - -
Enteric adenoviris - - - - 11 (3 to 18) 0 - -
Bacteria
Camplylobacter 14 (6 to 22) -
0 - -
0 0 -
6 (0 to 12) - -
Shigella 5 (0 to 10) -
0 -
3 (0 to 7) 3 (0 to 8)
5 (0 to 11) 2 (0 to 7)
2 (0 to 5) 1 (0 to 2)
6 (0 to 13) -
1 (0 to 3) -
Salmonella 8 (2 to 13) -
0 -
0 0
0 0
0 0 (0 to 2)
0 -
3 (0 to 7) -
Vibrio cholerae 0 - - - -
0 0 (0 to 1) -
0 -
*All Enteropathic E coli - upper estimate
25 (16 to 35) -
27 (17 to 36) -
23 (7 to 35) 11 (1 to 20)
91 (84 to 99) 33 (19 to 48)
63 (50 to 76) 57 (50 to 64)
37 (23 to 50) - -
*All Enteropathic E coli - lower estimate
18 (9 to 26) -
15 (4 to 26) 5 (0 to 12)
68 (56 to 80) 31 (17 to 45)
35 (24 to 47) 33 (26 to 39)
13 (3 to 22) - -
EHEC - - - - 0 0 -
Parasites
Giardia lamblia 9 (3 to 15) -
4 (2 to 8) -
18 (6 to 29) 13 (2 to 24)
0 0
20 (12 to 29) 8 (5 to 10)
14 (4 to 24) -
7 (2 to 12) -
Crytosporidium 11 (4 to 18) -
0 -
0 0
25 (6 to 36) 5 (0 to 11)
18 (9 to 27) 5 (3 to 8)
6 (0 to 13) -
0 -
Entomoeba histolytica 3 (0 to 6) -
0 -
3 (0 to 7) 0
0 0
0 1 (0 to 2)
0 -
0 -
At least one pathogen - 36 (26 to 46) -
55 (40 to 70) 37 (22 to 52) -
88 (80 to 95) 74 (68 to 80) - -
95% confidence intervals in parentheses, comparison group without diarrhoea in italics
Bangladesh 1991 - ETEC Bangladesh 1992a - ETEC, EAggEC, EPEC *Bangladesh 1992b - EAggEC India 1989 - ETEC, EPEC, EHEC, EAggEC India 1992 - ETEC, EPEC, EAggEC India 1995a - ETEC, EPEC India 1995b - EPEC, ETEC, EIEC Cambodia 1992 - ETEC, EPEC, EIEC Vietnam 1992 - ETEC, EPEC Brazil 1990 - ETEC, EPEC, EIEC Brazil 1995 - EAggEC, EPEC, ETEC Brazil 2000 - ETEC, EPEC, EAggEC Guatemala 1992 -ETEC, EPEC, EAggEC *Also tested for ETEC, data on ETEC not presented as the number of cases tested for ETEC was different than for the other E coli types
E coli types tested for
Table 5: Number of studies, total sample size, and percentage of cases testing positive for different pathogens by WHO Region
South East Asia Western Pacific The Americas All Studies Sample Mean Range Studies Sample Mean Range Studies Sample Mean Range Studies Sample Mean Range Virus Rotavirus 5 885 2% 0 - 6% 2 162 12% 5 - 19% 4 194 11% 9 - 20% 11 1241 5% 0 - 20% Enteric adenoviris 1 92 1% - 0 - - 1 66 11% - 2 158 5% 1 - 11% Bacteria Camplylobacter 7 1184 7% 0 - 11% 2 162 7% 0 - 14% 3 147 2% 0 - 6% 12 1493 7% 0 - 4% Shigella 8 1213 7% 0 - 14% 2 162 2% 0 - 5% 5 322 3% 2 - 6% 15 1697 6% 0 - 4% Salmonella 5 576 6% 0 - 16% 2 162 4% 0 - 8% 5 322 0% - 12 1060 4% 0 - 16% Vibrio cholerae 3 710 2% 0 - 3% 1 79 0% - 2 177 0% - 6 966 1% 0 - 3% *All Enteropathic E coli - upper or only estimate 7 1026 26% 6 - 76% 2 162 26% 25 - 27% 4 212 57% 23 - 88% 13 1400 31% 6 - 91% *All Enteropathic E coli - lower or only estimate 7 1026 24% 6 - 76% 2 162 22% 18 - 27% 4 212 35% 13 - 68% 13 1400 25% 6 - 76% EHEC 1 92 0% - 0 - - 1 51 0% - 2 143 0 - Parasites Giardia lamblia 8 1164 6% 2 - 20% 2 162 6% 4 - 9% 5 322 12% 0 - 20% 15 1648 7% 0 - 20% Crytosporidium 4 482 1% 0 - 2% 2 162 6% 0 - 11% 5 305 10% 0 - 25% 11 949 5% 0 - 25% Entomoeba histolytica 8 811 3% 0 – 9% 2 162 1% 0 - 3% 5 322 0% 0 - 3% 15 1295 2% 0 - 9% At least one pathogen 2 273 70% 51 - 88% 1 83 36% - 2 112 76% 55 - 88% 6 468 65% 36 - 88%
E coli types tested for
Bangladesh 1992a - ETEC, EAggEC, EPEC *Bangladesh 1992b - EAggEC India 1989 - ETEC, EPEC, EHEC, EAggEC India 1992 - ETEC, EPEC, EAggEC Brazil 1990 - ETEC, EPEC, EIEC Brazil 1995 - EAggEC, EPEC, ETEC Brazil 2000 - ETEC, EPEC, EAggEC
*Also tested for ETEC, data on ETEC no presented as the number of cases tested for ETEC was different than for the other E coli types
Table 6: Number of studies, total sample size, and percentage of cases and comparisons testing positive for different pathogens in South-East Asia and the Americas
South-East Asia
The Americas All Studies
Persistent diarrhoea
No diarrhoea Studies
Persistent diarrhoea
No diarrhoea Studies
Persistent diarrhoea
No Diarrhoea
Sample Mean Sample Mean Sample Mean Sample Mean Sample Mean Sample Mean Virus Rotavirus 4 496 2% 501 1% 3 162 12% 162 7% 7 658 5% 663 3% Enteric adenoviris 1 92 1% 92 2% 1 66 11% 82 0% 2 158 5% 174 1% Bacteria Camplylobacter 4 489 8% 375 9% 2 96 0% 80 0% 6 585 6% 455 8% Shigella 4 496 5% 501 3% 3 184 3% 520 2% 7 680 4% 1021 2% Salmonella 3 326 6% 337 0 3 184 0% 520 0% 6 510 4% 857 0% Vibrio cholerae 2 317 1% 373 1% 1 88 0% 440 0% 3 405 0% 813 1% *All Enteropathic E coli - upper estimate 4 386 32% 329 27% 3 164 63% 75 38% 7 550 41% 604 30% *All Enteropathic E coli - lower estimate 4 386 26% 328 22% 3 164 41% 275 25% 7 550 31% 603 23% EHEC 1 92 0 92 0 1 51 0% 51 0% 2 143 0% 143 0% Parasites Giardia lamblia 5 504 8 540 7% 3 184 14% 522 7% 8 688 10% 1062 7% Crytosporidium 2 232 1% 280 4% 3 167 16 369 5% 0 399 7% 649 4% Entomoeba histolytica 5 504 2% 541 4% 3 184 1% 522 1% 5 688 2% 1063 3% At least one pathogen 1 92 73% 92 22% 2 112 76% 233 60% 3 204 75% 325 43%
E coli types tested for
Cambodia 1992 - ETEC, EPEC, EIEC Bangladesh 1992a - ETEC, EAggEC, EPEC Vietnam 1992 - ETEC, EPEC *Bangladesh 1992b - EAggEC Brazil 1990 - ETEC, EPEC, EIEC India 1989 - ETEC, EPEC, EHEC, EAggEC Brazil 1995 - EAggEC, EPEC, ETEC India 1992 - ETEC, EPEC, EAggEC Brazil 2000 - ETEC, EPEC, EAggEC India 1995a - ETEC, EPEC Guatemala 1992 -ETEC, EPEC, EAggEC India 1995b - EPEC, ETEC, EIEC
*Also tested for ETEC, data on ETEC not presented as the number of cases tested for ETEC was different than for the other E coli types
