Professional Documents
Culture Documents
Bayeh Abera1, Tadesse Hailu1, Laway Beza2, Wondemagen Mulu1, Endalew Yizengaw1, Megbaru
ABSTRACT
Diarrhea in children is the second cause of morbidity and mortality in the Sub-Saharan Africa. This
study was conducted among 344 children with diarrhea aged below 5 years to determine protozoan
and bacterial etiology of diarrhea with antimicrobial usages. G. lamblia and E. histolytica
trophozoites were identified using direct wet mount microscopy. Cryptosporidium oocysts were
identified using modified Ziehl-Neelson methods. Pathogenic bacterial species were identified by
standard microbiological culture methods. The median age of children and duration of diarrhea
were 20 months and 2 days, respectively. Overall, 47.1% (95% CI, 41.9-52.4%) of children with
diarrhea had either protozoan or bacterial pathogens. Cryptosporidium species was were detected in
12.8%, while bacterial pathogens was were isolated in 13.1% of children. Prevalence of G. lamblia
and E. histolytica/dispar were 11.3% and 9.9%, respectively. ProtozoalProtozoan diarrhea was
significantly more common among males compared with females (P=0.03). Cotrimoxazole with
51.7% was the most frequently prescribed drug for diarrheic children. Empirically, 77% of
cotrimoxazole and ciprofloxacin were prescribed for laboratory unconfirmed diarrheic children.
Among antimicrobials tested, 55.6% and 48.9% of bacterial isolates revealed resistance to
pathogens are prevalent in diarrhea diarrheic children in the study area. However, in the routine
health center service these pathogens are not identified. Therefore, empirical treatments were not
appropriate for majority of diarrheic children. Laboratory capacity for diarrheic stool culture for
bacteriological data and modified Ziehl-Neelsen stool stain at health care services are essential to
1. Introduction
Among wide spectrums of waterborne diseases, acute diarrhea is a major public health problem
worldwide (Scrimgeour and Lukaski, 2008). According to the world World health Health
organization Organization reports, 4 million cases and 1.8 million deaths occur in children under-
five years of age annually worldwide. Acute diarrhea is one of the leading causes of morbidity and
mortality in children under 5 years of age (UNICEF/WHO, 2009). In sub-Saharan Africa, mortality
caused by acute diarrhea reaches 37% of all deaths during the first year of life (Lorna, 2005).
Ethiopia like other sub-Saharan African countries has high morbidity and mortality linked
with acute diarrhea. The Ethiopian Demographic and Health Survey (EDHS) conducted in 2016
from acute community diarrhea in two-weeks was 12.0% (EDHS, 2016). Likewise, studies
conducted at community levels in the northwest Amahara such as Bahir Dar City, West Gojjam and
North Gondar zones reported high prevalence of two-week childhood diarrhea (Dessalegn et al.,
Entamoeba histolytica (Efstratiou et al., 2017) and bacteria like Campylobacter jejuni, Escherichia
coli O157, Shigella species and Vibrio cholera (Wang and Cao., 2014). In Ethiopia, Giardia
lamblia, Entamoeba histolytica and Cryptosporidium parvum are the major enteric protozoan
causing acute diarrhea (Alemu et al., 2011; De Lucio et al., 2016; Mohammed et al., 2016; Fentie et
al., 2013). Among bacterial diarrhea, Shigella species and E. coli 0157:H7 were commonly reported
and tertiary level). The primary health care unit is composed of one health center and five satellite
health posts (Amhara Regional Health Bureau, 2017). In the primary health care facilities, data on
pathogen-specific acute diarrhea are limited due to lack of laboratory facilities. In this regard,
diarrheal pathogens such as Cryptosporidium species, Shigella spp., Escherichia coli 0157:H7 and
Salmonella spp., are not identified. Thus, empirical therapy with antimicrobial is a common
practice for children with acute diarrhea. This leads to the emergence and wide spread of
antimicrobial resistances. This study was therefore conducted to determine the protozoalprotozoan
and bacterial etiology of acute diarrhea in children under five years of age at the health centers.
Furthermore, empirical antimicrobial usage for acute diarrhea and antimicrobial resistances were
determined.
The ethical approval to carry out this study was obtained from Institutional Review Board (IRB) of
college of medicine Medicine and health Health sciencesSciences, Bahir Dar University. Moreover,
written consent was obtained from the guardian and parents of children.
A cross-sectional study was carried out in June, 2017 at four governmental (public) health centers
in Bahir Dar City, Ethiopia. Study participants comprised of children under 60 months of age with
acute diarrhea attending health centers. Health professional working at health centers filled the
questionnaires on the following variables: Sex, age, residence, duration of diarrhea, prescribed
antimicrobials, clinical presentations and stool features from children’s mothers or guardians. The
sample size was calculated using Epi info version 7.0.1 (public domain software
http.//www.cdc.gov) by considering 95% confidence level, with acceptable margin of error 0.05%
and 50% prevalence of bacterial and protozoalprotozoan diarrhea. Thus, a total of 344 children with
diarrhea was were determinedincluded. We took 86 children with diarrhea using convenient
Stool specimens were collected from each child at health centers. At the health center’s
laboratories, G. lamblia, E. histolytica/dispar and helminths infections were detected using a wet
mount of fresh stool samples prepared in physiological saline. The trophozoites and cysts stages of
G. lamblia, E. histolytica/dispar and ova of helminths were identified first under low power (10X)
and then at high power (40X) microscopic objectives. The remaining part of fresh fecal specimens
were kept in icebox and transported to the Amhara Public Health Institute, laboratory for isolation
The Cryptosporidium species was were detected using modified Ziehl-Neelson method upon
formol-ether concentration technique. Smears were performed on slides using sediments obtained
from formal-ether concentraerted samples. Modified Ziehl-Neelson method was done on smears
after methanol fixation (Arora, 2005). Cryptosporidium species oocysts were identified under oil
For the selective enrichment of Shigella and Salmonella spp., stool samples were inoculated
in Selenite Cysteine F broth and incubated for 6 hours. Then, stool samples were inoculated into
MacConkey agar and Xylose Lysine Deoxycholate (XLD) Agar (Oxoid, England). For isolation of
E. coli 0157:H7 samples were plated on Sorbitol MacConkey agar (Oxoid, England). For both, the
plates were incubated under aerobic atmosphere at 37oC and examined after 24 hours. Typical
colonies on MacConkey agar and XLD agar for Shigella spp., and Salmonella spp., were further
Data were analyzed using the Statistical Package for Social Sciences version 20 software
(IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Armonk, NY: IBM Corp). Chi-
square and Fisher exact tests were used where appropriate for categorical variables.
3. Results
The median age and weight of children were 20 months and 10 kg, respectively. Male to
female ratio of the participants was 1.26. The majority (79.7%) of diarrheic children were from
urban residence. All study participants were outpatients (non-hospitalized). The median duration of
diarrhea was 2 days. Vomiting, fever and dysentery were the major clinical signs observed in
diarrheic children. inIn contrast, only 3 children with diarrhea showed dehydration.
Overall, 47.1% (95% CI, 41.9-52.4%) of children with diarrhea were infected either with
protozoa or bacteria (Table 1). The prevalence of protozoan parasites was 34.0%. Overall, protozoa
parasites were significantly more common among male compared with female children (P=0.031).
in males than females (P=0.04 and P=0.005, respectively). Cryptosporidium species was were the
major protozoalprotozoan parasites detected in 12.8% of children. Bacterial pathogens was were
isolated in 13% of children with acute diarrhea. E. coli 0157:H7 and Salmonella spp., were the most
Table 2 shows microscopic stool exam reports at the health centers compared with stool
culture and modified Ziehl-Neelson results. A routine microscopic stool exam showed that 31.4%
and 23% were reported as many bacteria per high power filed (HPF) and many pus cells/HPF,
respectively. However, stool culture and modified Ziehl-Neelson methods revealed that
Cryptosporidium species and bacterial pathogens were detected in 26% of stools. In this regard,
12.8%, 10.2%, 1.7% and 1.2% of children with Cryptosporidium species, E. coli 0157:H7,
Salmonella spp., and Shigella spp., respectively were not identified at the health centers.
Table 3 depicts the frequency and types of empirically prescribed drugs for diarrhea at the health
centers. The most frequently prescribed antibiotic for diarrhea was cotrimoxazole (51.7%). After
stool culture results, a retrospective analyses of empirical prescription of drugs showed that only
46.7% of children with bacterial diarrhea received appropriate antimicrobials. In this regards,
16.7% (1/6) and 50% (2/4) of children with Salmonella and Shigella were empirically treated
Antimicrobial susceptibility tests exhibited that bacterial pathogens from diarrhea were
resistant to commonly prescribed drugs. In this study, 55.6% of resistance was shown against
cotrimoxazole. All Salmonella and Shigella spp., were susceptible to chloramphenicol. However,
5/6 (83%) of Salmonella spp., showed resistance to cotrimoxazol. E. coli 0157:H7 showed low
4. Discussion
In the primary health cares, the actual burden of cryptosporodian and bacterial diarrhea are
not documented due to lack of laboratory facilities like stool culture and modified Ziehl-Neelson
staining. For instance, in this study 31.4% and 23% of the microscopic examined stools were
reported as many bacteria per high power filed /HPF and many pus cells/HPF, respectively. Thus,
all children who had a report of many bacteria/HPF were treated empirically with
inappropriate (WHO, 2013). Moreover, prescribing drugs based on a report of many bacteria/HPF
is no guarantee for clinical decision for antimicrobial therapy due to the presence of complex
stools samples that were reported as many bacteria/HPF and many pus cells/HPF were positive
either for Cryptosporidium species or bacterial pathogens. In this regard, 12.8% and13.1% of
children with acute diarrhea had Cryptosporidium species and bacterial pathogens (E. coli 0157:H7,
Salmonella spp., and Shigella spp.,), respective. These indicate that such amount of protozoan and
Routine use of antimicrobials for acute diarrhea was compared with stool culture results.
Therefore, 46.7% of diarrheic children with bacterial pathogens was threated empirically with
cotrimoxazole/ceftriaxone while 22.2% was not treated with drugs. Most importantly, 77.0% and 4
(1.2%) children without bacterial pathogen were treated empirically with ciprofloxacin and
cotrimoxazole, respectively. These unnecessarily prescribed antimicrobial may enhance the spread
of antimicrobial resistance. Therefore, this might be the reason why we observed high level of
antimicrobial resistance against cotrimoxazole. These indicate that without data from local
antibiogram and bacterial etiology, empirical prescription of antimicrobials for acute diarrhea
Overall, the prevalence of enteric protozoan parasites was significantly higher among male
children compared with female children (P=0.031). Specifically, Cryptosporidium at the p-value
=0.04 and E. histolytica at p-value=0.005 were significantly higher among males than females.
Among enteric protozoan parasite, Cryptosporidium species oocyst (12.8%) and G. lamblia
trophozite (11.3%) were the major protozoan parasites. Cryptosporidium species prevalence among
children with diarrhea in this study is comparable to the 12.2% prevalence in Eastern Ethiopia
(Ayalew et al., 2008). In contrast, lower prevalence of Cryptosporidium species which ranges from
2.2 to 9% were documented in different part of Ethiopia (Berhe et al., 2008; Mersh et al., 1992;
7.8% Salmonella and 9.5 % Shigella prevalence were reported in the same study area (Admassu et
al., 2015). Likewise, frequency of E. coli O157:H7 isolate was lower than 28.9% E. coli O157:H7
prevalence (Adugna et al., 2015). This low prevalence of bacterial-diarrhea might be attributed to
improvement of child care practices, safe drinking water supplies and sanitations. The major
limitation of this study was that risk factors for etiology of diarrhea were not investigated due to a
In conclusion, Cryptosporidium species and enteric bacterial pathogen are prevalent among
children with diarrhea in the study area. However, the burden of these pathogens are not identified
at the routine health centers services. Thus, empirical prescription of drugs was not appropriate for
children with diarrhea. Bacterial isolates from diarrheic stool exhibited high levels of antimicrobial
stool culture and modified Ziehl-Neelsen methods facilities at health cares to avoid empirical
prescription of antimicrobials.
Acknowledgments
This research was financed by the College of Medicine and Health Sciences, Bahir Dar University
(Grant number: We acknowledge the parents/guardians of the children who participated in this
study. We acknowledge the technical support of staffs of the Amhara Public Health Institute
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Table 1
Isolation rates of protozoan and bacterial etiology with sex and age groups of diarrheic children
aged below 5 years, June 2017.
Isolation P
rate Female Male valu Age (months)
Pathogens n (%) (n=152) (n=192) e
49-60
0-24 13-24 25- 36 37-48 (n=23
(n=91) (n=122) (n=75) (n=33) )
Cryptosporidium spp., 44 (12.8) 14 (9.2) 30 (15.6) 0.04 6 (6.6) 21 (17.2) 12 (16) 4 (12) 1 (4.3)
G.lamblia trophozoites 39 (11.3) 19 (12.5) 20 (10.4) 0.54 3 (3.3) 11 (9.0) 12 (16) 7 (21) 3 (8.6)
0.00
34 (9.9) 8 (5.3) 26 (13.5)
E. histolytica/dispar 5 3 (3.3) 7 (5.7) 9 (12) 4 (12.1) 4 (17)
0.03
117 (34.0) 38 (11) 68 (20)
Total protozoan 1 12 (13) 39 (32) 34 (45) 15 (45) 8 (35)
Shigella spp., 4 (1.2) 1 (0.3) 3 (0.9) NA 1 (1.1) 1 (0.8) 0 2 (6) 0
Salmonella spp., 6 (1.7) 4 (1.2) 2 (0.6) NA 2 (2.2) 2 (1.6) 1 (1.3) 1 (3) 0
E. coli 0157:H 35 (10.2) 15 (4.4) 20 (5.8) NA 15 (16.5) 6 (4.9) 7 (9.3) 4 (12.1) 3 (13)
Total bacterial 45 (13.1) 20 (5.8) 25 (7.3) 0.91 18 (20) 9 (7.4) 10 (13) 6 (18) 3 (13)
44(58.6
162 (47.1) 58 (38) 93 (48.4)
Overall isolates 30 (33) 48 (39) ) 21 (63.6) 11(48)
Table 2
Microscopic stool exam reports at health centers versus stool culture and Ziehl-Neelsen results,
2017
Microscopy stool exam reports at health centers Stool culture MZN stain Total n (%)
Frequency n (%) Cryptosporidiu
Shigella Salmonella E. coli 0157:H7 m
G. lamblia 39 (11.3) 0 0 6 3 NA
E. histolytica 34 (9.9) 0 0 2 3 NA
Helminths 7 (2.0) 0 0 1 3 NA
Positive for intestinal parasites 70 (20.3) 0 0 0 0 NA
108 (31.4) 33 (30.5)
Many bacteria/HPF 1 1 13 18
79 (23.0) 15 (19.0)
Many pus cells/HPF 2 2 6 5
93 (27.0) 23 (24.7)
No parasites found 1 3 7 12
Negative for pathogens n (%) 280 (81.4) 4 (1.2) 6 (1.7) 35 (10.2) 44 (12.8) 89 (26.0)
Prescribed drugs
Microscopy results at health center Metronidazole Cotrimoxazole Ciprofloxaci Ceftriaxon No drugs Percentage of
n e appropriate
drugs
G. lamblia 39 0 0 0 0 100
E,histolytica 34 0 0 0 0 100
Many bacteria /HPF (n=108) 6 106 0 2 0 NA
Many pus cells /HPF (n=79) 2 67 4 2 0 NA
No parasites found (n=93) 0 5 0 0 88 NA
Total drugs prescribed 81 (23.5) 178 (51.7) 4 (1.2) 4 (1.2) 88 (94.6) NA
Isolated pathogens at research lab Empirically prescribed drugs N (%) Percentage of
appropriate
drugs
Metronidazole Cotrimoxazole Ciprofloxaci Ceftriaxon No drugs
n e
Shigella spp (n=4) 1 2/4 (50) 0 0 1/4 (25) 50
Salmonella spp (n=6) 2/6 1/6 0 0 3/6 (50) 16.7
E. coli 0157:H7 (n=35) 9/35 17/35 (48.6) 0 1/35 (2.8) 6/35 (17) 51.4
Total 12/41 (29.6) 20/35 (57) 0 1/35 (2.8) 10/45 46.7
C. parvum (n=44) 6/44 (13.6) 21/44 (47.7) 0 2/44 (4.5) 6 (13.6) 74.4
Drugs for unconfirmed etiologies 8/61 (13) 137/178 (77.0) 4/4 (100) 2/4 (50) 0
Table 4
Antimicrobial resistance profiles of bacterial pathogens from acute diarrhea, 2017.
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