Prevalence and Outcome of Community-Acquired Clostridium Difficile Infection Among Children Admitted with Diarrhoea at Mulago Hospital

doi:10.21203/rs.3.rs-4710072/v1

Download PDF

Research Article

Prevalence and Outcome of Community-Acquired Clostridium Difficile Infection Among Children Admitted with Diarrhoea at Mulago Hospital

https://doi.org/10.21203/rs.3.rs-4710072/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background: Clostridium difficile infection (CDI) is a potentially serious emerging infectious disease. CDI is usually considered to be hospital-acquired, causing diarrhoea in as high as 15% of children in healthcare settings during non-outbreak and 64% during outbreak periods in the US. However, recent studies show that the infection is increasingly being acquired from the community even without prior antibiotic use. Globally, few studies have been done to show the contribution of C. difficile in community-onset diarrhoea, and in sub–Saharan Africa, we have not come across any published data on C. difficile in community-onset childhood diarrhoea.

Study objective: This study was to determine the prevalence of CDI and to describe the outcome and factors associated with this infection among children aged 2-36 months with diarrhoea at Mulago National Referral Hospital (MNRH).

Methods: This was a descriptive cross-sectional/prospective cohort study among children aged 2-36 months admitted with diarrhoea at MNRH. The stool was examined for clostridium difficile toxin A and B using an enzyme-linked immune fluorescent assay.

Findings: Out of the 322 children admitted with diarrhoea during the study period, 168 were enrolled. Of the 168 children, there were 107(63.7%) males. The male-to-female ratio was 1.8:1. Their median age was 9.0 months with an Interquartile range of 7-14.

The prevalence of CDI in this study was 4/168(2.4%).

Three of the children with CDI were male. All the four children with CDI were below 12 months of age. On admission, the mean frequency of diarrhoea was 8.8 (SD 5.6) in children with CDI compared to 6.4 (SD 3.8) in children without. Children with CDI were hospitalised for a longer duration; 7.5 (SD6.5) vs. 4.5 (SD4.5) days in children without. The known risk factors such as HIV, antibiotic use, malnutrition, and age were not significantly associated with CDI.

Conclusion:The prevalence of CDI in children with community-acquired diarrhoea at MNRH is low, so routine testing is not necessary. The outcomes of children with CDI did not differ from those without, and the study lacked statistical power to make recommendations on factors associated with CDI.

Community-acquired clostridium difficile

prevalence

outcomes.

Childhood diarrhoea remains a common cause of illness in low-income settings and accounts for 17% of deaths among children aged less than 5 years in developing countries[1]. Clostridium difficile is among the most common causes of antibiotic-associated, hospital-acquired infectious diarrhoea [1–3]. It is an anaerobic, Gram-positive spore-forming toxin-producing bacillus responsible for various diseases. Clostridium difficile produces two exotoxins which contribute to the pathogenesis. Toxin A (TcdA) is a potent enterotoxin that is thought to cause most clinical symptoms due to induced fluid secretion and mucosal damage[4]. Toxin B (TcdB) is approximately 1000 times more cytotoxic than toxin A. The effect of TcdB is thought to depend on initial tissue damage by TcdA, suggesting that both toxins work synergistically[5]. The result of colonization by C. difficile may be separated into four categories: 1) asymptomatic carriage, 2) acute and protracted diarrhoea, 3) colitis (pseudomembranous colitis [PMC], fulminant colitis, toxic megacolon, and non-PMC colitis), and 4) recurrent infections[2]. Several Clostridium difficile-associated diarrhoea (CDAD) outbreaks since 2003 have been associated with the emergence of a more virulent strain that exhibits increased production of toxins A and/or B and a third binary toxin[6]. There is reason to believe that children with inflammatory bowel disease (IBD) may have an increased risk for CDAD. However, this claim is supported by a single study at a paediatric IBD centre in Italy[7].

While Clostridium difficile-associated disease has been well described in North America and Europe, there are very few studies on the prevalence of CDAD in south-east Asia and no studies published in the African population to date. Whether the persistent diarrhoea in some children is due to undiagnosed C. difficile or to unresponsiveness to treatment is unclear. Recent studies show that paediatric CDI is on the increase and the infection is increasingly being acquired from the community even without a preceding history of antibiotic use[8].

Epidemiology

The frequency of paediatric C. difficile varies by age. Neonates (birth through one month) have high colonization rates (up to 64%) but are usually asymptomatic carriers[9]. In children under 2 years old, the prevalence ranges from 3–62%. In older children (3–18 years), the prevalence is like that of adults (5–8%), with similar proportions of symptomatic and asymptomatic carriage. Children under 2 years, excluding neonates, are more likely to show symptoms compared to older children and adults[10].

Acquisition of Clostridium difficile

Individuals can become colonized or infected with C. difficile. The organism is introduced through the faecal-oral route by ingesting infected material, such as water, food, and soil. C. difficile spores are resistant to stomach acidity and germinate in the small intestine, developing into vegetative bacteria that release toxins causing colitis[11].

Risk factors

Risk factors for paediatric C. difficile disease can be categorized into several areas, including disruption of the normal microflora, host factors, and concurrent infections or medical conditions.

Antibiotic-associated disruption of the normal colonic microflora can lead to paediatric cases of CDI, typically occurring 4 to 18 days after the first antibiotic dose[12]. Symptomatic CDI is the most common cause of infectious diarrhoea and antibiotic-associated diarrhoea in healthcare facilities but may also occur as sporadic cases or outbreaks. The primary symptoms are watery diarrhoea, fever, abdominal pain, and/or tenderness[13]. Historically, Clostridium difficile-associated diarrhoea has usually been mild but could in rare cases result in emergency bowel surgery or death[14] .

We aimed to determine the prevalence and outcome of Clostridium difficile infection among children presenting with diarrhoea in the acute care unit (ACU) of Mulago Hospital.

Study design: A descriptive cross-sectional/prospective cohort study was conducted at Mulago Hospital, a national referral and teaching Hospital for Makerere University in Kampala, Uganda, between December 2012 and March 2013.

Inclusion criteria: All children between 2-36 months who presented at ACU Mulago with diarrhoea regardless of co-morbid condition during the study period and required admission. Children with co-morbid conditions were not excluded because we thought that they were more likely to get infected with clostridium since co-morbid conditions may be a risk factor and tend to have severe complications. Children whose parents or caretakers were available to give informed consent. Neonates were not included because they are normally asymptomatic and other previous studies have reported that the prevalence of CDI diarrhoea reduces with increasing age after 3 years.

Exclusion criteria: Children who had been treated with metronidazole or vancomycin for the current episode of diarrhoea. Antibiotic use was verified by examining prescription notes and containers of antibiotics especially syrups the parents /attendants came with.

Sample size estimation: By Kish Leslie formulae[15] . The sample size was estimated at 123 Children. This was the minimum number of children required however, 168 children were studied.

Ethical approval: This study received approval from the Uganda National Council for Science and Technology (UNCST) in Kampala Uganda, per the UNCST act, and ethical clearance was obtained from the Makerere University College of Health Sciences Institutional Review Board. Written informed consent was obtained from the mothers of children who were eligible. Children found to have toxins A and B in their stool were initiated on treatment with metronidazole which was more readily available in the hospital than vancomycin. The children who were found HIV positive were initiated on cotrimoxazole prophylaxis and referred to Baylor College of Medicine Centre of Excellence for paediatric HIV care in Mulago Hospital, to be initiated on antiretroviral therapy.

Data collection and Variables: All children between the ages of 2 and 36 months who presented at ACU with diarrhoea were approached. The objectives of the study were explained to the caretakers and informed consent was sought from them. Enrolment into the study was done on acceptance to participate and a written informed consent was obtained.

Demographic data, history of current illness, treatment history, past medical history, and histories of feeding and immunisation were obtained. General clinical examination including anthropometry was done on all children enrolled in the study. Measurements were taken by the same person to minimize inter-observer variation. The children were undressed and weighed using a standardized weighing scale while height was measured using a stadiometer to the nearest centimetre. Using the midpoint between the acromion and olecranon process, the mid-upper arm circumference was measured using a non-stretch fibreglass tape (Butterfly, China). This information was recorded on a structured questionnaire. Aseptic methods of stool collection using a rectal tube inserted into the rectum and connected to a syringe were used. The stool samples were put in clean leak-proof containers without preservatives or media to avoid interference with C. difficile toxin A and B assay results. Samples were kept at a temperature of 2°- 8°C in the ACU laboratory while awaiting transportation to MBN laboratories. They were kept at a temperature of -20°C for 3-5 days and analysed as a batch.

However, the samples could be held for up to 3 days (from the time of collection) at 2-8°C before processing without large losses in detectable C. difficile toxins A and B. The specimen may be stored at -25±6°C for one month and then one month after at -70°C or colder temperatures. HIV antibody test was done on the blood of all children whose parents /caretakers consented to the test in the ACU laboratory. Those found HIV positive by antibody test and were below 18 months of age were referred to Baylor College of Medicine Paediatric AIDS Foundation, Uganda for DNA PCR. All children who tested HIV positive were initiated on cotrimoxazole prophylaxis and referred to Baylor College of Medicine Centre of Excellence for paediatric HIV care in Mulago Hospital. Those who did not consent to HIV tests were not excluded from the study.

A semi-structured questionnaire was used to collect the required information. Study variables or measurements Information obtained included age, sex and duration of diarrhoea, prior use of antibiotics, HIV, and anthropometry. Each study participant was assigned a study number for identification. A study card was given which they could present whenever they came to ACU during the study period to ensure that a patient was not recruited more than once.

Outcome measures were:

The proportion of children with diarrhoea who tested positive for Clostridium difficile.
Change in frequency of diarrhoea during the period of hospitalisation. Diarrheal frequency was as reported by the parent /caretaker daily for a period of 24 hrs. The frequency was recorded from day zero (admission date) to discharge if less than 10 days or up to day 10 of hospitalisation.
Duration of hospitalisation of children with clostridium difficile and children without clostridium difficile.

Patients’ follow-up:

Enrolled children were assessed and managed according to the Uganda Ministry of Health protocols for managing diarrheal diseases. They were followed up and evaluated by the principal investigator daily until the day of discharge. During follow-up, they were assessed for the frequency of diarrhoea, abdominal tenderness, body temperature and hydration status. The results of the investigations once available were communicated to the clinical care team and explained to the parents and/or caretaker by the principal investigator. Those children found to have clostridium difficile toxins in their stool were treated with oral metronidazole for 10-14 days is readily available in the Hospital. All children were followed up until discharge from the hospital. The frequency of diarrhoea was assessed daily until discharge. Children who had already been discharged but were CDI positive were called back and given oral metronidazole to avoid future recurrences of diarrhoea due to untreated clostridia infection. Studies have shown that cure rates on metronidazole are more than 90%40, therefore it was anticipated that there would be very few treatment failures. Vancomycin, the second-line drug is not readily available in Mulago Hospital and was to be purchased for those few who may not have responded to metronidazole.

Laboratory analysis: Determination of C. difficile toxins: The stool samples were kept in a refrigerator at 2-8°^C while still in ACU and then -20°^C at the MBN laboratories and examined within 3-5 days for C. difficile toxin. MBN Clinical Laboratory is a Medical Laboratory Diagnostic facility in Kampala, Uganda. The laboratory is a centre of excellence in Microbiology, PCR Diagnostics, Chemistry, and haematology. MBN as a research Laboratory offers research facilities ranging from culture and sensitivity to complex tests such as HLA typing. MBN offers professional laboratory services performed according to Good Clinical Laboratory Practices (GCLP) standards. MBN performs internal quality testing and participates in quality assurance programs, making it an ideal laboratory for testing in patient care and research. An enzyme immune assay method was used for analysis. Tests were performed on the Vidas instrument (from Biomeurex in France) to determine toxins A and B. The VIDAS C .difficile toxin A and B assay is an automated test for use on the VIDAS family of instruments for the qualitative detection of C .difficile toxin A and B in stool specimens using the enzyme-linked fluorescent assay. The VIDAS C .difficile toxins A and B aid in the diagnosis of CDAD. The assay principle combines a two-step enzyme immunoassay sandwich method with a final fluorescent detection. The four reaction steps are performed automatically by the instrument. The fluorescence increases according to the quantity of toxins A and /or B in the sample. When the VIDAS C .difficile toxin A and B test is completed, the results are analysed automatically by the instrument, a test value is generated, and the results are printed for each sample. The reagents for the assay are ready to use and are pre-dispensed in the sealed reagent strips. They are in KITS, and each is for 60 tests imported from France. Technicians were trained in using the Kits and the VIDAS instrument. Quality control in the laboratory: Two positive controls and one negative control are included in each VIDAS C. difficile toxin A and B kits. These controls were performed immediately after opening the new kit to ensure that reagent performance was not altered. Each calibration was checked using these controls. The controls were processed like patient samples and pipetted directly into the sample wells of VIDAS C. difficile toxin A and B reagent strips. A reading of < 0.13 was taken to be a negative test whereas a reading of more than 0.13 was regarded as positive.

Statistical analysis: Epi data version 3.1 with inbuilt consistency checks was used. Data analysis: Data was exported to SPSS version 19.0 for analysis.

The prevalence was obtained as a proportion of children with diarrhoea who had clostridium difficile toxins A and B of the studied population. The change in frequency of diarrhoea was obtained as a difference in diarrhoea frequency between the date of admission and date of discharge if less than 10 days or up to day 10 of hospitalisation. The outcome in terms of duration of hospitalisation was obtained as the mean number of days spent in the hospital for both the children with Clostridium difficile and children without C. difficile.

Statistical tests between dependent and independent variables were done using Fisher’s Exact Tests for categorical data. Cross tabulations were used to calculate the Fisher’s exact test for cells less than five as a measure of association and statistical significance was determined using a p-value of < 0.05.

Tabular trend analysis was used to study the change in the frequency of diarrhoea and duration of hospitalisation among the C. difficile-positive and the Clostridium difficile-negative children. Continuous variables such as age, diarrhoeal frequency, and duration of hospitalisation were analysed using measures of central tendency of the mean and standard deviation. Some variables were further coded categorically and analysed using frequency and cross-tabulations.

Quality control: The questionnaires were pre-tested and research assistants were trained before commencing data collection. The data collection tools were checked daily for completeness. The weighing scale was calibrated before use. Axillary temperatures were recorded daily using a digital thermometer in degrees Celsius. Stool samples were collected using a rectal tube which was inserted into the rectum with a syringe connected. The sample was collected by suction and the contents of the syringe were emptied into clean leakproof stool containers without preservatives. Stool samples were taken off and kept at a temperature of 2-8 0C while in ACU and sent to MBN laboratory where they were stored at - 20°C for 3-5 days and then analysed as a batch. Recruitment was 24 hours a day both weekdays and weekends this was to ensure that no child was missed.

A total of 168 children aged 2 to 36 months were recruited consecutively following consent by their parents /caretakers. Children on metronidazole were excluded. None of the screened children had used vancomycin. Shown in Figure 1.

Description of study participants.

Out of 168 children, 107 (63.7%) were males. The average age was 10.9 months (SD 6.2), with roughly half of the participants being between seven and twelve months old. Overall, most of the children were under one year old, accounting for 115 out of 168 (68.5%). In the previous two months, all children with CDI had experienced some form of illness, and only one out of the four children had been hospitalized due to the illness. Out of the 168 children studied, only one had never been breastfed. All children had diarrhoea of varying severity at admission The common concurrent symptoms were fever and vomiting. Children with persistent diarrhoea were more likely to test positive for C. difficile. Fever was the most common sign in children with C. difficile and those without. The rest of the baseline characteristics, details of signs and symptoms are shown in Table 1. At the end of the document.

Table 1: Demographic, nutrition, immunization characteristics, signs and symptoms.

Characteristic

Frequency(%)

N=168

CDI Positive

N=4(%)

CDI Negative

N=164(%)

P-value

Fisher’s Exact test

Age

<6months

7-12 months

>12 months

32(19.1)

83(49.4)

53(31.5)

1(25)

3(75)

0(0)

31(18.9)

80(48.8)

53(32.3)

0.309

Sex

Male

Female

107(63.7)

61(36.3)

3(75)

1(25)

104(63.4)

60(36.6)

0.540

Nutritional status

Well-nourished

Moderate malnutrition

Severe malnutrition

99 (59.0)

32 (19.0)

37 (22)

2(50)

0(0)

2(50)

97(59.1)

32(19.5)

35(21.3)

1.000

Immunization status

Not immunized

Immunized (n=165)

Fully immunized

Partially immunized

3(2.0)

165 (98)

78 (47.3)

87(52.7)

0(0)

1(25)

3(75)

3(1.83)

77(47.0)

84(51.2)

Breastfeeding status

Breastfed.

<6months(n=167)

≥6months(n=167)

167(99.4)

65(38.9)

102(60.1)

4(100)

1(25)

3(75)

163(99.3)

64(39.0)

99(60.4)

0.986

Current feeding method

Exclusive breastfeeding

Mixed

Replacement

Complementary

Other feeds other than breast milk

18(10.7)

5(3.0)

2(1.2)

104(61.9)

39(23.2)

1(25)

3(75)

18(10.9)

4(2.4)

2(1.22)

101(61.1)

39(23.8)

Watery Diarrhoea

Less than 2 weeks

2 weeks and more

142(84.5)

26(15.5)

2(50)

140(85.4)

24(14.6)

0.05

Bloody diarrhoea

12(7.1)

0(0.0)

12(7.32)

0.74

Fever

139(82.7)

4(100)

135(82.3)

0.47

Vomiting

142(84.5)

4(100)

138(84.1)

0.51

Prior antibiotic use

72(42.9)

2(50)

70(42.7)

0.77

Children sick in the previous 2 months.

82 (48.8)

4(100)

78(47.6)

0.05

Treated with antibiotics in previous 2 months

76(45.2)

1(25)

75(45.7)

Known chronic medical illnesses

12(7.1)

0(0.0)

12(7.31)

Temperature (⁰C) >37.5

<37.5

99(58.9)

69(41.1)

2(50)

97(59.1)

67(40.9)

0.54

Abdominal tenderness

0(0.0)

Abdominal distension

1(0.6)

0(0.0)

1(0.6)

Hydration status

No dehydration

Some dehydration

Severe dehydration

29(17.3)

79(47.0)

60(35.7)

1(25)

3(75)

0(0.0)

28(17)

76(26)

60(37)

0.679

Children whose weight for height /length Z score was up to -2SD were regarded as being well nourished. Moderate malnutrition was WHZ-score was between -2 and -3 SD. Severe malnutrition was WHO Z- score -3SD. There were no factors found significantly associated with CDI among the demographic and nutritional characteristics above.

Chronic medical illnesses included sickle cell disease, cerebral palsy, HIV, and congenital heart disease. Some of the children were admitted because they had other serious concurrent illnesses but were not dehydrated.

World Health Organization standards were used to determine nutritional status[16].

Laboratory findings of all study participants

Out of 168 children, 4 tested positive for C. difficile toxin A and B. The prevalence of CDI among children aged 2-36 months with diarrhoea was 2.4%, with a 95% confidence interval of 0.0 - 4.9%. 138 children were tested for HIV, and DNA PCR was performed for those under 18 months old who tested antibody positive. The results are in Table 2 below.

Table 2: Laboratory findings of participants.

Result

Total (168)

Percentage(%)

C. difficile positive (Toxin A &B)

2.4

C. difficile negative (Toxin A &B)

164

97.6

HIV status (antibody test)

Positive

Negative

Not Tested

126

7.1

18.5

HIV status (DNA PCR for children < 18 months).(n=12)

Positive

Negative

4.2

3.0

Of the 12 HIV antibody-positive children, seven were confirmed HIV infected by DNA–PCR. Thirty-one children were not tested for HIV because the parents/attendants declined the test. All the antibody-positive children were below 18 months. The prevalence of HIV among the study participants was 7/138 (5.1%).

Seventy-two (72) children had previously received treatment with antibiotics upon admission. The most used antibiotics for diarrhoea were gentamicin, followed by cotrimoxazole. The details are shown in Figure 2.

Case summaries of children with C. difficile infection

Case 1: A nine-month-old female admitted with watery diarrhoea which had lasted for more than two weeks. The diarrhoea was associated with fever and vomiting. The estimated frequency of diarrhoea at admission was 3. This child had been given ampicillin and gentamicin before hospital admission. The child had no chronic medical illnesses. She was partially immunised and had been exclusively breastfed for 6 months. On admission, she was febrile with a temperature of 38.0°C and had mild pedal oedema without signs of dehydration. The anthropometric measurements were as follows; WT=5.9Kg, L=66.3 cm, (WL Z score between -2 &-3SD) and MUAC=11.5cm. The blood smear was negative for malaria parasites, and she was HIV-negative. The child was hospitalised for 4 days and discharged with neither diarrhoea nor oedema. She was to continue nutritional rehabilitation as an outpatient and follow up at the Nutrition Rehabilitation Unit (Mwana Mugimu) at Mulago.

Case 2: A seven-month-old male was admitted with watery diarrhoea which had lasted ten days. The estimated frequency of diarrhoea per day was 16, associated with fever and vomiting. He had not received antibiotics before admission. He had no chronic medical illness. His immunisation status was up-to-date, and he was on complementary feeding following exclusive breastfeeding for 6 months. Physical examination revealed the following: a temperature of 38.0°C, and some dehydration. WT= 8.0kg, L=70.0cm (WL Z score 0 SD), MUAC=14.0Cm. He had no malaria parasites in the peripheral blood smear, and he tested negative for HIV. He was hospitalised for 6 days and had no diarrhoea at discharge.

Case 3 A four-month-old male child was admitted with acute watery diarrhoea with an estimated frequency of 10 bowel movements per day. The diarrhoea was accompanied by vomiting but no fever. His other systems were normal. He had not received any antibiotics before admission, and his immunizations were up to date. The mother did not report any chronic medical illnesses. The infant was no longer exclusively breastfed and was on mixed feeding. The physical examination findings were as follows; temperature =36.8°C, some dehydration, WT =8.0kg, L =69.0, WL Z score between 0 &-1SD. He was not tested for HIV because the caretaker did not consent. The infant was discharged after three days of hospitalisation.

Case 4: A 10-month-old male baby was admitted with non-bloody diarrhoea which had lasted more than two weeks with a frequency of 6 per day. He also had a history of vomiting but no fever. The caretaker reported a 3-day history of body swelling with normal micturition habits. Other systems were unremarkable, and he had no known chronic medical illnesses. He had been treated with cotrimoxazole before hospitalisation. He was fully immunised and was on other foods other than breast milk. He had been exclusively breastfed for six months. Physical examination at admission revealed temperature = 36.6°C, some dehydration and pedal oedema but no sacral pad. WT =6.0 kg, L =65.0cm (WL Z score -3SD), MUAC =10.5cm. The HIV Test was negative. He was treated with ReSoMal, zinc gluconate, and nutritional rehabilitation initially with F-75 and later with F-100. Although he was hospitalised for 17 days, diarrhoea stopped on day five. The child was discharged to continue nutritional rehabilitation as an outpatient. In summary, two of the 4 children with CDI-associated diarrhoea had been treated with antibiotics before hospitalisation and had severe acute malnutrition.

Outcome of children with clostridium and children without clostridium difficile infection.

The duration of hospitalization was longer for children who tested positive for Clostridium difficile compared to those who tested negative. However, this difference was not found to be statistically significant. See Table 3.

Table 3: Outcome of children with and without CDI.

Variable

Clostridium positive

Mean 95% CI

Clostridium Negative

Mean 95% CI

P. value.

Fisher's exact test.

Duration of Hospitalisation (Days)

7.5 (2.8-17.8)

4.5 ( 3.8-5.2)

0.194

Change in frequency of diarrhoea.

8.8

6.4

0.187

P-values less than 0.05 were considered statistically significant.

Change in diarrhoea frequency over time between children with clostridium difficile and children without clostridium difficile.

The average rate of diarrhoea was initially higher in children with C. difficile but with time it dropped to 1.5 by the time of discharge. While in the C. difficile negative children their initial average rate of diarrhoea frequency was lower as shown in Figure 3.

The prevalence of clostridium difficile infection(CDI) among children aged between 2-36 months admitted with diarrhoea at Mulago Hospital, Kampala, Uganda was 2.4%. The recovery of toxigenic strains of C .difficile implies the possibility of faecal oral contamination from the environment or human and animal contacts. However, this is a low prevalence compared to what other authors have reported among children with diarrhoea in low-income countries. In two studies conducted in Brazil among children with acute diarrhoea, the prevalence was 5.5% and 13.8% respectively[17]. This research was conducted on hospitalized children with acute diarrhoea in three different hospitals. The study used a cross-sectional method and included a comparison group of children without diarrhoea, aged 0-5 years. None of the children without diarrhoea tested positive for C. difficile. The study involved toxicogenic assays for toxins A and B, as well as additional tests such as cytotoxicity on cells (African green monkey kidney) tissue culture and PCR. The results showed that the toxins had minor effects on the Vero cells, indicating that their presence was not a significant cause of diarrhoea in these children. It's important to note that in the current study, only a toxin assay was conducted, so we cannot directly link the presence of toxins to the diarrhoea in these children.

In a Turkish study, the prevalence was 6.9% in patients with nosocomial diarrhoea[18]. The study was carried out on admitted patients using cytotoxin assay as the gold standard. No previous studies in Sub-Saharan Africa were available for comparison. The low prevalence in this study may be due to a low prevalence of clostridia in the environment or human contacts, or differences in the study population. The study excluded neonates below two months, known to have a high colonisation rate. They were purposely excluded because they are likely to be asymptomatic carriers[9].

Most of the children in our study were breastfed, and breastfeeding is known to protect against CDI. This may have helped to lower the prevalence of C. difficile in our study compared to other studies conducted in settings where formula feeding is more common. In the present study, children were enrolled upon admission, suggesting that the diarrhoea was not acquired in the hospital but rather in the community. This may have contributed to a lower prevalence of clostridia in the community compared to hospital-acquired disease.

All children with Clostridium difficile had non-bloody diarrhoea. Half had acute diarrhoea, while the other half had persistent diarrhoea, and were more likely to have used antibiotics, a known risk factor for CDI in the study by Ferreira et al[17].

Studies suggest that adult CDI risk factors such as antibiotic and healthcare exposure may not be as important for children who acquire CDI in the community[19]. Fever is a common symptom of CDI, and a temperature above 38.5°C indicates severe disease. The association in the current study was not significant because of the small numbers. The hydration status of children with CDI varied from no to some dehydration, implying that the patients had mild disease. The level of dehydration in CDI depends on the frequency of diarrhoea. Some children with CDI may experience high frequency, while others may have low frequency, depending on the species of Clostridium difficile[20]. The study did not specify the C. difficile species, and due to the limited number of positive results, it was not possible to conclude the connection between the species and the severity of the disease. In the present study, none of the children with C. difficile exhibited abdominal tenderness or distension. However, pseudomembranous colitis may complicate the infection, as it can lead to the destruction of the gastrointestinal mucosa and the lumen of the large intestine, which may present as abdominal tenderness[20].

The children with CDI had no significant clinical features compared to children without CDI, most likely due to the small number of positive cases.

Demographic and nutritional characteristics

All infants with CDI were under 12 months old. This is not surprising as most studies have reported the highest frequency in infancy[10]. The current study cannot report an association between CDI and age group due to small numbers, and these results cannot be compared to studies that included older children and adolescents. The study population was predominantly male. Most of the children with C. difficile were males, but this gender difference was not statistically significant. The higher number of males may have been due to chance or possibly due to selective factors. In Africa, male children tend to receive more care than female children when they are sick. This was supported by Marte Eline et al. in Tanzania, who reported that female patients had more often been treated at home before hospital admission than males[21]. The preference for male children in many African countries likely influences gender selection in seeking care. In a study conducted by Alexander R. Marra et al., gender was not found to be a predictor of infection or death due to Clostridium difficile[22].

Although the numbers were small, half of the children with CDI had severe acute malnutrition. This might have been a coincidence but should not be dismissed entirely.

A study conducted in adults reported an association between nutritional status and infection with CDI [23]. Breastfeeding is said to be protective against CDI[24]. Most of the study participants had ever breastfed. This may have protected most of these children from getting Clostridium difficile, and even those who got infected from getting severe disease. Studies indicate that infants who are not breastfed face a higher risk of dying from infectious diseases in the first two months of life, including diarrhoea, compared to those who are breastfed[25].

Outcome of children with Clostridium difficile infection

Children with CDI had a higher rate of average frequency of diarrhoea per day compared to those who did not have clostridium difficile. There was resolution of diarrhoea in all the children with CDI. Other researchers have reported that specific treatment may be unnecessary, especially in mild CDI without systemic signs[26]. In certain instances, resolution occurs upon discontinuation of the offending drug or agent[26] .Children in this study had mild disease, which may have contributed to their faster recovery. Children with Clostridium difficile had a longer hospitalization period compared to those without the infection. The duration of hospitalization did not correlate with the resolution of diarrhoea. This could mean that the length of hospital stay was influenced by concurrent conditions such as acute severe malnutrition.

Factors associated with Clostridium difficile infection.

In this study, persistent diarrhoea, antibiotic use, and severe acute malnutrition were only weakly associated with CDI. The lack of significance for most of the factors could be explained by the low prevalence and the small numbers. Children with persistent diarrhoea were more likely to have used antibiotics. However, antibiotic use may not be important in children who acquire CDI from the community.

Strength: This study is the first documented report of CDI among Ugandan children with diarrhoea. It provides us with a snapshot of the prevalence of CDI in children aged 2-36 months. However, the study design is limited as it cannot establish a cause-and-effect relationship. The laboratory test to confirm CDI is compared favourably with the gold standard, cytotoxic assay. The enzyme immunoassay (EIA) has a high specificity of 93-100% and a reasonably high sensitivity of 63-99%.

LIMITATIONS

Recall bias, particularly among illiterate parents, may have influenced the results by causing under-reporting or over-reporting of the number of bowel movements and antibiotic use. Informants' limited and unreliable recall may compromise studies that rely solely on this[27]. This study aimed to reduce bias by using a diary to track each time the child had a bowel movement. However, this method couldn't account for the number of diarrheal occurrences recorded at admission. For antibiotic use, we used filled prescriptions presented, and remaining drugs/drug bottles. Also due to cost constraints, other investigations like stool microscopy for other infestations were not done so one cannot comment on the co-infections associated with C. difficile. The limited studies on the prevalence of C. difficile in Africa possess a factor of no comparison with what is happening in the region. Due to the lack of power of the study, associated factors could not be ascertained. Prior use of metronidazole or vancomycin would have been better verified by examining serum concentrations, a test that was beyond the scope of this study. It is possible that some of the enrolled children had taken metronidazole but had no documentary evidence. This implies that some children could have been inappropriately included contributing to the low positivity of the Clostridium difficile test and low prevalence.

The study found that the prevalence of Clostridium difficile infection in children aged 2-36 months who were admitted with community-acquired diarrhoea at Mulago National Referral Kampala is low. This suggests that routine testing for CDI in these children may not be necessary. However, the immediate outcomes of children with CDI were like those without it. It's important to note that the study did not have enough statistical power to make conclusions or recommendations on factors associated with CDI.

RECOMMENDATIONS

Further studies are needed to determine the prevalence of CDI in children hospitalized for at least 72 hours, in those hospitalized in the past 10 weeks, and in high-risk groups such as malnutrition, persistent diarrhoea, and prior antibiotic use.

ACU: Acute Care Unit

C. difficile: Clostridium difficile

CCNA: Cell Cytotoxicity Neutralization Assay

CDAD: Clostridium difficile associated diarrhoea

CDI: Clostridium difficile infection

EIA: Enzyme Immunoassay

GI: Gastrointestinal HAART: Highly Active Antiretroviral Therapy

HIV: Human Immunodeficiency Virus

IBD: Inflammatory Bowel Disease

IMCI: Integrated Management of Childhood Illnesses

MUAC: Mid-Upper Arm Circumference

PCR: Polymerase Chain Reaction

TcdA: Toxin A

TcdB: Toxin B

UK: United Kingdom US: United States

WBC: White Blood cell Count

WT: Weight

MNRH: Mulago National Referral Hospital.

OPERATIONAL DEFINITIONS:

CDAD case: A patient with diarrhoea and a positive C. difficile cytotoxin assay on the stool sample.

Treatment failure: Occurs when diarrhoea recurs with a positive stool specimen for C. difficile or when a physician orders a second course of treatment for the organisms.

Previous antibiotic use is an antibiotic prescribed for at least 48 hours in the 60 days before CDAD.

Duration of hospitalization is the difference in days between admission and discharge.

Positive clostridium toxin assay: when the test value by Vidas C. difficile toxin A and B assay is >0.13.

Negative clostridium toxin assay: When the test value by Vidas C. difficile toxin A and B assay is <0.13.

ACKNOWLEDGEMENT

Special thanks to Makerere University for providing administrative clearance and all research assistants for the role played in data collection.

COMPETING INTERESTS

Authors have no competing interests to declare.

AUTHOR CONTRIBUTIONS

Authors participated in the proposal development, interpretation, critical review of content, write‐up, and approval of the final manuscript.

HUMAN ETHICS AND CONSENT TO PARTICIPATE

This study received approval from the Uganda National Council for Science and Technology (UNCST) in Kampala Uganda, per the UNCST act, and ethical clearance was obtained from the Makerere University College of Health Sciences Institutional Review Board. Written informed consent was obtained from the mothers of children who were eligible. To ensure confidentiality and anonymity, numerical identifiers were used in the patient database, which was only accessible to the principal investigator (PI). The PI kept All charts under lock and key, and the information obtained was solely for this study.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Data will be available upon reasonable request from the corresponding author.

FUNDING

The study was supported by the Belgium Embassy. The study sponsor had no role in the design, execution, interpretation, or writing of the study.

Nelson DE, Auerbach SB, Baltch AL, Desjardin E, Beck-Sague C, Rheal C, Smith RP, Jarvis WR: Epidemic Clostridium difficile-associated diarrhea: role of second-and third-generation cephalosporins. Infect Control Hosp Epidemiol 1994, 15(2):88-94.
Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, Pepin J, Wilcox MH: Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol 2010, 31(5):431-455.
Pépin J, Valiquette L, Alary ME, Villemure P, Pelletier A, Forget K, Pépin K, Chouinard D: Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. Cmaj 2004, 171(5):466-472.
Lyerly DM, Lockwood DE, Richardson SH, Wilkins TD: Biological activities of toxins A and B of Clostridium difficile. Infect Immun 1982, 35(3):1147-1150.
Lyerly DM, Saum KE, MacDonald DK, Wilkins TD: Effects of Clostridium difficile toxins given intragastrically to animals. Infect Immun 1985, 47(2):349-352.
McFarland LV: Update on the changing epidemiology of Clostridium difficile-associated disease. Nat Clin Pract Gastroenterol Hepatol 2008, 5(1):40-48.
Pascarella F, Martinelli M, Miele E, Del Pezzo M, Roscetto E, Staiano A: Impact of Clostridium difficile infection on pediatric inflammatory bowel disease. J Pediatr 2009, 154(6):854-858.
Pant C, Deshpande A, Altaf MA, Minocha A, Sferra TJ: Clostridium difficile infection in children: a comprehensive review. Curr Med Res Opin 2013, 29(8):967-984.
Cerquetti M, Luzzi I, Caprioli A, Sebastianelli A, Mastrantonio P: Role of Clostridium difficile in childhood diarrhea. The Pediatric infectious disease journal 1995, 14(7):598-602.
McFarland LV, Brandmarker SA, Guandalini S: Pediatric Clostridium difficile: a phantom menace or clinical reality? Journal of pediatric gastroenterology and nutrition 2000, 31(3):220-231.
Carrico R, Archibald L, Bryant K, Dubberke E, Fauerbach L, Garcia J, Gould C, Koll B, Mayfield J, Pang X: Guide to the Elimination of Clostridium difficile in Healthcare Settings. Association for Professionals in Infection Control & Epidemiology 2008:1-66.
Ferroni A, Merckx J, Ancelle T, Pron B, Abachin E, Barbut F, Larzul J, Rigault P, Berche P, Gaillard J-L: Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service. European Journal of Clinical Microbiology and Infectious Diseases 1997, 16:928-933.
Walbrown MA, Aspinall SL, Bayliss NK, Stone RA, Cunningham F, Squier CL, Good CB: Evaluation of Clostridium difficile-associated diarrhea with a drug formulary change in preferred fluoroquinolones. Journal of Managed Care Pharmacy 2008, 14(1):34-40.
Fordtran JS: Colitis due to Clostridium difficile toxins: underdiagnosed, highly virulent, and nosocomial. In: Baylor University Medical Center Proceedings: 2006: Taylor & Francis; 2006: 3-12.
Kish L, Sons. JWa: Survey Sampling; 1965 New York.
De Onis M, Monteiro C, Akré J, Glugston G: The worldwide magnitude of protein-energy malnutrition: an overview from the WHO Global Database on Child Growth. Bulletin of the World Health Organization 1993, 71(6):703.
Ferreira CE, Nakano V, Durigon EL, Avila-Campos MJ: Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in Sao Paulo city, Brazil. Memorias do Instituto Oswaldo Cruz 2003, 98:451-454.
Söyletir G, Eskitürk A, Kiliç G, Korten V, Tözün N: Clostridium difficile acquisition rate and its role in nosocomial diarrhoea at a university hospital in Turkey. European journal of epidemiology 1996, 12:391-394.
BOENNING DA, FLEISHER GR, CAMPOS JM, HULKOWER CW, QUINLAN RW: Clostridium difficile in a pediatric outpatient population. The Pediatric Infectious Disease Journal 1982, 1(5):336-338.
Keel MK, Songer J: The comparative pathology of Clostridium difficile-associated disease. Veterinary pathology 2006, 43(3):225-240.
Ween-Velken ME, Ween-Velken NC: Children with suspected malarial infection admitted to Kolandoto hospital, Tanzania: Exploring statistical methods. 2007.
Marra AR, Edmond MB, Wenzel RP, Bearman GM: Hospital-acquired Clostridium difficile-associated disease in the intensive care unit setting: epidemiology, clinical course and outcome. BMC infectious diseases 2007, 7:1-6.
Wong S .S, Driscoll J.O, M W: A pilot study to evaluate the prevalence of malnutrition in patients with Clostridium difficile(C. diff) infection. proceedings of the nutritional society. . 2009, 19(68).
Tullus K, Aronsson B, Marcus S, Möllby R: Intestinal colonization with Clostridium difficile in infants up to 18 months of age. European Journal of Clinical Microbiology and Infectious Diseases 1989, 8:390-393.
Medhin G, Hanlon C, Dewey M, Alem A, Tesfaye F, Worku B, Tomlinson M, Prince M: Prevalence and predictors of undernutrition among infants aged six and twelve months in Butajira, Ethiopia: the P-MaMiE Birth Cohort. BMC Public Health 2010, 10:1-15.
Kyne L, Merry C, O'Connell B, Kelly A, Keane C, O'Neill D: Factors associated with prolonged symptoms and severe disease due to Clostridium difficile. Age and ageing 1999, 28(2):107-113.
Melo MCNd, Taddei JAdA, Diniz-Santos DR, May DS, Carneiro NB, Silva LR: Incidence of diarrhea: poor parental recall ability. Brazilian Journal of Infectious Diseases 2007, 11:571-579.

No competing interests reported.

Download PDF

Reviewers agreed at journal
11 Sep, 2024
Reviewers agreed at journal
09 Sep, 2024
Reviewers agreed at journal
04 Sep, 2024
Reviewers agreed at journal
29 Jul, 2024
Reviewers agreed at journal
27 Jul, 2024
Reviewers invited by journal
26 Jul, 2024
Editor invited by journal
16 Jul, 2024
Editor assigned by journal
11 Jul, 2024
Submission checks completed at journal
11 Jul, 2024
First submitted to journal
09 Jul, 2024

You are reading this latest preprint version

Prevalence and Outcome of Community-Acquired Clostridium Difficile Infection Among Children Admitted with Diarrhoea at Mulago Hospital

Status:

Version 1

Abstract

Figures

INTRODUCTION

Epidemiology

Acquisition of Clostridium difficile

Risk factors

METHODS AND MATERIALS

RESULTS

DISCUSSION

STRENGTHS AND LIMITATIONS OF THE STUDY.

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1