Cryptosporidiosis and Infantile Diarrhoea in Calabar, Nigeria
N.G. Etim ,
M.F. Useh ,
The involvement of Cryptosporidium in infantile diarrhoea was investigated among 200 children with diarrhoea who attended the Children Emergency Ward of the University of Calabar Teaching Hospital (UCTH) and Out-Patients Department of the General Hospital, Calabar, Nigeria. Another subset of 100 apparently healthy non-diarrhoeic children of comparable age range were used as controls. Stool samples collected from the test and control groups were examined for the presence of intestinal parasites including oocyst of Cryptosporidium using standard parasitological procedures. Cryptosporidium oocyst was found in 52 (26%) of the diarrhoeic stool samples and 8 (8%) of the non diarrhoeic control samples respectively (p<0.05). Cryptosporidium occurred as a single infection in only 32 (16%) of the 200 diarrhoeic subjects examined. The highest prevalence of cryptosporidiosis occurred among subjects aged 2/12-1 year. Infection was not detected in those aged ≥8 years. Other intestinal parasites detected were Ascaris lumbricoides (19%), Trichuris trichiura (2%), Hookworm (1%) and Entamoeba histolytica (1%). This study has demonstrated the involvement of Cryptosporidium as one of the aetiologic agents of infantile diarrhoea in Calabar, Nigeria. The study has made a case for the routine screening of stool samples of children with diarrhoea for the presence of Cryptosporidium before administrating prompt and appropriate therapy.
Cryptosporidiosis is an opportunistic diarrhoeal disease caused by an intracellular spore forming protozoan parasite known as Cryptosporidia. About 21 species of Cryptosporidium have been identified but the main specie responsible for human infection is C. hominis formerly known as C. parvum human genotype or C. parvum type 1 (Loureiro et al., 1989; NiChito et al., 1998). The infection is transmitted by ingestion of the oocyst in faecally contaminated food and water from animals to humans and through person to person contact. The probability of transmission from just a small amount of contamination is fairly high. It is often transmitted in day care setting to care givers, older children and adults who come in contact with infected children at home (Alpert et al., 1984) or hospital acquired (Baxby, 1983; Koch et al., 1985). A recent study has shown that the 50% infective dose (LD50) of C. parvum is only 132 oocysts for healthy persons with no previous serological immunity to cryptosporidiosis (ODonoghue et al., 1995)
The commonest symptom of cryptosporidiosis is diarrhea, which is usually watery stool. It is accompanied by abdominal cramps, nausea, vomiting, fever, headache and loss of appetite (Khan, 2000). The symptoms differ greatly in immunocompetent and immunocompromised individuals. In the former the infection is acute, yet a self limiting diarrhoeal illness whereas the illness is much more severe in the later. The incubation period may range from 1 to 12 days (Juranek, 1995).
Cryptosporidiosis is endemic in the tropics with high prevalence in less developed countries (Gatei et al., 2003) Children are more vulnerable in developing Cryptosporidium-associated diarrhoea (Abdel-Messih et al., 2005). It has been shown that children less than 12 months of age are 2.4 times more likely to be infected with Cryptosporidium while children aged between 12 and 23 months are 1.9 times more likely to be infected as compared with older ones (Abdel-Messih et al., 2005). Molbak et al. (1997) reported increased carriage of cryptosporidiosis in malnourished children. Studies conducted in Brazil, Peru and Guinea-Bissau have associated cryptosporidiosis with impairment of growth, physical fitness and cognitive function (Checkley et al., 1998; Guerrant et al., 1999; Molbak et al., 1997).
There are isolated studies on the epidemiology of cryptosporidiosis in Nigeria. In one of these studies, Useh and Jonah (1998) reported a prevalence of 4.4% for C. parvum amongst diarrhoeal subjects of all ages in Calabar, South Eastern Nigeria. In the same setting, Alaribe et al. (1998) reported a prevalence of 5.6% among diarrhoeic patients. Infection was not reported among healthy control subjects with no history of diarrhoea. Elsewhere in North Central and South Western Nigeria, higher prevalence of cryptosporidiosis of 4.8 and 14% were reported among undernourished children and diarrhoeic patients by Ikeh et al. (2006) and Nwabuisi (1998) respectively. Although these studies have confirmed the presence of cryptosporidiosis in Nigeria, this organism is not sought for routinely even in Teaching Hospitals like ours. It is likely that this organism may be playing a very important role particularly as an opportunistic pathogen among the HIV/AIDS patients in Nigeria. The paucity of information on the prevalence and significance of Cryptosporidium parvum as an agent of infantile diarrhoea necessitated this study. It is envisaged that the findings of this study may herald some radical decisions on the diagnosis and control of diarrhoea of Cryptosporidium origin.
MATERIALS AND METHODS
Study area: The subjects were Children who presented with diarrhoea
and were attended to at the Children Emergency ward (CHER) of the University
of Calabar Teaching Hospital (UCTH) and the Out-Patient Department (OPD) of
the General Hospital, all in Calabar between April and September, 2006. The
UCTH is a tertiary health institution which caters principally for residents
of Cross River and Akwa Ibom states and others referred from other states in
Nigeria and the neighboring Republic of Cameroon. The states mentioned above
are located in the South-eastern rainforest belt of Nigeria which experiences
a typical tropical climate. Calabar and its immediate environs lack adequate
supply of portable water. The drainage system is poor. There is inadequate management
of domestic waste. These may provide effective media for the transmission of
Subjects and consent: Two hundred children aged between 2 months and 8 years diagnosed of diarrhoea and 100 healthy non-diarrhoiec control subjects of the same age range who lived within the University community were examined. Diarrhoea was defined as the presence of three or more loose or watery stools within a 24 h period (Sherries, 1989). Stool samples were collected from both patients and test subjects in clean universal containers and processed within 30 min in the Parasitology Department of the UCTH.
A written permission was sought and obtained from the Ethical Committee in the two hospitals. Verbal consent was sought and obtained from parents to enable their children participate in the study.
Examination of samples: The standard method of stool examination as described by WHO (1991) was adopted for the study. These included macroscopy, microscopic examination of wet mounts in saline and Lugols iodine and the formal ether concentration technique.
Formal ether concentration technique: All the samples were concentrated using Formal ether concentration technique. One milliliter of a well mixed stool sample was put in a tube containing 4 mL of 10% formalin. Three milliliter of the 10% formalin was again added and mixed by shaking. The suspension was sieved using a coffee strainer into a centrifuge tube. Three milliliter of diethyl ether was added and stoppered. It was then mixed vigorously for 1 min. The stopper was removed and the suspension centrifuged for 1 min at 400 g. The entire column of the fluid below the faecal debris and ether was carefully removed using a pasteur pipette and transfer into another centrifuge tube. Ten percent formalin was added to the transferred suspension to make up to 10 mL. It was then centrifuge at 1000 g for 10 min. The supernatant was decanted and the bottom of the tube tapped to re-suspend the deposit. The deposit was examined using the 10 and 40x objectives for the presence of cyst or ova of parasite.
Modified Ziehl Nelson Staining Technique for Oocyst: The modified Ziehl Nelson technique was used for the demonstration of oocyst of Cryptosporiduim. A methanol fixed thin smear of faeces was stained in cold carbol fuchsin for 10 min. Differentiation was done with 1% hydrochloric acid-ethanol while 0.25% methylene blue was utilized as counterstain. Crypotosporidium oocyst appeared as bright rose-pink spheres on a pale blue background (Current, 1989).
Data analysis: The data obtained from the study were analysed using Chi-square test to determine the influence of age and sex on the prevalence of infection.
Table 1 is on the distribution of Cryptosporidiosis by age of subjects examined. A prevalence of 26% was observed among the diarrhoiec subjects whereas control subjects had a prevalence of 8%. There was a statistically significant difference in prevalence rate of cryptosporidiosis between the diarrhoeic and non-diarrhoeic subjects (χ2 = 13.5, df = 1, p<0.05). Among the diarrhoeic group, subjects within the age group 2 months to 1 year had the highest prevalence of cryptospodiosis (38%).
The distribution of cryptospodiosis by sex of subjects examined is shown on Table 2. Female subjects had a higher prevalence rate of infection (26.9%) than the males (25%) but there was no statistical significant difference between the infection rate in both sexes (χ2 = 0.09, df 1, p>0.05).
The distribution of parasites detected as single infection among diarrhoeic
subjects examined by age is shown on Table 3. Cryptosprodium
parvum was found as a single infection in 16% of the total diarrhoeic subjects
examined while Ascaris lumbricoides was found as a single infection in
Figure 1 shows the distribution of other intestinal parasites detected among diarrhoeic and the non diarrhoeic control subjects examined. Ascaris lumbricoides was the most frequently encountered intestinal parasite 19 and 14% in both the diarhoeric and control subjects, respectively. Other parasites detected among diarrhoeic and the non diarrhoeic control subjects were Hookworm 1 and 4%, Trichurus trichiura 2 and 2%, and Entamoeba histolytia 1 and 2%, respectively.
|| Distribution of cryptosporidiosis by age of subjects examined
|0 denotes absence of infection
|| Distribution of Cryptosporidiosis by sex of subjects examined
||Distribution of parasites detected as single infection observed
among diarrhoeic subjects examined by age
||Distribution of other intestinal parasites detected among
diarrhoeic subjects examined
The present study has shown a very strong association between cryptosporidiosis and infantile diarrhea in Calabar, Nigeria. The prevalence rates of 26 and 8% reported for diarrhoeic and non-diarrhoeic healthy controls respectively is reasonably high. This confirms Cryptosporidium as one of the leading causes of diarrhoea in infants in Calabar. This rate is higher when compared to previous reports in the same area by Alaribe et al., (1998) (5.6%) and Useh and Jonah (1998) (4.4%). However it is within the prevalence of 3-29% summarized by Fayer and Ungar (1986) for this part of the world. The factors that may account for this high prevalence include poor personal and environmental hygiene, lack of safe and adequate drinking water, inappropriate methods of disposal of human waste, increased urbanization and poor drainage system. These factors may have created the enabling environment for the transmission and sustainable of the life cycle of the parasite.
Although, we did not confirm the HIV status of these subjects, it is likely
that the lowered immunity precipitated by the infection may have been responsible
for the high prevalence since Cryptosporidium is present in this area
(8% in control subjects). Currently HIV/AIDS is a serious public health problem
in Nigeria. The national prevalence rate is 4% (UNAIDS, 2004) exception in Cross
River State where infection level rose from 4% in 1993-1994 to 12% in 2003 (Federal
Ministry of Health, Nigeria, 2004).
The highest prevalence of cryptosporidiosis (38%) was reported among subjects aged 2/12 to 1 year. This agrees with the findings of Abdel-Messih et al. (2005) that younger age children aged less than 12 months were many times more likely to develop infection compared to older ones. The tendency and risk for children of this age to be exposed to the factors that leads to acquisition of infection are higher. However Cryptosporidiosis was not detected among children aged above 7 years. This is in accordance with the report of Abdel-Messih et al. (2005) who observed less risk factor in the disease among older children. Their poor immune status might also play a prominent role. Female subjects had a higher prevalence rate of infection (26.9%) than the males (25%) though the difference in prevalence was not statistically significant (p>0.05). It is possible that females were more exposed to risk factors of infection.
Ascaris lumbricoides was the most frequently encountered intestinal parasite (19%). Others were Trichurus trichiura (2%), Entamoeba histolytia (2%) and Hookworm (1%). The age of ≥8 years had the highest prevalence rate of infection with Ascaris lumbricoides (40%) and Entamoeba histolytia (20%). There was a statistically significant difference in the frequency of intestinal infection between the test and the control subjects (p<0.05) confirming their role in diarrhoeic causation. One of the limiting factors in this study is the fact that we did not screened our subjects for other aetiologic agents of diarrhoea like bacteria and viruses which could cause diarrhoea in those with cryptosporidiosis and others who had no oocyst detected in their stools. Other conditions such as immunosuppressive ailments like AIDS and cancer (Tanyuksel and Doganci 1995), solid organ transplantation (Campos et al., 2000, Gerber et al., 2000), malnutrition (Macfarlane and Horner-Bryce 1987), diabetes (Chan et al., 1989) may enhance to this infection even though these aetiologies were not sought for among our study subjects. In conclusion, this study has confirmed the involvement of Cryptosporidiosis in infantile diarrhoea in Calabar. There is a justified need for routine screening of stool samples of children with diarrhoea for Cryptosporidium which is not being currently done to ensure prompt and appropriate management of the disease.
The authors are grateful to all the physicians who contributed to the success of this study. We appreciate the co-operation we received from all the nurses and the parents of the subjects.
Abdel-Messih, I.A., T.F. Wierzba, R. Abu-Elyazzed, A.F. Ibrahim and S.F. Ahmed et al
Diarrhoea associated with Cryptosporidium parvum
among young children of the Nile River Delta in Egypt. J. Trop. Paediatr., 10: 1093-1105.
Alpert, G., L.M. Bell and C.E. Kirkpatrick, 1984.
Cryptosporidiosis in a day-care centre. N. Eng. J. Med., 311: 860-861.
Alaribe, A.A.A., S.J. Utsalo and E.N.U. Ezedinachi, 1998.
Prevalence of cryptosporidiosis among diarrhoiec patients in Calabar. Global J. Pure Applied Sci., 4: 423-425.
Campos, M., E. Jouzdani, C. Sempoux, J.P. Buts, R. Reding, J.B. Otte and E.M. Sokal, 2000.
Sclerosing cholangitis associated with cryptosporidiosis in liver-transplanted children. Eur. J. Pediatr., 159: 113-115.
Chan, A.W., I.A. MacFarlane and J.M. Rhodes, 1989.
Cryptosporidiosis as a cause of chronic diarrhoea in a patient with insulin-dependent diabetes mellitus. J. Infect., 19: 293-293.
Checkley, W., L.D. Epstein, R.H. Gilman, R.E. Black, L. Cabrera and C.R. Sterling, 1998.
Effects of Cryptosporidium parvum
infection in Peruvian children: Growth faltering and subsequent catch-up growth. Am. J. Epidemiol., 148: 497-506.
Current, W.L., 1989. Cryptosporidiosis
. In: New Strategies in Parasitology. Proceedings of an International Symposium on Frontiers of Infectious Disease, pp: 257-272.
Fayer, R. and B.L.P. Ungar, 1986. Cryptosporidium
spp. and cryptosporidiosis. Microbiol. Rev., 50: 458-483.
Federal Ministry of Health, Nigeria, 2004.
2003 National HIV sero-prevalence sentinel survey. Technical Report, Federal Ministry of Health Abuja.
Gerber, D.A., M. Green, R. Jaffe, D. Greenberg, G. Mazariegos and J. Reyes, 2000.
Cryptosporidium infection after solid organ transplantation in children. Pediatr. Transplant., 4: 55-55.
Guerrant, D.I., S.R. Moore, A.A. Lima, P.D. Patrick, J.B. Schorling and R.L. Guerrant, 1999.
Association of early childhood diarrhea and cryptosporidiosis with impaired physical fitness and cognitive function four-seven years later in a poor urban community in northeast Brazil. Am. J. Trop. Med. Hyg., 61: 707-713.Direct Link |
Gatei, W., J. Greensill and R.W. Ashford et al
Molecular analysis of the 18S rRNA gene of Cryptosporidium
parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil. The United Kingdom and Vietnam. J. Clin. Microbiol., 41: 1458-1462.
Ikeh, E.I., M.O. Obadofin, B. Brindeiro, G. Baugher and F. Frost et al
Intestinal parasitism in rural and urban areas of North central Nigeria: An update. Internet J. Microbiol., 2: 1-12.Direct Link |
Juranek, D.D., 1995.
Cryptosporidiosis: Sources of infection and guidelines for prevention. Clin. Infect. Dis., 21: S57-S61.
Khan, P.A., 2000.
A review of Cryptosporidium. Department of International Health Report, pp: 1-15.
Koch, K.L., D.J. Phillips, R.C. Aber, W.L. Current, 1985.
Cryptosporidiosis in hospital personnel, evidence for person to person transmission. Ann. Intern. Med., 102: 593-596.
Loureiro, E.C.B., A.C. Linhares and L. Mata, 1989.
Criptosporidisoe em criancas de 1 a 2 anos de idade, com diarreia aguda em Belem, Para, Brasil. Mem. Inst. Oswaldo Cruz., 84: 117-122.
Macfarlane, D.E. and J. Horner-Bryce, 1987.
Cryptosporidiosis in well-nourished and malnourished children. Acta Paediatr. Scand., 76: 474-477.
Molbak, K., M. Andersen P. Aaby, N. Hojlyng, M. Jakobsen and A.P. da Silva, 1997. Cryptosporidium
infection in infancy as a cause of malnutrition: A community study from Guinea-Bissau, West Africa. Am. J. Clin. Nutr., 65: 149-152.Direct Link |
Nchito, M., P. Kelly, S. Sianongo, N.P. Luo, R. Feldman, M. Farthing and K.S. Baboo, 1998.
Cryptosporidiosis in urban Zambian children: An analysis of risk factors. Am. J. Trop. Med. Hyg., 59: 435-437.PubMed |
Nwabuisi, C., 1998.
Cryptosporidiosis among diarrhea patient in Ilorin, Nigeria. Nig. Med. Practioner, 35: 39-41.
O'Donoghne, P.J., 1995. Cryptosporidium
and cryptosporidiosis in man and animal. Int. J. Parasitol., 25: 139-195.
Sherries, J.C., 1989.
Medical Microbiology: An Introduction to Infectious Disease. 2nd Edn., Butterworth, Cambridge, pp: 577-596
Tanyuksel, M., H. Gun and L. Dogancoi, 1995.
Prevalence of Cryptosporidium
spp. in patients with neoplasia and diarrhoea. Scand. J. Infect. Dis., 27: 69-70.
Report on the global AIDS epidemic. Geneva, Switzerland.
Useh, M.F. and B.U. Jonah, 1998.
Cryptosporidiosis among diarrhoeic subjects in Calabar. Mary Slessor J. Med., 1: 20-24.
Basic Laboratory Methods in Medical Parasitology. World Health Organization, Geneva, ISBN- 13: 9789241544108, pp: 9-25