348 South African Journal of Science 104, September/October 2008 Research in Action

sites constitute the largest group of Prevalence of asymptomatic parasites known to be associated with diarrhoea in humans. The incidence of intestinal coccidian parasite protozoan with diarrhoea has been on the increase, following the advent infections among non-diarrhoeic of HIV infection and the acquired im- mune deficiency syndrome (AIDS) pan- HIV-positive children in Zaria, demic. Common diarrhoeal protozoan Nigeria pathogens are Giardia lamblia, Entamoeba histolytica and coli and, lately, coccidian parasites like belli, M. Aminu*‡ and Y.E. Yakubu* parvum, Microsporidia and cayetanensis.3–5 Coccidian parasites are protozoans PPORTUNISTIC COCCIDIAN PARASITES, likely to have been infected with coccidian belonging to the phylum Apicomplexa6 amongst other infections, frequently parasites than the control children (odds and were known historically to be patho- Ocomplicate human immunodeficiency ratio = 13.0, 95% CI = 3.9–42.6). Cyclospora virus (HIV) infection by causing chronic diar- cayetanensis was the most prevalent parasite genic mainly to some animal , in- rhoea. The magnitude of these parasitic infec- identified in the study (36%). Dual infections cluding insects, birds and non-human tions in HIV-positive patients requires caused by C. cayetanensis and C. parvum were primates.7,8 Today, these opportunistic careful attention in developing countries. found in 17% of HIV-positive patients. Female parasites cause chronic diarrhoea in There have been inadequate studies address- children were found to have more (53%: 25/47) humans, especially in those who are ing this problem in Nigeria. The investiga- coccidian oocysts in their stools than the boys immunocompromized.4,5,8,9 The parasites tion reported here was conducted at Ahmadu (49%: 20/41) (P > 0.05). We found an increase are transmitted to humans through Bello University Teaching Hospital, Zaria, in parasite prevalence with age of the patient. Nigeria, with the objective of determining This study indicates that coccidian parasites contaminated drinking and recreational the prevalence of these parasitic infections may be important opportunistic infection water, food, and contact with infected among HIV-positive children. Eighty-eight agents in non-diarrhoeic HIV-infected chil- animals and persons.8 stool samples were collected during the wet dren. The prevalence of these parasites and Cryptosporidium, Cyclospora and Isospora humid months of July and August 2006 their potential for compounding the health have been shown to complicate HIV from 60 non-diarrhoeic, HIV-positive and 28 problems of HIV-infected patients suggest HIV-negative children less than 10 years old. that the diagnosis and treatment of coccidian infection by causing chronic diarrhoea. 9–14 The samples were examined for intestinal parasites should be a part of routine HIV care. This facilitates progression to AIDS. coccidian parasites by microscopy and modi- Diarrhoea occurs in up to 80% of persons fied Kinyoun’s acid fast staining methods. Introduction with HIV infection.15 The prevalence of Coccidian parasites, , The World Health Organization reported coccidian parasitic infection is high and Isospora belli, in 1998 that 33% of global deaths are a and widely distributed in sub-Saharan were identified in 51% (45/88) of all the stool consequence of infectious and parasitic Africa, where the majority of HIV cases samples examined. The parasite oocysts were 16 identified in 68% (41/60) of the HIV-positive diseases, whereas the effect of mortality are located. Few studies on the associa- patients presenting at the hospital and in 14% and morbidity are as a result of some tion of coccidian parasites and HIV infec- (4/28) of the controls (P < 0.01). The HIV parasitic infections.1 Parasitic infections tion have been carried out in Nigeria.17 patients were found to be thirteen times more caused by protozoan pathogens impose a Thus, there is need for further studies to *Department of Microbiology, Faculty of Science, Ahmadu substantial health and economic burden examine the association of coccidian Bello University, Zaria, Nigeria. ‡ on tropical, poor countries where such parasites with HIV infection, an associa- Author for correspondence. E-mail: [email protected] infections are prevalent.2 Protozoan para- tion that can lead to chronic diarrhoea Research in Action South African Journal of Science 104, September/October 2008 349

and consequent weight loss, exacerbating Table 1. Prevalence of coccidian parasites detected in non-diarrhoeic stools of HIV-positive (n = 60) and HIV-negative (n = 28) children less than 10 years old in Zaria, Nigeria. the illness of HIV/AIDS individuals in the 17 country. Patients Coccidian parasites (%) Materials and methods Cyclospora cayetanensis Cryptosporidium parvum Isospora belli See Appendix. HIV-positive 29 (48)* 21 (35)* 1 (2) Results HIV-negative 3 (11) 1 (4) 0 (0) Coccidian parasite oocysts were recorded Total (n = 88) 32 (36) 22 (25) 1 (1) in 45 (51%) of the 88 non-diarrhoeic stool *P = 0.001 samples analysed in total. Oocysts were recorded in 68% (41/60) of the HIV-positive than the boys (49%: 20/41); this was not children. This indicates a correlation children and in 14% (4/28) of the control statistically significant (P > 0.05). The age between coccidian pathogens and HIV children. The parasites were significantly of the children ranged from six to 108 infection and implies that infections by associated with HIV-positive children months. There was an increase in parasite these parasites increase during HIV infec- (P < 0.01). The HIV patients were thirteen load with patient age; with the highest tion as has already been suggested by times more likely to have been infected prevalence in children in the age group others.5,11 Detection in both patients and with coccidian parasites than the controls 97–120 months (100%: 7/7) (Table 2). controls could be a reflection of poor envi- (odds ratio = 13.0, 95% CI = 3.9–42.6). Children of less than 24 months had ronmental and personal hygiene prac- Cyclospora cayetanensis was the most fewer stool oocysts, with none found in tices that facilitate the parasites’ mode of prevalent parasite found in the study children of less than 12 months. The only transmission. (36%: 32/88), followed by Cryptosporidium Isospora belli oocyst detected was in the Our study also shows C. cayetanensis to parvum (25%: 22/88) (Fig. 1). Cyclospora stool of an 84-month-old child. No signifi- be the most prevalent pathogenic coccidian cayetanensis and C. parvum were found cant association between patient age and parasite found (36%). This is in contrast to to be significantly associated with HIV the detection of parasitic agents was a previous report from Nigeria4 in which infection (P < 0.01), with C. cayetanensis found (P > 0.05). Cryptosporidium was identified as the occurring with the highest frequency most prevalent protozoan . In (48%: 29/60). Isospora belli was detected in Discussion agreement with other reports,4,11,12 C. only one HIV-positive child (Table 1). This study shows that coccidian para- cayetanensis and C. parvum were found to Cyclospora cayetanensis and C. parvum sites are common in children in Zaria in be significantly associated with HIV infec- were detected in three and one HIV- view of their rate of detection (51%) in tion, implying that HIV predisposes negative stools, respectively. Dual infec- non-diarrhoeic stools. This prevalence is patients to these parasitic infections. The tions comprising C. cayetanensis and C. higher than previously reported in a oocysts of coccidian parasites have been parvum occurred in only 17% (10/60) of similar study16 and even from studies found more commonly in the stools of HIV-positive patients. using diarrhoeic stools.4,9,11,17,19 The para- older children, with all children within Girls were found to have more (53%: site oocysts were found in 68% of the the age group 97–120 months being 25/47) coccidian oocysts in their stools HIV-positive and in 14% of the control infected. We attribute this to the more active nature of older children, and the consequences of increased risk of infec- tive contact with contaminated food and water. Combined coccidian parasitic infection comprising C. cayetanensis and C. parvum is shown to be common in HIV patients and absent in the control group. Another Nigerian study4 has drawn similar infer- ences and indicates the facilitated infec- tion by parasites in immunocompro- mised patients. Co-infection with Cyclo- spora or Cryptosporidium and Isospora belli was not detected in this study, in contrast 4,19 Fig. 1. Percentage prevalence of coccidian parasites detected in stool samples of non-diarrhoeic children less to previous reports. than 10 years old in Zaria, Nigeria. The result of this and other studies4,17 shows a steady increase in the reported

Table 2. Percentage distribution of coccidian parasites by age group among non-diarrhoeic children less than 10 years old in Zaria, Nigeria.

Age group Study population HIV-positive HIV-negative (months) Total no. of samples No. positive (%) Total no. of samples No. positive (%) Total no. of samples No. positive (%)

0–24 11 4 (36) 7 3 (43) 4 1 (25) 25–48 34 15 (44) 23 15 (65) 11 0 (0) 49–72 24 13 (54) 17 13 (76) 7 0 (0) 73–96 12 6 (50) 8 5 (63) 4 1 (25) 97–120 7 7 (100) 5 5 (100) 2 2 (100) Total 88 45 (51) 60 41 (68) 28 4 (14) 350 South African Journal of Science 104, September/October 2008 Research in Action

incidence of Cryptosporidium and Cyclo- 2. Ochoa C.I. and Martinez A.R. (2001). Uniform adults is strongly associated with acute or chronic staining of coccidian oocysts by modified acid fast diarrhea. Am. J. Trop. Med. Hyg. 73, 54–57. spora infections in Nigeria. This may have staining techniques. J. Clin. Microbiol. 35, 730–733. 20. Cheesbrough M. (2005). Parasitological tests. In arisen from greater awareness of these 3. Wilson M.L. (1999). Emerging infections: the once District Laboratory Practice in Tropical Countries, pathogens among clinicians and labora- and future diseases. Am. J. Clin. Pathol. 112, Pt 1, 2nd edn, pp. 178–309. Cambridge University tory scientists. Another reason could 595–596. Press, Cambridge. 4. Alakpa G.E and Fagbenro-Beyioku A.F. (2002). be the HIV/AIDS pandemic, which is Cyclospora cayetanensis and intestinal parasitic Appendix claimed to increase coccidian parasite in- profile in stool samples in Lagos, Nigeria. Acta fection.11 Protozool. 41, 221–227. Material and methods 5. Wiwanitkit V.(2006). Intestinal parasitic infection The Ethical and Research Committee of the In summary, our results make an impor- in HIV infected patients. Curr. HIV Rev. 4, 87–96. tant contribution to the detection and 6. Visvesvara G.S., Moura H., Kovacs E.N. and Ahmadu Bello University Teaching Hospital, identification of coccidian parasites in Wallace S. (1997). Staining and identification of Zaria, Nigeria, approved the study. Sixty coccidian parasites in persons with HIV infection. asymptomatic HIV-positive children receiving non-diarrhoeic stool samples of HIV- Am. J. Trop. Med. Hyg. 63, 231–235. antiretroviral (ARV) drugs at the hospital’s positive children in Zaria, Nigeria. Our 7. Marshall M.M., Naumavitz D., Ortega Y. and Pediatric HIV Clinic were selected as study study also shows that Cyclospora cayeta- Sterling C.R. (1997). Waterborne protozoan participants, while controls were 28 HIV-nega- pathogens. Clin. Microbiol. Rev. 10, 67–85. nensis is the most prevalent intestinal tive children attending the same hospital. 8. Norman J.P., Fernando J.B., Susan B.S. and coccidian parasite in these patients. We Alexandre J.D. (2006). Prevalence of intestinal The children were aged <10 years and com- have identified a need to collect more parasites in HIV infected patients. Emerg. Inf. Dis. prised 41 males and 47 females. Stool samples information on asymptomatic infections 23, 4–5. were collected according to WHO standard 9. Kumar S.S, Ananthan S. and Saravanan P. (2002). 18 with these emerging pathogens and their procedure. Non-diarrhoeic stool samples were Role of coccidian parasites in causation of placed in labelled leak-proof, screw-capped relation to HIV/AIDS, and will continue diarrhea in HIV infected patients in Chennai. In- plastic containers during the wet humid investigations. dian J. Med. Res. 116, 85–89. 10. Mansfield L.S and Gajadhar A.A. (2004). months of July and August 2006. A single stool Our recommendations are that health Cyclospora cayetanensis, a food and water borne sample was collected from each child and practitioners should receive more inten- coccidian parasite. Vet. Parasitol. 126, 73–90. transported to the laboratory of the Depart- sive education on emerging diarrhoeal 11. Pinlaor S., Mootsikapum P.,Pinlaor P.,Pipitgool V. ment of Microbiology, Faculty of Science, pathogens and the importance of targeting and Tuangnadee R. (2005). Detection of opportu- Ahmadu Bello University, for analysis within nistic and non-opportunistic intestinal parasites 1.5 hours of collection. Patient details such as these common infections while treating and liver flukes in HIV-positive and HIV-negative age, sex and reason for visiting the hospital HIV-positive patients for opportunistic subjects. Southeast Asian J. Trop. Med. Publ. Hlth 36, were provided by caregivers, who signed the infections. Asymptomatic infection with 841–845. 12. Hunter G., Bagshawe A.F., Baboo K.S., Luke R. consent forms. these parasites requires only time before and Prociv P. (1992). Intestinal parasites in Zam- Stool samples were first concentrated by the symptomatic conditions develop, leading bian patients with AIDS. Trans R. Soc. Trop. Med. formalin-ether sedimentation method.20 Hyg. 86, 543–545. to chronic diarrhoea that may facilitate Smears were stained by the modified Kinyoun 13. Chintu C., Luo C., Baboo S., Khumalo-Ngwenya 6 HIV infection and progression to clinical B., Mathewson J., DuPont H.L. and Zumla A. carbol-fuchsin staining procedure and exam- AIDS. Routine examination of stool (1995). Intestinal parasites in HIV-seropositive ined at ×400 magnification using a Nikon light samples for coccidian parasites will also Zambian children with diarrhoea. J. Trop. Pediatr. microscope fitted with an eye-piece microme- benefit HIV-infected individuals signifi- 41, 149–152. ter that had been calibrated with a stage 14. Tarimo D.S., Killewo J.Z., Minjas J.N. and cantly through reduction of morbidity micrometer. Oocyst sizes were thus measured Msamanga G.I. (1996). Prevalence of intestinal under this magnification. Identification of the and improving quality of life. The need parasites in adults patients with enteropathic parasites was based on the size and shape of for intervention measures at community AIDS in northeastern Tanzania. E. Afr. Med. J. 73, 397–399. the oocysts. Cryptosporidium parvum (4–6 µm in level, with the purpose of reducing risk 15. Bowers M. (1997). Diarrhea. Bull. Expt. Treat. AIDS diameter) is round in shape and contains 4 factors of acquiring coccidian diarrhoeal 6, 1–14. sporozoites within a thick-walled oocyst; diseases, is emphasised through screen- 16. Houpt E.R, Bushen O.Y, Sam N.E. and Kohli A. Cyclospora cayetanensis (8–10 µm in diameter) is (2005). Asymptomatic infec- round to oval in shape, whereas Isospora belli ing for the presence of parasites in both tions among HIV infected patients in Tanzania. patients and controls. Afr. Med. J. 31, 520–522. (25–30 µm in diameter) is oval in shape. 17. Nwokediokwo S.C and Ozumba U.C. (2005). Coccidian species stained pinkish-red against a We thank W.N.Ogalla, O. Ogunrinde, L.W.Umar, and Intestinal helminthes in relationship to chronic uniform green background. Oocysts stained the nurses at the Pediatric HIV Clinic of ABUTH for diarrhea in HIV seropositive adults in Enugu. Nig. red and were easily recognized against a green involving their patients in this study. Post Grad. Med. J. 9, 88–91. background of yeast and faecal debris. 18. World Health Organization (1991). Basic Labora- Data were analysed with the SPSS 11.0 ver- 1. World Health Organization (1998). The state of tory Methods in Medical Parasitology. Geneva. world health. In The World Health Reports 1998. Life 19. Certad G., Arenas-Pinto A., Pocaterra L., Ferrara sion statistical package. Differences with P-val- in the 21st century: a vision for all, pp. 57–58. G., Castro J., Bello A. and Nunez L. (2005). ues >0.05 were considered insignificant at 95% WHO, Geneva. Cryptosporidiosis in HIV-infected Venezuelan confidence intervals (CI).