HIGH PREVALENCE OF HOOKWORM INFECTION AND APPARENT ABSENCE OF ASCARIS LUMBRICOIDES: A CASE STUDY AT THE KOMFO ANOKYE TEACHING HOSPITAL IN GHANA

HIGH PREVALENCE OF HOOKWORM INFECTION AND APPARENT ABSENCE OF ASCARIS LUMBRICOIDES: A CASE STUDY AT THE KOMFO ANOKYE TEACHING HOSPITAL IN GHANA

Sammy C. K. Tay ¹, Stephen Y. Gbedema*² and Thomas K. Gyampomah ³

Department of Clinical Microbiology, School of Medical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology ¹, Kumasi, Ghana

Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology ², Kumasi, Ghana

Department of Clinical Microbiology, Komfo Anokye Teaching Hospital ³, Kumasi, Ghana

ABSTRACT

Background: Parasitic helminth infection is one of the major risk factors underlying the high rates of anaemia and malnutrition in many third world countries due to the poor socio-economic and environmental conditions of the people and these play a very significant role in their transmission.

Method: 2000 stool specimens collected between May and October 2008 at the Komfo Anokye Teaching Hospital in Ghana were screened using the Kato-Katz and formol-ether concentration methods for the presence of intestinal helminthes. Prior to sample collection, histories of any antihelmentic drug usage in the last three months preceding this study were collected. Hookworm positive stool specimens were further cultured by the modified Harada-Mori test-tube technique for the identification of the hookworm filariform larvae.

Results: Hookworm (Necator americanus) was the most prevalent helminth parasite (2.9%) found in the study. Other parasitic helminthes detected were Dicrocoelium dendriticum (2.1%), Strongyloides stercoralis (2.1%), Schistosoma mansoni (1.8 %), Hymenolepis nana, (1.4%) Taenia species (0.6%) and Trichuris trichuria (0.1%). S. mansoni infection was however high among patients aged between 1 to 15 years. Ascaris lumbricoides was not detected in any of the specimens examined.

Conclusion: In this study therefore, gender and dewormer usage did not play any significant role (p> 0.05) in the rate and level of helminth infection. Further studies to assess the quality and efficacy of the various types of antihelmentic drugs on the Ghanaian market and the factors contributing to absence of A. lumbricoides must be considered

Anaemia

INTRODUCTION: Helminthes are complex eukaryotic organisms with large genomes, endowing some species with the ability to live for decades in human host ¹. There are about 20 major helminth infections of humans, affecting more than one third of the world's population ² and the most common ones that account for most of the global helminth disease burden in many sub-Saharan African countries including Ghana are ascariasis, trichuriasis, strongyloidiasis, taeniasis, schistosomiasis and hookworm infections ^3-8.

It is common for the host-parasite interplay to seem harmonious, and clinically asymptomatic helminth carriers act as long term reservoirs for transmission. About 7 % of the helminth-infected patients suffer severe and permanent impairments of immunopathological complications such as granulomatous disease and organ failure ^{9, 10}.  Helminth infections have also been shown to play a significant role in anaemia and childhood malnutrition, which often leads to growth retardation, poor intellectual development and impaired cognitive function, resulting in a poorer quality of life and less ability to contribute to society ^11-13.

Additionally, immune suppression by helminth infections has well been documented:  Schistosome-infected children have been shown to exhibit lower levels of skin reactivity to allergens than uninfected classmates, indicating a helminth-mediated down modulation of the immune system ¹⁴.

In Ghana, there are not enough available data on the prevalence of these infections in most of the poor communities which are heavily burdened with these infections. This study was therefore designed to evaluate and provide data on the prevalence of some of the major intestinal helminth infections reported at The Komfo Anokye Teaching Hospital in Kumasi.

MATERIALS AND METHODS:

Study area and population: The study was carried out at the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana. The geographical location of this hospital and commercial nature of Kumasi make the hospital accessible, and a referral hospital for the middle and northern parts of Ghana. A non-selective sample of 2000 consented in- and out-patients aged between 1-90 years were recruited for the study between May 2008 to October 2008.

Ethical Issues: The study protocol was approved by the Committee on Human Research, Publications and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, in collaboration with the KATH management which also granted the permission to undertake the study.

Specimen Collection: Specimen collection and handling were carried out in accordance with standard protocols reported by Booth et al., ¹⁵, NCCLS ¹⁶, Melvin and Brooke ¹⁷ and WHO ¹⁸. Demographic data and history of antihelmentic drug usage in the last three months preceding this study were collected prior to the sample collection.

Parasitological Examination: Parasitological screening methods such as Kato-Katz and Formol-ether concentration were employed. Each specimen was first examined macroscopically and its consistency or nature was recorded in accordance with the description by Ash and Orihel ¹⁹, Estevez and Levine ²⁰ and Smith and Schad ²¹. The test procedures were carried out in accordance with standard protocols as described by Garcia ^{22, 23} and WHO ^{19, 24}.

Helminth Infection Intensity Determination by Kato Katz Technique: The faecal sample was pressed through a Kato nylon screen mesh (HelmR test kits, Belo Horizonte, Brazil) of size 200μm and the sieved stool was transferred into the 6 mm diameter hole of the cardboard template on a microscope slide. The template was then removed and the remaining sample (approximately 42 mg) was covered with the glycerol soaked cellophane strip of size 25×35 mm and 50μm thick. The prepared slide was microscopically examined and the eggs of each of the helminth species were recorded. The eggs per gram (epg) of faeces was calculated and used to estimate the infection intensity of the parasites based on the classification reported by WHO ²⁵ for major soil-transmitted helminths and S. mansoni infections. Loose and watery stool specimens were not analyzed by the Kato-Katz method because of the technical difficulties that limit analysis of such specimens by this method ^{15, 26}.

Cultivation and Detection of Hookworm Larvae from Stool samples: Hookworm ova positive stool samples in the Formol-ether concentration method were cultured by a modified Harada-Mori technique ^27-29. The culture tubes were incubated upright in darkness for 10 days at 25^oC and then heated in a water bath at 50^oC for 15 minutes to kill the infective larvae. This was followed by centrifugation at 1000 rpm for 5 minutes and after aspirating the supernatant, the sediment was stained with a drop of Lugol’s iodine. A slide of the sediment was then prepared and examined microscopically for the presence of larvae as described by Wu and Peng ³⁰ (1965), Yoshida ³¹ and Little ³². The length of each larva and sheath were measured and recorded. All the laboratory procedures were carried out in accordance with standard protocols ^16-18.

RESULTS AND ANALYSIS:

Demographic Characteristics: A total of 2000 patients comprising of 1,185 (59.25%) females and 815 (40.75%) males, with ages ranging between 1 to 90 years were studied. About 71% of the patients were aged between 16 and 40 years while the 41 years and above patients constituted 13.5% (Fig. 1). Antenatal care attendants were the highest, followed by patients whose conditions were not clearly diagnosed (Table 1).

Antihelmentic Drug Usage: A total of 620 patients of the studied population reported of having taken some form of antihelmentic drug in the 3 months preceding the study. The highest antihelmentic drug usage was found among the 26-30 years age group, followed by those in the 31-35 age group. The age groups 1-5 and those over 55 years had the least number of persons who had dewormed in the 3 months preceding the study (Fig. 2).

FIG. 1: DEMOGRAPHIC DATA OF PATIENTS STUDIED FOR HELMINTH INFECTIONS AT KATH

FIG. 2: REPORTED ANTIHELMENTIC DRUG USAGE AMONG THE STUDIED POPULATION

Organoleptic Characters of Stool Specimens: Most of the stool specimens (75.7%) were normal in nature, while 120 were diarrhoeal specimens and 39 contained blood and/or mucus (Table 1).

TABLE 1: RELATIVE FREQUENCIES OF THE TYPES OF CONSISTENCY OF THE STOOL SAMPLES

Helminth Parasites Prevalence: The prevalence of hookworm was 2.9 % and the overall prevalence rate of helminth parasites in the population studied was 11.1% (Table 2).

TABLE 2: PREVALENCE OF INTESTINAL PARASITES AMONG THE 2000 PATIENTS EXAMINED

Helminth Infection Intensities: The intensity of helminth infection was measured on the basis of egg counts per gram (epg) of stool ³³, determined by the Kato-Katz method. Infection intensities for the soil transmitted helminths (STHs) and S. mansoni cases detected in the study were classified in accordance with the criteria prescribed by WHO ²⁵.  All cases of STHs found in the study were of low intensity (Table 3). It was evidenced that a significantly high number of individuals infected with medium and high S. mansoni burden belonged to 10-15 age group (p<0.05).

TABLE 3: INFECTION INTENSITY OF STHS AND S. MANSONI USING THE KATO-KATZ

Species identification of Hookworm Filariform Larvae: The criteria for species identification of hookworm larvae cultivated by the Harada-Mori technique were based on distinct morphological characteristics of larval nematodes described by Wu and Peng ³⁰, Yoshida ³¹ and Little ³². All the hookworm larvae species recovered morphological features of Necator americanus (Fig. 3).

FIG. 3: PICTURES OF HOOKWORM LARVAE RECOVERED FROM THE STOOL SPECIMENS (A: WHOLE LARVAE, B: ROUNDED HEAD OF LARVAE AND C: SHARPLY POINTED END OF LARVAE)

DISCUSSION: Poor nutrition in general and anaemia in particular are the main underlying causes of poor pregnancy outcomes in developing world ³⁴. Hence stool examination for parasitic intestinal infections is one of the routine medical examinations for pregnant women who attend antenatal clinics in health institutions in Ghana and this accounted for the high numbers of the female participants (59.3%) in the study. Antihelmentic drug usage within the three preceding months of the study period was reported in only 31 % of the patients. This did not significantly (p<0.05) support the speculation that many people regularly take antihelmentic drugs.

However, children aged between 6 to 15 years who had dewormed were more than those who had not dewormed. This observation is probably a reflection of the national deworming programme for school children in Ghana under the School Health Education Programme ^35-37 but there was no significant association between dewormer usage and the prevalence of helminth parasites realized from the study. This appears to provide strong indications for further studies to assess the efficacies of the various types of medicines commonly used to treat helminth infections in Ghana. The study in addition, did not show any significant association (p<0.05) between gender and helminth infection reported.

Out of the overall prevalence (11.1%) of parasitic helminthes in the studied population, hookworm was the highest (2.9%) and these supported our preliminary survey (unpublished data) which identified hookworm as the predominant parasite among school children in the Ayigya community of Kumasi, Ghana. Earlier epidemiological studies have also reported the predominance of hookworm infections in some communities in Ghana ^{38, 39}, Mali ⁴⁰, Kenya ⁴¹ and Nigeria ⁴². As reported by Hotez et al., ⁴, hookworm infection is still a significant public health problem in Sub-Saharan Africa.  Hookworm infection is associated with high intestinal blood loss; ingestion of 0.15 ml per worm per day can occur and these can results in severe iron- and or protein- deficiency anaemia ^{43, 44}. Species identification is important in a community as its transmission influences the burden of iron deficiency anaemia in that community and the efficacy of antihelmentic therapy also depends on the infecting species ^{45, 46}. In susceptible children, hookworms cause intellectual, cognitive and growth retardation, intrauterine growth retardation, prematurity and low birth weight among neonates born to infected mothers.

Out of a total of 226 specimens that showed the presence of helminthes, 25.7% were positive for hookworm (table 2).  Necator americanus was the only prevalent species detected (Fig 3) and it was of light intensity (table 3). Other studies conducted by Tay et al., ³⁸ at Kintampo in the Brong Ahafo Region of Ghana also revealed high prevalence of hookworm in the country. These findings supported the reports that N. americanus predominates in developing countries of the tropics and subtropics ^{47- 49}.  However, because only 58 stool samples were cultured for larvae, the data available cannot be considered sufficient enough to conclude the absence of Ancylostoma duodenale infection in Ghana.  N. americanus infection is acquired almost exclusively by active penetration of the skin ^50-53.

Other parasitic helminthes such as Dicrocoelium dendriticum (2.1 %), Strongyloides stercoralis (2.1%), Schistosoma mansoni (1.8%), Hymenolepis nana (1.4%), Taenia species (0.6%) and Trichuris trichuria (0.1%) which were earlier reported in Ghana by Odei ⁵⁴ and Wolfe ⁵⁵ are still prevalent in the country (Table 2).  These parasitic helminthes were also recently reported in other parts of the country ^{38, 39}. Our findings therefore, seem to agree with reports by Odei ⁵⁴, King ⁵⁶, Manga-Gonzalez et al., ⁵⁷ and Pillai and Kain ⁵⁸ that dicrocoeliasis is a zoonotic infection. Schistosoma mansoni infection was predominant among the 1 to 15 years age groups, with those in the 10-15 years age group accounting for most of the moderate and heavy infections.

Ascaris lumbricoides infection was not detected in any of the specimens examined and this disagreed with the previous reports of 2.1% prevalence in the Brong Ahafo Region of Ghana ³⁸. Ascariasis occurs through ingestion of food and or water contaminated with faeces containing Ascaris eggs. Its transmission and distribution is largely determined by inadequate sanitary practices and improper faeces disposal ^{8, 25, 59}. The absence could be due to the fact that adult worms of Ascaris are regularly found in stool of infected individuals who then self-medicate with antihelmentics ¹² after seeing these worms in their stool, in addition to improvement in prevailing social environment and behaviours of people in the communities ^{60, 61}.

CONCLUSION: In this study, a true association between dewormer usage and not being infected could not be statistically established (p>0.05).  Also, hookworm infections are still prevalent in Ghana. Hence studies to assess the quality and efficacy of the various types of antihelmentic drugs on the Ghanaian market are necessary. Further, studies are also needed to determine the contributing factors for the absence of A. lumbricoides and confirm its absence.

REFERENCES:

1. Muller R. In Worms and animal disease. CABI Publishing, Wallingford, UK 2002; 1-161
2. Awasthi S, Bundy DAP, Savioli L. Helminth infections. British Medical Journal 2003; 23: 431–433.
3. Bethony J, Brooker S, Albonico M, Geiger SM, Loukas A, Diemert D, Hotez PJ. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 2006; 367: 1521–1532.
4. Hotez PJ, Bethony J, Bottazzi ME, Brooker S, Diemert D and Loukas A. New technologies for the control of human hookworm infection. Trends Parasitol 2006; 22: 327–331.
5. De Silva NR, Brooker S, Hotez PZ, Montresor A, Engles D, Savioli L. Soil-transmitted helminth infections: updating the global picture. Trends Parasitol 2003; 19:547-551.
6. Brooker S, Clements ACA, Bundy DAP Towards an atlas of human helminth infection in sub-Saharan Africa: the use of geographical information systems (GIS). Parasitol. Today 2000; 16:303-307.
7. Annan A, Crompton DWT, Walters DE, Arnold SE. An investigation on the prevalence of intestinal parasites in pre-school children in Ghana. Parasitology 1986; 92:209–217.
8. Feachem RG, Guy MW, Harrison S, Iwugo KO, Marshall T, Mbere N, Muller R, and Wright AM. Excreta disposal facilities and intestinal parasitism in urban Africa: preliminary studies in Botswana, Ghana and Zambia. Transactions of the Royal Society of Tropical Medicine and Hygiene 1983; 77:515–521.
9. Chan MS, Medley GF, Jamison D, Bundy DAP. The evaluation of potential global morbidity attributed to intestinal nematode infections. Parasitology1994; 109: 373 – 387.
10. Bundy DAP, Chan MS, Medley GF, Jamison D, Savioli L. Intestinal nematode infections. In Health Priorities and Burden of Disease Analysis: Methods and Application from Global, National and Sub-National Studies. Cambridge, USA, Harvard University Press for the WHO and World Bank 2000; 1-122.
11. Crompton DWT, Savioli L. Handbook of helminthiasis for public health. Boca Raton, CRC Press 2007; 362.
12. Adams EJ, Stephenson LS, Latham MC, Kinoto SN. 1994. Physical activity and growth in Kenyan school children with hookworm, Trichuris trichiura and Ascaris lumbricoides infections are improved after treatment with albendazole, Journal of Nutrition 1994;124:1199–1206.
13. Drake LJ, Jukes MCH, Sternberg RJ, Bunday DAP. Geohelminth infections (ascariasis, trichiuriasis, and hookworm): cognitive and development impacts. Sem Paediatr Infect Dis 2000; 11:245-51.
14. Maize RM, Yandanbakhsh M. Immune regulation by helminth parasites: cellular and molecular mechanisms, Nature reviews. 2003;3:733–745
15. Booth M, Vounatsou P, N’Goran EK, Tanner M and Utzinger J (2003). The influence of sampling effort and the performance of the Kato–Katz technique in diagnosing Schistosoma mansoni and hookworm co-infections in rural Côte d’Ivoire, Parasitology 2003;127:525–531.
16. NCCLS (1997). Procedures for the Recovery and Identification of Parasites from the Intestinal Tract. Approved guideline M28-A. National Committee for Clinical Laboratory Standards, Wayne, PA.1997;
17. Melvin D M and Brooke MM. Laboratory Procedures for the Diagnosis of Intestinal Parasites, U.S. Department of Health, Education, and Welfare publication no. (CDC) 85-8282. U.S. Government Printing Office, Washington, D.C. 1985; 163–189.
18. World Health Organization. Basic Laboratory Methods in Medical Parasitology. Geneva 1991.
19. Ash LR, Orihel TC. Parasites: A Guide to Laboratory Procedures and Identification. Chicago: American Society of Clinical Pathologists 1991.
20. Estevez EG and Levine JA. Examination of preserved stool specimens for parasites: lack of value of the direct wet mount. J Clin Microbiol 1985; 22:666–667.
21. Smith G, Schad GA (1990). Ancylostoma duodenale and Necator americanus: effect of temperature on egg development and maturity. Parasitology1990; 99:127–132.
22. Garcia LS (). Diagnostic Medical Parasitology, 4th ed. ASM Press, Washington, D.C. 2001; 1-723.
23. Garcia LS (1999). Practical Guide to Diagnostic Parasitology. ASM Press, Washington, D.C. 1999;1-645
24. World Health Organization, WHO (1994). Bench aids for the diagnosis of intestinal parasites. Geneva 1994.
25. World Health Organization. Prevention and Control of Schistosomiasis and Soil-Transmitted Helminthiasis. WHO Technical Series Report 912. Geneva 2002. www.who.int/ctd/para/disease.php. (Accessed 20 November 2008).
26. Siegel DL, Edelstein PH, Nachamkin I. Inappropriate testing for diarrheal diseases in the hospital. JAMA 1990; 263:979–982.
27. Harada Y, Mori O. A new method for culturing hookworm Yonago Acta Med 1955; 1:17.
28. Sasa M, Hayashi S, Tanaka H, Shirasaka R. Application of test-tube cultivation method on the survey of hookworm and related human nematode infection. J. Exp. Med. 1958; 28:129–137.
29. Hsieh HC. Employment of a test-tube filter-paper method for the diagnosis of Ancylostoma duodenale, Necator americanus and Strongyloides stercoralis. Geneva: World Health Organization, mimeograph AFR/ANCYL/CONF/16. Annex VI 1961; 37-41.
30. Wu ZX, Peng JM. Studies on the morphological differentiation of the infective larvae of Ancylostoma duodenale and Necator americanus. Acta Parasitol Sin1965; 2: 280-290.
31. Yoshida Y. Morphological differences between Ancylostoma duodenale and Necator americanus in the 4th larval stage. Journal of Parasitology 1966; 52:122–126.
32. Little MD. Comparative morphology of six species of Strongyloides (Nematoda) and redefinition of the genus. Parasitol. 1966; 52:69–84.
33. Beaver PC (1950). The standardization of faecal smears for estimating egg production and worm burden. Parasitol 1950; 36:451-6.
34. Fuseini G, Edoh D, Kalifa BG, Hamid A, Knight Parasitic infections and anaemia during pregnancy in the Kassena-Nankana district of Northern Ghana, Journal of Public Health and Epidemiology 2010;2(3):48-52.
35. Brooker, S., Marriot, H., Hall, A., Adjei, S., Allan, E., Maier, C., Bundy, D. A., Drake, L. J., Coombes, M. D., Azene, G., Lansdown, R. G., Wen, S. T., Dzodzomenyo, M., Cobbinah, J., Obro, N., Kihamia, C. M., Issae, W., Mwanri, L., Mweta, M. R., Mwaikemwa A, Salimu M, Ntimbwa P, Kiwelu VM, Turuka A, Nkungu D R, Magingo J. Community perception of school‐based delivery of antihelmentics in Ghana and Tanzania. Tropical Medicine and International Health 2001; 6:1075‐1083.
36. Nock IH, Aken’Ova T, Galadima M. Deworming: adding public health education to the equation. Trends in Parasitology 2006; 23:7–8.
37. PCD [Partnership for Child Development]. The cost of large‐scale school health programmes which deliver anthelmintics in Ghana and Tanzania. Acta Tropica, 1999; 73:183‐204.
38. Tay SCK, Twum WA, Bosompem KM. Epidemiological survey of soil transmitted helminthes in occupational risk groups and non-school going children in the Kintapo north district of the Brong Ahafo Region of Ghana. Proceedings of International Conference on Research and Development, Universite Nationale Du Benin. 2009; 2(39): 22 – 39.
39. Bosompem KM, Asigbee JRK, Otchere J, Haruna A, Kpo KH, Kojima S. Accuracy of diagnosis of urinary schistosomiasis: comparison of parasitological and a monoclonal antibody-based dipstick method. Parasitology International 1998; 47:211-217.
40. Behnke JM, De Clerq D, Sacko M, Quattara DB, Vercrysse J. The epidemiology of human hookworm infections in the southern region of Mali, Tropical Medicine and International Health 2000; 5:343-354.
41. Brooker S, Bethony J and Hotez PJ. Human Hookworm Infection in the 21st Century Advances in Parasitology 2004; 58:19 –288.
42. Adenusi AA and Ogunyomi EOA. Relative prevalence of the human hookworm species, Necator americanus and Ancylostoma duodenale in an urban community in Ogun State, Nigeria. African Journal of Biotechnology 2003; 2(11):470-473.
43. Albonico M, Stoltzfus RJ, Savioli L, Tielsch JM, Chwaya HM, Ercole E, Cancrini G. Epidemiological evidence for a differential effect of hookworm species, Ancylostoma duodenale or Necator americanus, on iron status of children. Int J Epid 1998; 27:530–537.
44. Pawlowski ZS, Schad GA, Stott GJ. Hookworm Infection and Anaemia: Approaches to Prevention and Control. World Health Organization, Geneva 1991.
45. Horton J. Global antihelmentic chemotherapy programs: learning from history. Trends in Parasitology 2003; 19:405–409.
46. Crompton DWT, Savioli L. Handbook of helminthiasis for public health. Boca Raton, CRC Press 2007; 362.
47. Salem SN, Truelove SC. Hookworm disease in Immigrants. British Medical Journal 1964; 1:1074–1077.
48. Davies HD, Sakuls P, Keystone JS. Creeping eruption. A review of clinical presentation and management of 60 cases presenting to a tropical disease unit. Arch dermatol 1993; 129:588–591.
49. Hochedez P, Caumes E. Hookworm – related cutaneous Larva migrans. Journal of Travel Medicine 2007; 14(5):326 – 333.
50. Yu SH, Jian ZX, Xu LQ (1995). Infantile hookworm disease in China. A review. Acta Tropica. 1995; 59:265–270.
51. Nwosu AB. Human neonatal infections with hookworms in an endemic area of Southern Nigeria. A possible transmammary route. Tropical and Geographical Medicine 1981; 33:105–111.
52. Rim HJ, Lee BS, Seo BS and Lim JK. Antihelmintic effects of pyrantel pamoate and bephenium hydroxynaphtoate against Necator americanus Korean J. Parasit., Abstract 1971; 9:22.
53. Reynoldson JA, Behnke JM, Pallant LJ, Macnish MG, Gilbert F, Giles S, Spargo RJ and Thompson RC. Failure of pyrantel in treatment of human hookworm infections (Ancylostoma duodenale) in the Kimberley region of North West Australia. Acta Tropica 1997; 68:301–312.
54. Odei MA. A note on dicrocoeliasis and Fasciola gigantica infection in livestock in Northern Ghana, with a record of spurious and of genuine Dicrocoelium infections in man. Ann Trop Med Parasitol 1966; 60:215–8.
55. Wolfe MS. Spurious infection with Dicrocoelium hospes in Ghana. Am J Trop Med Hyg 1966; 15:180–182.
56. King EV. Human infection with Dicrocoelium hospes in Sierra Leone. Journal of Parasitology 1971; 57:989.
57. Manga-Gonzalez MY, Gonzalez-Lanza C and Cabanas, C. Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum. Parasitology 2001; 123:91–114.
58. Pillai DR, Kain KC. Common Intestinal Parasites. Current Treatment Options in Infectious Diseases 2003; 5:207–217.
59. Anderson RM. The dynamics and control of direct life cycle helminth parasites. Biomath 1980; 39:278 – 322.
60. Montresor A, Crompton DWT, Hall A, Bundy DAP, Savioli L. Guidelines for the evaluation of soil-transmitted helminthiasis and schistosomiasis at community level. A guide for management of control programmes. WHO, Geneva 1998;1-45

Warren KS. An integrated system for the control of the major human helminth parasites. Acta Leiden 1990; 59:433-442