INTRODUCTION

Five species of filarial parasites have been reported in Nigeria, namely Wuchereria bancrofti, Onchocerca volvulus, Loa loa, Mansonella perstans and M. streptocerca1,2. Studies on these infections have been carried in most parts of Nigeria, including south-eastern parts of the country3-12. These studies have shown that these infections are widespread, and that the prevalence of microfilaraemia and clinical manifestations vary from one locality to another in south-eastern Nigeria, except for M. streptocerca.

In the Imo River Basin, both endemic onchocerciasis (EO) and sporadic onchocerciasis (SO) have been reported in the Upper and Lower parts respectively4,5,8. The EO area is known to support the breeding of 11 species of blackflies including the Simulium damnosum complex, which is the main vector of endemic onchocerciasis in south-eastern Nigeria13. In addition, bancroftian filariasis, mansonellosis and loiasis are all widespread and endemic in both parts of the basin 3, 9-11.

There have been few reports of co-infections of filarial worms in literature most of which have focused on the diagnostic difficulties of parasitic co-infections14, while others have bothered on the contribution of bacteria to inflammatory disease pathogenesis and the use of antibiotic therapy as a novel method of treatment15-18.

Studies on co-infections involving the four human filarial infections have not yet been undertaken. This study is therefore aimed at determining the prevalence and patterns of multiple filarial species infections between EO and SO populations in the Imo River Basin, Nigeria.

MATERIAL & METHODS

Study area and population

The study was conducted in two parts of the Imo River Basin. Endemic onchocerciasis is found in the Upper IRB5, an area with undulating plains and fast flowing streams. Southerly and closer to the shore is the Lower IRB, an area with relatively slow flowing rivers, swamps and sporadic onchocerciasis8. Farming is the main occupation in both parts of the IRB, but fishing is also a major occupation in the Lower IRB. Fish-smoking, a local traditional way of preserving fish is a regular practice among the older women.

Blood sampling and examination

From every consenting person of one year and above, 50 µl of day and night blood samples were collected and processed respectively with Haemotoxylin and Giemsa as vital stains. Detailed description of the collection and processing day and night blood samples for parasitological examination has been reported10. Identification was according to the keys in Learning Bench Aid No. 3 (Tropical Health Technology).

Skin snipping

Two skin snips (one each from the shoulder and the buttocks) for parasitological examination were taken from each individual during day time using a Walser corneoscleral punch. Processing for microscopy was as described elsewhere5,8.

Ethical approval

Ethical clearance was given by the Ministry of Health of both Okigwe and Emohua Local Government Areas, whose field staff actually mobilized the community and assured them of our compliance with ethical standards.

RESULTS

General observations

The occurrence of W. bancrofti, O. volvulus, M. perstans, and L. loa microfilaraemia separately, or together in double or multi-species microfilaraemia in both the EO and SO populations was analyzed. A total of 500 individuals were positive for microfilaraemia of at least one of these four filarial species in the EO area, while it was 419 individuals in the SO area. Individuals who were 10 yr old and above constituted 91.8% of microfilaraemic cases in EO area as against 94.5% in the SO area (Table 1).

In the EO area, 432 (84.6%) had single species microfilaraemia; 61 (12.2%) had double species microfilaraemia; while 7 (1.4%) had triple species microfilaraemia. Similarly, in the SO area, 345 (82.3%) had single species microfilaraemia; 71 (16.9%) had double species microfilaraemia; while three (0.7%) had triple species microfilaraemia. The major difference observed between the two populations was that those positive for the three categories of infection (single, double and triple) were dominated by those in the 10+ yr age group in the SO population while in the EO population, those under 10 yr of age featured almost equally in the single and double species microfilaraemia categories (Fig. 1). There were no cases of quadruple species microfilaraemia in both the populations. The occurrence of the four microfilarial species as single, double and triple infections in both the populations was analyzed (Table 2). In the EO population, single species microfilaraemia constituted most of the microfilaraemic cases as follows: O. volvulus (83.9%), W. bancrofti (79.1%), L. loa (53.6%), and M. perstans (51.6%). Double species microfilaraemia was dominated by M. perstans (43.7%), whereas in triple species microfilaraemia L. loa (21.4%) presented the highest occurrence. Similarly, in the sporadic onchocerciasis study population, single species microfilaraemia presented were as follows: O. volvulus (85.2%), W. bancrofti (75%), M. perstans (66.3%) and L. loa (64%). L. loa (29.8%) occurred most in double species microfilaraemia, whereas it was M. perstans (1.8%) for triple species microfilaraemia.

Relationships between the filarial species

In the following analyses of relationships between the different microfilarial species, only individuals of 10 yr and above were included, because microfilaraemia among children below 10 yr of age was not common.

Cluster I: Co-infections among those positive for W. bancrofti microfilaraemia

The co-occurrence between W. bancrofti microfilaraemia and microfilaraemia due to other human filarial species in the EO population was calculated (Table 3) and the overall prevalence of W. bancrofti microfilaraemia was 5.1%. The overall prevalence of W. bancrofti microfilaraemia was lower than the prevalence of W. bancrofti among those who had L. loa microfilaraemia (3.6%), M. perstans microfilaraemia (2.7%), or O. volvulus microfilaraemia (0.3%). Due to low number of microfilaraemic individuals, these differences were not analyzed statistically. The equivalent analyses for the SO area showed that overall prevalence of W. bancrofti microfilaraemia was 7.7%.

The W. bancrofti mf prevalence in SO population among those positive for L. loa microfilaraemia (10.1%) was the highest and was significantly higher than both the W. bancrofti mf prevalence among those positive for O. volvulus microfilaraemia (1.9%), and those positive for M. perstans microfilaraemia (1.9%) (χ2-test; p <0.01, for both the tests).

In the SO population, the overall W. bancrofti mf GMI was 199 mf/ml, and this was significantly lower than the W. bancrofti mf GMI among both those who had M. perstans microfilaraemia (396 mf/ml) and those who had L. loa microfilaraemia (251 mf/ml) (t-test; p <0.001 for both the tests).

Cluster II: Co-infection among those positive for O. volvulus microfilaraemia

Interaction between O. volvulus microfilaraemia and other microfilarial species in the EO and SO populations was assessed. The overall prevalence of O. volvulus microfilaraemia was 42.9% (Table 4). In this cluster in the EO, the prevalence of O. volvulus microfilaraemia was comparable among those positive for M. perstans microfilaraemia (41.1%; χ2-test; p > 0.05), but significantly lower among those who had L. loa microfilaraemia and W. bancrofti microfilaraemia (17.9 and 2.4%, respectively; χ2-test; p < 0.01 for both tests). The overall O. volvulus mf GMI was 16 mf/skin snip, which was comparable with the O. volvulus mf GMI among those who had M. perstans microfilaraemia (15 mf/snip; t-test; p >0.05), but was significantly higher than O. volvulus mf GMI among individuals who had L. loa microfilaraemia (25 mf/snip; t-test; p <0.001).

In the SO population, the overall prevalence of O. volvulus microfilaraemia was 4.3%, and was comparable to the prevalence of O. volvulus microfilaraemia among those positive for M. perstans (3.8%) (χ2-test; p >0.05), but significantly lower among those (in the cluster) positive for W. bancrofti microfilaraemia (1.1%) (χ2-test; p <0.05). In this cluster, the overall mf GMI was 22 mf/ snip, which is comparable to O. volvulus mf GMI among individuals positive for M. perstans microfilaraemia (21 mf/snip; t-test; p >0.05).

Cluster III: Co-infections among those positive for M. perstans microfilaraemia

The overall prevalence of M. perstans microfilaraemia in the EO area was 14% (Table 5). This was comparable to the prevalence of M. perstans microfilaraemia among those who had O. volvulus microfilaraemia (13.1%), but considerably lower among those who had W. bancrofti microfilaraemia (7.3%) and L. loa microfilaraemia (3.6%). The overall M. perstans mf GMI was 105 mf/ml, which was significantly higher among those positive for W. bancrofti microfilaraemia (184 mf/ml) and O. volvulus microfilaraemia (132 mf/ml) (t-test; p < 0.05 for both the tests).

In the SO area, the overall mf prevalence of M. perstans was 12.7%, and was significantly lower than the prevalence of M. perstans microfilaraemia among those positive for L. loa microfilaraemia (24.3%; χ2-test; p <0.001); comparable to that of those positive for O. volvulus microfilaraemia (11.1%; χ2-test; p >0.05), but significantly higher than the prevalence of M. perstans microfilaraemia among those positive for W. bancrofti microfilaraemia (3.2%; χ2-test; p <0.01). The overall M. perstans mf GMI was 121 mf/ml, which was significantly higher than the M. perstans mf GMI of among those positive for W. bancrofti microfilaraemia (460 mf/ml), and that among those positive for O. volvulus microfilaraemia (192 mf/ml) (t-test; p <0.05 for both tests), but significantly lower than the M. perstans mf GMI among those positive for L. loa microfilaraemia (80 mf/ml) (t-test; p <0.05).

Cluster IV: Co-infection among those positive for L. loa microfilaraemia

The overall prevalence of L. loa in the EO area was 3.4% (Table 6), and this was lower than the prevalence of L. loa microfilaraemia among those positive W. bancrofti microfilaraemia (2.4%), O. volvulus microfilaraemia (1.4%) and M. perstans microfilaraemia (0.9%). These differences were not analyzed statistically due to low number of microfilaraemic individuals in these groups. The overall L. loa mf GMI was 231 mf/ml and was significantly lower than the L. loa mf GMI among those who had O. volvulus microfilaraemia (665 mf/ml; t-test; p< 0.001).

In the SO population, the overall prevalence of L. loa microfilaraemia was 13.5%. This was significantly lower than the prevalence of L. loa microfilaraemia among individuals positive for M. perstans microfilaraemia (26.3%; χ2-test; p <0.05), and but comparable to that of those positive for W. bancrofti microfilaraemia (18.3%) (χ2-test; p>0.05). There was no positive case of L. loa microfilaraemia among individuals positive for O. volvulus microfilaraemia in the sporadic onchocerciasis population. The overall L. loa mf GMI was 154 mf/ml, which was significantly lower than the L. loa mf GMI among those who had W. bancrofti microfilaraemia (183 mf/ml; t-test; p < 0.001), but higher than that among those positive for M. perstans microfilaraemia (114 mf/ml; t-test; p < 0.05).

Triple species co-infections

The triple filarial infections were not statistically analyzed because the number of cases in the sporadic onchocerciasis area was small.

DISCUSSION

Prevalence of single, double and triple species filarial infection

Wuchereria bancrofti, M. perstans, and L. Loa are endemic in all the parts of the Imo River Basin. Endemic onchocerciasis is restricted to the Upper Imo River Basin regarded as EO area in this study. The proportions of microfilaraemia of different categorizations in both the EO and SO populations were to large extent comparable and similar to reports of filarial species co-infections reported elsewhere19.

Of all the four filarial species examined in this study, O. volvulus and W. bancrofti exhibited greatest tendencies towards single species microfilaraemia in both the study populations, whereas M. perstans and L. loa showed greatest proclivity towards double and triple species microfilaraemia in both EO and SO areas. This is suggestive of relatively higher probability of concomitant infection involving M. perstans and L. loa than involving O. volvulus and W. bancrofti in either populations.

Possible case of antagonistic interaction

The difference between the overall prevalence of W. bancrofti and the prevalence of W. bancrofti microfilaraemia among those positive for O. volvulus was statistically significant in both the study populations. Similarly, the prevalence of O. volvulus microfilaraemia among those who were positive for W. bancrofti microfilaraemia was not only lower than the overall prevalence, but was also lowest of the prevalences among those positive for mf microfilaraemia of any other human filarial species in both the study populations. There is need for further studies to ascertain if there is possible heterologous antagonistic interaction between the two microfilarial species.

Multi-parasitism is a common feature in tropical countries19,20 and many examples of heterologous interaction among other parasitic infections have been reported. Studies on interactions between different filarial species and other parasite species in arthropod vectors have shown that secondary infection with B. pahangi microfilariae by intrathoracic inoculation reduced the development rate of a pre-existing Plasmodium gallinaceum infection, both in susceptible and refractory strains of Ae. aegypti21. Coinfections of helminth and P. falciparum infections have been shown to have clinical importance22. Interestingly, co-infection with Trypanosoma cruzi protects mice from early death and P. berghei co-infected mice survived longer, symptoms of cerebral malaria were absent, and breakdown of brain blood barrier was less pronounced in mice co-infected with T. cruzi23. The intensity of P. falciparum infection tends to be reduced in children with concomitant infection with measles or influenza, while infection with Bordetella pertussis was found not to influence the intensity of P. falciparum infection. It has been observed that W. bancrofti and P. falciparum influenced each other in a given host during concomitant infection19, and this was probably because of induction of inflammatory cytokines during both malaria infection24, and lymphatic filariasis infection25. Such cytokines may affect the course of both helminth and protozoa infections26. With concomitant microfilaraemia, P. falciparum followed a more benign course in monkeys27 and susceptibility to cerebral malaria was down-regulated in mice simultaneously infected with the filarial parasite Brugia pahangi28. It is evident from these studies that cytokine production induced by W. bancrofti may play an important role in its interaction with other parasites, such as O. volvulus.

Co-infection with combinations of helminths and malaria parasites are reportedly common. Such infections may have considerable health consequences, leading to more severe clinical symptoms and pathology than for infection with single parasite species29. They further concluded that interaction of malaria and helminth infections increases the severity of anaemia and organomegaly observed in schoolchildren and thus may potentially create a great challenge for disease control in the tropics. Some findings suggest that co-infection with multiple parasites could alter the immune responses30.

Some evidence from experimental models point to the possible existence of heterologous interaction. All who had triple species microfilaraemia in this study were >10 yr old, while most of the infected children had single species microfilarial infection. This is consistent with the observed age-related patterns of filarial infection with prevalence increasing with age. The period between first exposure and first appearance of microfilariae in the blood is between five and eight years for W. bancrofti31-34. The probability for multi-species filarial infection in children is therefore minimal.

Different stages of filarial worms stimulate different Th-cell subsets35. The microfilariae of O. volvulus and W. bancrofti have different surface characteristics, and it is known that microfilariae show higher levels of AChE activity than adult worms36,37. Since antigens of O. volvulus microfilariae can induce non-specific suppressor cells activity in vitro38, we can surmise that the larval stages could be the most important phase for study on any possible heterologous interaction between O. volvulus and W. bancrofti.

Cases of no definite interaction

Wuchereria bancrofti microfilaraemia was the most common among those who had L. loa microfilaraemia in both the EO and SO populations, but due to small sample sizes, definite conclusions could not be drawn on whether there exists any synergistic interaction between these two filarial species. However, there was a higher W. bancrofti microfilarial intensity among those with M. perstans microfilaraemia than among those positive for any of the other filarial species in both the study populations. Similarly, the intensity of M. perstans microfilaraemia among those positive for W. bancrofti exceeded the overall intensity of M. perstans in both the study populations at statistically significant levels. The existence of any synergistic interaction between W. bancrofti and M. perstans is a subject for further studies.

In both the study populations, the prevalence and intensity of O. volvulus microfilaraemia among those positive for M. perstans microfilaraemia were comparable to the overall levels of O. volvulus microfilaraemia. It is interesting to note that the prevalence of M. perstans microfilaraemia among those positive for O. volvulus was in the same range as the overall prevalence of M. perstans microfilaraemia in both the study populations. This may be indicative of a possible lack of interaction between O. volvulus and M. perstans. This requires verification using immunological methods.

The prevalence of M. perstans microfilaraemia among the cluster positive for L. loa was significantly lower than the overall prevalence of M. perstans microfilaraemia in the onchocerciasis endemic population, but significantly higher than the overall prevalence of M. perstans microfilaraemia in the sporadic onchocerciasis population. Loa loa and M. perstans are the most widespread and ubiquitous of all the human filarial species in Nigeria, most times occurring in the same areas. It can be inferred that perhaps, negative interaction does not exist between the two species. The common concomitant infections between the two species could partly be attributed to their transmission ecologies which are sympatric. The vectors of both the infections bite mostly outdoors in the forest. However, the pertinent questions, which constitute the kernel of a planned future study are: why are concomitant infections between the two species significantly low in EO area but significantly higher in SO area? Does onchocerciasis endemicity antagonize concomitant infection involving L. loa and M. perstans? As of now, what is known is that antigens of O. volvulus microfilariae can induce non-specific suppressor cells activity in vitro38.

The overall prevalence of L. loa microfilaraemia was significantly lower than the prevalence of L. loa among those positive for W. bancrofti microfilaraemia in the EO population, but in the SO population, both the prevalences were comparable. But what could explain these? There are many questions than there are answers.

The pertinent question to be asked is "Since co-infection with multiple strains may reduce the total amount of parasite ('parasitic load') in an infected person, will parasitic load increase if a person is infected with only two strains instead of all four?"39. It might be necessary to add: was mf intensity observed to be significantly reduced in this study? The answer is that there was no evidence of a definite pattern of mf density established in this study whether in the EO or SO populations.

In conclusion, we can learn from concomitant infections involving non-filarial patients, where a different thread of observations has been reported. We can draw inference from the fact that in most regions where onchocerciasis is endemic, there could be "varied outcomes of onchocerciasis infection attributable to positive or negative regulatory effects of other pathogens harboured by the patients"23. Further studies are underway to unravel patterns of relationships between these human filarial infections in onchocerciasis endemic and sporadic populations.

ACKNOWLEDGEMENTS

We appreciate our field assistants Emma Nwaimo, Esther, Comfort Nwankwoala, Uzoma Christopher, Bentina David, and Bassey Cobham, as well as the entire staff of the Ministry of Health, Okigwe and Emohua LGAs for their assistance in village mobilization and sample collections.

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Author affiliation:

Emmanuel Uttah1 & Dominic C. Ibeh2

1 Department of Biological Sciences, Cross River University of Technology, Calabar; 2 Landmarks Hospital, Aba, Abia State, Nigeria

Author affiliation:

Correspondence to: Dr Emmanuel C. Uttah, Department of Biological Sciences, Faculty of Science, Cross River University of Technology, Calabar, Nigeria.

E-mail: drecuttah@yahoo.com

Received: 20 August 2011 Accepted: 25 November 2011