Elsevier

Acta Tropica

Volume 89, Issue 1, December 2003, Pages 25-32
Acta Tropica

Sensitive and specific serodiagnosis of river blindness using Onchocerca ochengi antigens

https://doi.org/10.1016/j.actatropica.2003.08.003Get rights and content

Abstract

Despite the large-scale application of ivermectin for the treatment of human onchocerciasis, this filarial disease has remained a public health hazard, requiring rapid and sensitive diagnostic methods for more efficient management. Several serological methods based on homologous native or recombinant diagnostic antigens have been developed. However, these antigens have remained scarce in the endemic areas. We report herein the preparation and evaluation of an Onchocerca ochengi total worm extract together with its low molecular weight (<31 kDa) and 14.4 kDa antigens for the diagnosis of human onchocerciasis. The 14.4 kDa antigen and the low molecular weight antigen fraction were identified by Western blot experiments. The antigens were employed for the detection of IgG4 in sera of 43 onchocerciasis patients, 35 normal Africans, 15 schistosomiasis patients, 11 ascariasis patients, 9 loaisis patients and 3 healthy Europeans. The diagnostic specificity values were 94.5, 87.5 and 100%, while the sensitivities were 90.7, 80 and 76.6% using the worm extract, low molecular weight antigen fraction and the 14.4 kDa antigen, respectively. We conclude that total worm extracts of O. ochengi can conveniently replace recombinant antigen cocktails in poor settings that do not have access to the latter.

Introduction

Onchocerca volvulus, the etiologic agent of onchocerciasis or river blindness infects an estimated 17 million people in endemic countries of tropical Africa, Latin America and the Yemen, with over 85 million persons at risk of infection (WHO, 1995). The disease is highly debilitating, with dermatitis, lymphadenitis, sub-cutaneous nodules and blindness being its main clinical manifestations. It is estimated that over 300,000 people are blind due to the disease (WHO, 1995).

Despite the large-scale application of ivermectin for the treatment of this filarial disease, it has remained a public health hazard, requiring rapid and sensitive diagnostic methods for more efficient management. The standard parasitological test, which is based on the detection of microfilaria in bloodless skin snips, suffers from low sensitivity and is highly invasive (Weiss and Karam, 1989). Several immunodiagnostic tests have been attempted in a bid to circumvent these problems with the encouraging ones relying on the use of homologous native or recombinant antigens (Bradley et al., 1993, Bradley and Unnasch, 1996, Harnett et al., 1998, Nde et al., 2002). Unfortunately, the latter are expensive and difficult to obtain by many laboratories in the poor endemic areas. The paucity of homologous native antigens is due mainly to the fact that O. volvulus worms are not easily obtainable from patients, and are difficult to propagate in the laboratory. A number of tests employing diagnostic antigens from related or heterologous parasites which can easily be obtained or maintained in the laboratory including Litosomoides sigmodontis (Klenk et al., 1984), Brugia malayi (Lujan et al., 1984), O. gibsoni (Cabrera and Parkhouse, 1986, Cabrera and Parkhouse, 1987) have therefore been attempted. The current opinion is that Onchocerca ochengi from cattle is the closest relative of O. volvulus (Hagen et al., 1995, Trees et al., 2000), and that in onchocerciasis, there is up-regulation and high specificity of the IgG4 sub-class (Lal and Ottesen, 1988, Cabrera et al., 1989, Gbakima, 1994). Increased specificity in IgG4 detection was observed even when whole worm preparation was used (Gbakima, 1994). In addition, antibodies to phosphorylcholine and carbohydrate determinants responsible for much of the cross-reactivity among nematodes are sub-class restricted in humans and absent in the IgG4 sub-class (Lal and Ottesen, 1988, Weil et al., 1990). Recent findings indicate that IgG4 detection appears to correlate well with active filariasis, with titres that decline after treatment with antifilarial drugs such as ivermectin and diethylcarbamazine (Mahanty et al., 1994, Atmadja et al., 1995). A number of studies have also shown that low molecular weight antigens and antigen fractions of Onchocerca are more species specific than the high molecular weight ones (Cabrera and Parkhouse, 1986, Cabrera and Parkhouse, 1987, Weiss and Karam, 1989). Thus, we have evaluated antigens from the readily available bovine O. ochengi as cheaper reagents in IgG4-capture diagnosis of human onchocerciasis.

Section snippets

Study subjects

After informed consent, subjects in onchocerciasis endemic areas of Mbonge and Tubah sub-divisions (Cameroon) were physically examined for onchocercal lesions and skin snips were taken from them and observed for microfilariae as previously described (Titanji et al., 1985). Blood films were also prepared for diagnosis of blood-dwelling helminth infections. Microfilaria positive patients (43) were subsequently retained for the study. Normal African controls comprised of 35 subjects from

Clinical and demographic profiles of study subjects

All onchocerciasis patients (n=43) were mf positive and the mean density of mf was 47.28±76.7 mf per snip. In this group, 4.7% of the subjects were positive for Loa loa, and 34.9% were females (Table 1). Control subjects (n=73) presented no evidence of onchocerciasis and some were tested positive for other parasitic infections (Table 1).

Diagnostic ELISA based on total extract of O. ochengi (OOTE)

The optimised assay was performed with plates coated at antigen concentration of 2 μg/ml and antisera (from 43 onchocerciasis patients, 35 normal Africans, 15

Discussion

In the present study, we have exploited previous findings pointing to high specificity of IgG4 in onchocerciasis, high sensitivity of parasite total extract, and the high cross-reactivity between O. ochengi and O. volvulus antigens in developing an affordable and a reliable immunoassay for river blindness. The data obtained for a 14.4 kDa antigen, and for low molecular weight antigens (<31 kDa) of the same parasite (both identified by Western blotting) have also been presented. The optimised

Acknowledgements

We are grateful to Dr. Ndamukong Kenneth and Ms Ayuk Mary of the Department of Life Sciences, University of Buea for providing schistosomiasis antisera. The work received financial support from the International Science Program, Sweden (PROJECT CAM 01) and from the European Union (INCO-DC Programme).

References (28)

  • A.A. Gbakima

    IgG4 subclass serology for diagnosis of onchocerciasis in Gabonese pediatric populations with multiple filarial infections

    Clin. Exp. Immunol.

    (1994)
  • H.E. Hagen et al.

    A simple field method for the purification of Onchocerca ochengi microfilariae from a mixed Onchocerca infection in cattle

    Trop. Med. Parasitol.

    (1995)
  • W. Harnett et al.

    Molecular and immunodiagnosis of human filarial nematode infections

    Parasitology

    (1998)
  • A. Klenk et al.

    Serodiagnosis of human onchocerciasis: evaluation of sensitivity and specificity of a purified Litomosoides carinii adult worm extract

    Tropenmedzin und Parasitologie

    (1984)
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