Use of affordable technology for the sensitive and specific diagnosis of onchocerciasis (river blindness)

Sammanfattning: Onchocerciasis or river blindness is the most common infectious cause of blindness. Even in the absence of blindness, the skin effects caused are of social stigma. It is the goal of the World Health Organization to eradicate filariasis (including onchocerciasis). Eradication of onchocerciasis is to be undertaken by mass treatment with tile microfilaricide ivermectin, combined with vector control. To date, the gold standard for diagnosis of onchocerciasis is through the parasitological identification of the organism, Onchocerca volvulus. As ivermectin treatment provokes a decrease in the microfilarial load of the individuals, the sensitivity of the assay for detection would need to be enhanced. Before and even after eradication is achieved, sensitive, specific, quick and affordable methods will be necessary for adequate diagnostics and epidemiological assessment. A number of recombinant proteins have been identified for use in diagnosis but the results have been disappointing. Using cocktails of recombinant proteins somewhat improved the sensitivity. However, production of recombinant material has proved to be a problem and costs have not even been discussed. Since onchocerciasis is a disease endemic in less welloff countries, the cost of reliable diagnostic methods is not a trivial matter. Therefore, there is definitely a need to re- address this problem of developing improved methods for monitoring the eradication programme in the context of sensitivity, specificity, availability and affordability. Using manual ion exchange chromatography, we have isolated a fraction of low molecular weight proteins of 0. volvulus (designated PakF), derived from adult female worms. This fraction, used in a Dot Blot Assay (DBA , ), proved to be highly sensitive and specific for the diagnosis of onchocerciasis in different geographic regions. We have tested the PakF-DBA in Guatemala (where no other filaria has been described), in Ghana (where other filariases are prevalent) and in an infectious Disease referral center in Sweden (a country not endemic for filaria). Additionally, we have tested for specificity using well-defined filarial sera from areas non-endemic for onchocerciasis. When used to analyze samples from different endemic scenarios, the PakF-DBA reached high levels of sensitivity (96%) and specificity (98%). Compared to methods that use recombinant material, the PakF-DBA offered similar or even better sensitivity and specificity values, but without the high costs involved in the production of recombinant proteins. The PakF-DBA was worked out so it could provide specific and sensitive results and yet provide an affordable alternative to the use of more expensive or sophisticated diagnostic methods. In this regard, the method was simplified to use eluted finger pricked blood instead of serum as the source of antibodies to the PakF mixture. Using this technique, a positive correlation was found between the results obtained with the "gold-standard" skin snip test and the PakF-DBA, suggesting that the PakFDBA could substitute, or at least provide a more sensitive tool when the parasitological examination is not possible, due to the reluctance of the individuals to undergo this procedure, or the lack of qualified personnel that performs the skin snip. A closer look at the biochemical properties of this mixture of native proteins revealed that PakF is stable at room temperature, which in some endemic settings could reach 30-35 C. IgG4 antibody response from infected individuals is infrequent and did not improve upon sensitivity, which adds an attractive feature in terms of costs, as anti-IgG4 reagents tend to be expensive. Its stability at relatively high working temperatures makes PakF attractive under field conditions. In addition, heat inactivation of sera or eluted blood to reduce the risk of labile blood borne viruses does not affect the response to PakF. The relatively simple production of PakF, together with its stability, makes the PakF-DBA a serious contender in the quest for a reliable, yet affordable method for the diagnosis of onchocerciasis in areas where costs arc an important issue arid the disease threatens with recrudescence, even after years of control. The results of this work speak for the potential of this protein mixture, PakF, as a contending diagnostic tool that, when put into the right format, could be a valuable tool for the monitoring of onchocerciasis control measures.