Detection of Loa loa-specific DNA in blood from occult-infected individuals

Accurate and specific diagnosis of human loiasis is of crucial importance in an endemic area where two-thirds of infected individuals are without circulating microfilariae (occult loiasis). By using the polymerase chain reaction (PCR) and specific primers to the repeat 3 region (15r3) of the gene coding for Loa loa 15-kDa polyprotein antigen, DNA was amplified from total blood lysate of occult-infected subjects. A 396-bp DNA fragment was specifically detected. We tested the specificity of this method by qualitative hybridization to PCR products using blood lysates of the following subjects: (1) from Gabon (80 individuals residing in L. loa endemic area where loiasis exists sympatrically with Mansonella perstans); (2) from Togo (12 individuals infected with Onchocerca volvulus and M. perstans); (3) from Tahiti (12 individuals infected with Wuchereria bancrofti); and (4) from Mali (12 individuals infected with O. volvulus and M. perstans). Samples from Gabon included 60 L. loa amicrofilaremics and 20 L. loa occult-infected subjects. Qualitative hybridization carried out at 50 degrees C on PCR products, using a 15r3-specific oligonucleotide probe, revealed hybridization with L. loa-infected samples from Gabon and four samples from Togo after 2 days exposure to the film. The positive samples from Togo were characterized by the use of nested PCR. Three nested PCR products have been sequenced. No differences were observed between the three sequences and they are 99.72% identical to L. loa 15r3. None of bancroftian-infected individuals from Tahiti, nor O. volvulus- and M. perstans-infected individuals from Mali reacted after 1 week's exposure (overexposure) to the film. This allows us to conclude first that our 15r3 PCR assay is specific for L. loa and secondly that L. loa infections occur in Togo. The sensitivity of this 15r3 PCR assay was further investigated with occult patients and field-collected amicrofilaremic samples. We found that 19 of the 20 occult-infected individuals were positive on Southern hybridization, whereas 35/60 amicrofilaremics were positive. These results have shown that the sensitivity of this assay in detecting unequivocal, parasitologically proven occult loiasis was 95%, while the specificity with regard to the sympatric M. perstans was 100%.