Microscopic detection of active phase of lymphatic filariasis is indicated by the presence of microfilaria in whole blood. This method is not sensitive and requires relatively large amount of blood sample. PCR allows very sensitive detection of the parasite DNA using a smaller amount of blood; and the use of dried blood spots facilitates sample transportation. Nevertheless, limited studies have been reported on PCR using dried blood spot for detection of Brugia malayi. In this study, we investigated the effects of concentrating whole blood genomic DNA sample and the amplification methods [conventional PCR (C-PCR) and real-time PCR] on the detection of B. malayi DNA from dried blood spots from a very low endemic area in Malaysia. Both C-PCR and real-time PCR detected 2 out of 18 (11%) samples as positive from non-concentrated genomic DNA preparations. After the DNA samples were pooled and concentrated, both C-PCR and realtime PCR detected B. malayi DNA amplifications in 7 out of 18 (39%) samples. However one sample which showed faint band in C-PCR was detected as highly positive in real-time PCR. In conclusion, both C-PCR and real-time PCR using dried blood spots from a low endemic area demonstrated equal sensitivity for detection of B. malayi DNA.
In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.