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Simple Sensitive Diagnosis and Genome Analysis of Wuchereria bancrofti microfilariae and Wolbachia endosymbiont Archived from Membrane Filters by Nested PCR
13th International Congress on Infectious Diseases Abstracts, Poster Presentations
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Seroprevalence of Toxoplasma gondi Antibodies in HIV/Aids Patients and Healthy Blood Donors in Port Moresby General Hospital, Papua New Guinea
Simple Sensitive Diagnosis and Genome Analysis of Wuchereria bancrofti microfilariae and Wolbachia endosymbiont Archived from Membrane Filters by Nested PCR
L. John 1,∗ , I.H. Kevau 2 , J. McBride 3 , K. Wilson 4 , J. Milan 5 , G. Tau 1 1 Port Moresby General Hospital, Port Moresby, Papua New Guinea 2 UPNG School of Medicine and Health Science, Port Moresby, Papua New Guinea 3 Cairns Base Hospital, Cairns, Australia 4 National Reference Serology Laboratory, Melbourne, Australia 5 National HIV/AIDS Council Secretariat, Port Moresby, Papua New Guinea
A seroepidemiological survey was conducted at Port Moresby General Hospital (PMGH) over a 24 month period (1st March 2003—31st March 2005). The aim of the study was to determine seroprevalance of toxoplasma gondi in HIV infected patients and seronegative healthy blood donors. The population was unmatched but complete data was obtained for some important sociodemographic characteristics. These were compared to see if there were any risk factors for acquisition of toxoplasmosis seropositivity. Blood samples of 181 consecutive HIV infected patients and 120 consecutive healthy blood donors were tested for toxoplasma IgG and IgM antibodies using the enzyme - linked immunoassay (ELISA) technique. The study showed that 59.7% of the HIV infected population and 40.1% of the Healthy Blood donors were positive for toxoplasma antibodies, giving the overall prevalence of 52.2%, for the study population. The two important independent risk factors that showed significant correlations with toxoplasma antibody positivity were regional grouping (Highlands origin) and exposure to cats. This study revealed that toxoplasma antibody is prevalent in the community and would be a major health problem amongst the immuno-compromised population, notably the HIV infected patients. Toxoplasma infection is anticipated to increase with the current HIV/AIDS epidemic in PNG. The Health Authorities need to be made aware of this problem, now, so they can formulate preventive and management policies for PNG, for the future. doi:10.1016/j.ijid.2008.05.996
A. Bhumiratana 1,∗ , P. Wiboonjak 1 , C. Sangkamanee 2 , S. Koyadun 2 , P. Yongyuth 3 , P. Mahannop 1 1
Department of Parasitology, Faculty of Public Health, Mahidol University, Bangkok, Thailand 2 Office of Disease Prevention and Control 11 (Nakhon Si Thammarat), Department of Disease Control, Ministry of Public Health, Nakhon Si Thammarat, Thailand 3 Thapput Hospital, Ministry of Public Health, Phangnga, Thailand Background: Membrane filtration technique (MFT) has been used for diagnosis of Wuchereria bancrofti in microfilaremic (Mf) patients either untreated or treated with diethylcarbamazine (DEC) or in a combination of albendazole (ABZ) or ivermectin, as parts of the Global Programme to Eliminate Lymphatic Filariasis (GELF). The MFT-based genomic DNA recovery of W. bancrofti Mf and Wolbachia endosymbiont has however never been developed for diagnostic purpose and genome analysis. Methods: Mf blood (1 to 3 ml) of individual patients, collected at time close to peak hour (0100 h) before and after DEC + ABZ treatment, was filtered using polycarbonate membrane, 5 um pore size and 25 um diameter. The filters retaining Mf were used for gDNA extraction or stored in 0.9% normal saline solution for months at cold temperature until use. Results: Parasite gDNAs archived from the membranes were efficiently amplified using primers specific for W. bancrofti beta-tubulin gene isotype 1 and of Wolbachia ftsZ gene by nested PCR. Also, all the samples can be amplified using primers specific for human beta-tubulin gene homologue (210 bp amplicon) for internal control. Amplification of W. bancrofti gave amplicon sizes 607 bp of the first reaction and 141 bp (exon 4) and 174 bp (exon 5) of the separate second reactions, as the Wolbachia 681 bp and 300 bp, respectively. Conclusion: Amplicons of W. bancrofti and Wolbachiaspecific nested PCR using filter-archived parasite gDNAs can be inserted into cloning plasmid and subsequently sequenced towards the corresponding exons that are associated with benzimidazole resistance involving amino acid substitutions. This will be beneficial for the GELF at country level to evaluate and monitor drug resistance of W. bancrofti populations in target areas and, in particular, it will help to standardize laboratory investigation performance in cost-effective manner in the endemic countries as the GELF partners. doi:10.1016/j.ijid.2008.05.997
e377
65.008
65.009
Seroprevalence of Toxoplasma gondi Antibodies in HIV/Aids Patients and Healthy Blood Donors in Port Moresby General Hospital, Papua New Guinea
Simple Sensitive Diagnosis and Genome Analysis of Wuchereria bancrofti microfilariae and Wolbachia endosymbiont Archived from Membrane Filters by Nested PCR
L. John 1,∗ , I.H. Kevau 2 , J. McBride 3 , K. Wilson 4 , J. Milan 5 , G. Tau 1 1 Port Moresby General Hospital, Port Moresby, Papua New Guinea 2 UPNG School of Medicine and Health Science, Port Moresby, Papua New Guinea 3 Cairns Base Hospital, Cairns, Australia 4 National Reference Serology Laboratory, Melbourne, Australia 5 National HIV/AIDS Council Secretariat, Port Moresby, Papua New Guinea
A seroepidemiological survey was conducted at Port Moresby General Hospital (PMGH) over a 24 month period (1st March 2003—31st March 2005). The aim of the study was to determine seroprevalance of toxoplasma gondi in HIV infected patients and seronegative healthy blood donors. The population was unmatched but complete data was obtained for some important sociodemographic characteristics. These were compared to see if there were any risk factors for acquisition of toxoplasmosis seropositivity. Blood samples of 181 consecutive HIV infected patients and 120 consecutive healthy blood donors were tested for toxoplasma IgG and IgM antibodies using the enzyme - linked immunoassay (ELISA) technique. The study showed that 59.7% of the HIV infected population and 40.1% of the Healthy Blood donors were positive for toxoplasma antibodies, giving the overall prevalence of 52.2%, for the study population. The two important independent risk factors that showed significant correlations with toxoplasma antibody positivity were regional grouping (Highlands origin) and exposure to cats. This study revealed that toxoplasma antibody is prevalent in the community and would be a major health problem amongst the immuno-compromised population, notably the HIV infected patients. Toxoplasma infection is anticipated to increase with the current HIV/AIDS epidemic in PNG. The Health Authorities need to be made aware of this problem, now, so they can formulate preventive and management policies for PNG, for the future. doi:10.1016/j.ijid.2008.05.996
A. Bhumiratana 1,∗ , P. Wiboonjak 1 , C. Sangkamanee 2 , S. Koyadun 2 , P. Yongyuth 3 , P. Mahannop 1 1
Department of Parasitology, Faculty of Public Health, Mahidol University, Bangkok, Thailand 2 Office of Disease Prevention and Control 11 (Nakhon Si Thammarat), Department of Disease Control, Ministry of Public Health, Nakhon Si Thammarat, Thailand 3 Thapput Hospital, Ministry of Public Health, Phangnga, Thailand Background: Membrane filtration technique (MFT) has been used for diagnosis of Wuchereria bancrofti in microfilaremic (Mf) patients either untreated or treated with diethylcarbamazine (DEC) or in a combination of albendazole (ABZ) or ivermectin, as parts of the Global Programme to Eliminate Lymphatic Filariasis (GELF). The MFT-based genomic DNA recovery of W. bancrofti Mf and Wolbachia endosymbiont has however never been developed for diagnostic purpose and genome analysis. Methods: Mf blood (1 to 3 ml) of individual patients, collected at time close to peak hour (0100 h) before and after DEC + ABZ treatment, was filtered using polycarbonate membrane, 5 um pore size and 25 um diameter. The filters retaining Mf were used for gDNA extraction or stored in 0.9% normal saline solution for months at cold temperature until use. Results: Parasite gDNAs archived from the membranes were efficiently amplified using primers specific for W. bancrofti beta-tubulin gene isotype 1 and of Wolbachia ftsZ gene by nested PCR. Also, all the samples can be amplified using primers specific for human beta-tubulin gene homologue (210 bp amplicon) for internal control. Amplification of W. bancrofti gave amplicon sizes 607 bp of the first reaction and 141 bp (exon 4) and 174 bp (exon 5) of the separate second reactions, as the Wolbachia 681 bp and 300 bp, respectively. Conclusion: Amplicons of W. bancrofti and Wolbachiaspecific nested PCR using filter-archived parasite gDNAs can be inserted into cloning plasmid and subsequently sequenced towards the corresponding exons that are associated with benzimidazole resistance involving amino acid substitutions. This will be beneficial for the GELF at country level to evaluate and monitor drug resistance of W. bancrofti populations in target areas and, in particular, it will help to standardize laboratory investigation performance in cost-effective manner in the endemic countries as the GELF partners. doi:10.1016/j.ijid.2008.05.997