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Human occult loaiosis: improvement in diagnostic sensitivity by using nested polymerase chain reaction
Oral Sessions I Parasitology International 47 (Suppl.) (1998) 133-281
232
( )-O-l2 1
O-0423 DETECTION OF Wuchereria bancrofi IN BLOOD SAMPLES FROM SORSOGON PROVINCE, THE PHlLJPPrNES
Hafalla JCR, Ram& BL, Gabriel I, Salazar F, Alamares IA. Pasay MCJ and Torres EP Molecular Biology Laboratory and Department of Parasitology and Medical Entomology, Research institute for Tropical Medicine, Muntinlupa City, Philippines The prevalence of Wuchereria bancmjii in Sorsogon province, a low-filariasis endemic region in the Philippines, was assessed using three methods for detecting microfdariae in human blood. Among 54 randomly collected blood samples, 2OuL ofwhole blood WBSsubjected to Giemsa thick smear, I mL whole blood to Nucleopore membrane filtration and 2OOuL blood clots for SspI PCR assay*. Using these three different methods, different prevalence estimates emerged: 5.6% (3/54), 7.4% (4154) and 13.0% (g/54), respectively. Combmed Giemsa and Nucleopore techniques yielded an estimated prevalence of 7.4% (4/54). The Sspf PCR assay detected microfilariae in all Giemsa-smear positive and Nncleopora-positive samples, with a limit of detection at 5 mf/mL of blood. These data show that blood-clot Sspi PCR assay has higher sensitivity for microfilariae detection than the other two methods combined Since tilaria case-finding in the Philippines is currently based on the latter methods, the prevalence of Wbancrojii in this country may be underestlmated * Zhonp el al ( 1996) AINJ Trop hfed Hyg 54.357-63
O-0422 OCCULT LOAIOSIS, IMPROVEMENT IN DIAGNOSTIC BY USING NESTED F’OLYhiERASE CHAIN REACTION F. S. Touti’, L. Kassambara’, T. Williams’, P. Millet’, 0. Bain-, A. J. Georges’, T. G. Egwang’. ‘Centre International de Recherches M6dicales de Franceville (CIRMF) BP769 Fanceville, Gabon. * Dbpartement GEpidBmiologie et des A&ctions Parasitaires, Cole Nationale de MQdecine et de Pharmacie BP. 1805 Bamaka, Mali,** Lsboratoire de bialogie Parasitaire Mu&um National d’Histoire NaturelIe, 61 rue Buffon 75231 Cedex 05 Paris France. The development of control strategies for loaiosis is of crucial importance in endemic areas and depends heavily on the accurate identi6cation of occult-infected individuals. Polymerase chain reaction @‘CR) and nested-PCR targeted on the repeat 3 region of the gene encoding a Loa loa 15 kDa polypratein were developed. The assays were performed on 20 blood samples from occult-infected subjects (OL), and 30 from field individuals (AMF). The size of the initial collected amicro6laremic PCR product is 366 basepairs. When this initial ampl&cation was carried out for 30 cycles, 3 out of the 20 OL and S/30 AMF were visualized by ethidium bromide staining. Subsequent Southern blotting and hybridization with the specific probe (15r3 probe) revealed hybridization in 19M0 OL and in 23/30 AMF samples but only after two days of exposure to the film. When nested PCR was carried out (product size 366 bp) the 19120 OL and 23130 AMF samples positive by Southern hybridization from the initial PCR products were also strongly positive by ethidium bromide staining. Qualitative Southern blotting on the nested PCR products using the same 15r3 probe, con&med the ethidium bromide staining results after a very short exposure time of 1 hours. These results demonstrate that the nested PCR ampl&cation product (a region of 366 base pairs of Loa km DNA) is specific and that the sensitivity in detecting occult loaioeis ia 95%. Thu approach has significant potential for improving the screening of large human populations for active loaios~s without the requirement for blotting and hybridization of the PCR products.
IfUh4AN
SENSITIVITY
PARASITOLOGY GNATHOSTOMOSIS hl Llaum~‘.
AND INMUNOLOGY
OF
R Lao’
*Research Center of Parawe an timgus diseases The Gnathostomosis it’s known srnce 1979
with epidemical characteristics in Ecuador
A( first it was described by distinguished dermatolo& as a traveller, L hWJal , and with the clintcal staff of Modular Shifting Paruculitis, Eosinofilic W Ollayue We have in our consulring room patient with the dental injury charactenstic of the clinical deep this cases we have 10 resalt one of a young man G Gnathostomosis in the left thigh We associate thts case with his mother who prooess. with a wide diferential diagnostic wIthout
shape of gnathostomoris In L 0 with the climcal staff of presents a pleuropulmow an ethiologcal detinmon
Both have ingest a fish cocktail twenty days before The mmunological study by intradertnoreaction antigen of Gnathostomosis manufactered by T Mmon.Japan. was positwe 10125 and 10145 respectively. with an eosinotilic of 27% and 32% verify the Gnathostomosls in both cases Another case -For a diagnonic research we received an speamen extract of the front camera of the left eye by ecuadonan oRalmologist. previously a surgtcal act. the patiern R C A of twenty years old of a rural population with three months of evolution We realized the micmmorfologrcal description of a Nematodes larva and with the characteristzcr that we saw we described as a third stage larvae of Gnathostoma spinlgerurn This observation was the 1% report 1” Ecuador of Ocular Gnahostomosts
232
( )-O-l2 1
O-0423 DETECTION OF Wuchereria bancrofi IN BLOOD SAMPLES FROM SORSOGON PROVINCE, THE PHlLJPPrNES
Hafalla JCR, Ram& BL, Gabriel I, Salazar F, Alamares IA. Pasay MCJ and Torres EP Molecular Biology Laboratory and Department of Parasitology and Medical Entomology, Research institute for Tropical Medicine, Muntinlupa City, Philippines The prevalence of Wuchereria bancmjii in Sorsogon province, a low-filariasis endemic region in the Philippines, was assessed using three methods for detecting microfdariae in human blood. Among 54 randomly collected blood samples, 2OuL ofwhole blood WBSsubjected to Giemsa thick smear, I mL whole blood to Nucleopore membrane filtration and 2OOuL blood clots for SspI PCR assay*. Using these three different methods, different prevalence estimates emerged: 5.6% (3/54), 7.4% (4154) and 13.0% (g/54), respectively. Combmed Giemsa and Nucleopore techniques yielded an estimated prevalence of 7.4% (4/54). The Sspf PCR assay detected microfilariae in all Giemsa-smear positive and Nncleopora-positive samples, with a limit of detection at 5 mf/mL of blood. These data show that blood-clot Sspi PCR assay has higher sensitivity for microfilariae detection than the other two methods combined Since tilaria case-finding in the Philippines is currently based on the latter methods, the prevalence of Wbancrojii in this country may be underestlmated * Zhonp el al ( 1996) AINJ Trop hfed Hyg 54.357-63
O-0422 OCCULT LOAIOSIS, IMPROVEMENT IN DIAGNOSTIC BY USING NESTED F’OLYhiERASE CHAIN REACTION F. S. Touti’, L. Kassambara’, T. Williams’, P. Millet’, 0. Bain-, A. J. Georges’, T. G. Egwang’. ‘Centre International de Recherches M6dicales de Franceville (CIRMF) BP769 Fanceville, Gabon. * Dbpartement GEpidBmiologie et des A&ctions Parasitaires, Cole Nationale de MQdecine et de Pharmacie BP. 1805 Bamaka, Mali,** Lsboratoire de bialogie Parasitaire Mu&um National d’Histoire NaturelIe, 61 rue Buffon 75231 Cedex 05 Paris France. The development of control strategies for loaiosis is of crucial importance in endemic areas and depends heavily on the accurate identi6cation of occult-infected individuals. Polymerase chain reaction @‘CR) and nested-PCR targeted on the repeat 3 region of the gene encoding a Loa loa 15 kDa polypratein were developed. The assays were performed on 20 blood samples from occult-infected subjects (OL), and 30 from field individuals (AMF). The size of the initial collected amicro6laremic PCR product is 366 basepairs. When this initial ampl&cation was carried out for 30 cycles, 3 out of the 20 OL and S/30 AMF were visualized by ethidium bromide staining. Subsequent Southern blotting and hybridization with the specific probe (15r3 probe) revealed hybridization in 19M0 OL and in 23/30 AMF samples but only after two days of exposure to the film. When nested PCR was carried out (product size 366 bp) the 19120 OL and 23130 AMF samples positive by Southern hybridization from the initial PCR products were also strongly positive by ethidium bromide staining. Qualitative Southern blotting on the nested PCR products using the same 15r3 probe, con&med the ethidium bromide staining results after a very short exposure time of 1 hours. These results demonstrate that the nested PCR ampl&cation product (a region of 366 base pairs of Loa km DNA) is specific and that the sensitivity in detecting occult loaioeis ia 95%. Thu approach has significant potential for improving the screening of large human populations for active loaios~s without the requirement for blotting and hybridization of the PCR products.
IfUh4AN
SENSITIVITY
PARASITOLOGY GNATHOSTOMOSIS hl Llaum~‘.
AND INMUNOLOGY
OF
R Lao’
*Research Center of Parawe an timgus diseases The Gnathostomosis it’s known srnce 1979
with epidemical characteristics in Ecuador
A( first it was described by distinguished dermatolo& as a traveller, L hWJal , and with the clintcal staff of Modular Shifting Paruculitis, Eosinofilic W Ollayue We have in our consulring room patient with the dental injury charactenstic of the clinical deep this cases we have 10 resalt one of a young man G Gnathostomosis in the left thigh We associate thts case with his mother who prooess. with a wide diferential diagnostic wIthout
shape of gnathostomoris In L 0 with the climcal staff of presents a pleuropulmow an ethiologcal detinmon
Both have ingest a fish cocktail twenty days before The mmunological study by intradertnoreaction antigen of Gnathostomosis manufactered by T Mmon.Japan. was positwe 10125 and 10145 respectively. with an eosinotilic of 27% and 32% verify the Gnathostomosls in both cases Another case -For a diagnonic research we received an speamen extract of the front camera of the left eye by ecuadonan oRalmologist. previously a surgtcal act. the patiern R C A of twenty years old of a rural population with three months of evolution We realized the micmmorfologrcal description of a Nematodes larva and with the characteristzcr that we saw we described as a third stage larvae of Gnathostoma spinlgerurn This observation was the 1% report 1” Ecuador of Ocular Gnahostomosts