Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films
Autor(a) principal: | |
---|---|
Data de Publicação: | 2016 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Digital do Instituto Evandro Chagas (Patuá) |
Texto Completo: | https://patua.iec.gov.br/handle/iec/3008 |
Resumo: | The study aimed to evaluate a protocol of nested PCR using archival Giemsa-stained thick blood smears (GTS)as source of Plasmodium DNA. A total of 138 GTS from patients of five municipalities from Pará State (Amazon Region, Brazil) was included in this survey. These samples were classified in three groups (group 1: 85 Plasmodium positive and negative GTS stored in plastic box during five years; group 2: 28 Plasmodium positive and negative GTS stored in wooden box during 10 years; and group 3: 25 Trypanosoma cruzi GTS negative for Plasmodium stored in plastic box during a month) and were submitted to DNA extraction with Chelex-100. Subsequently, extracted DNA samples were quantified and the integrity was verified by electrophoresis. Nested PCR protocol was performed to detect Plasmodium species. The results of nested PCR were compared to microscopy and statistic parameters were calculated by screening test. DNA samples from all groups had acceptable quantity and purity level, but the evaluation of integrity showed 19 degraded samples from group 2. By nested PCR, this group showed very low sensitivity (29.63%) and accuracy (32.14%), while nested PCR for samples from group 1 showed 100% of sensitivity and 97.65% of accuracy. The results of this research showed that samples stored until five years can be useful as Plasmodium DNA source for nested PCR to identify Plasmodium species, being an important alternative to support retrospective studies. |
id |
IEC-2_7b47d00b61bc7926b9ffe0dfc8acdc5d |
---|---|
oai_identifier_str |
oai:patua.iec.gov.br:iec/3008 |
network_acronym_str |
IEC-2 |
network_name_str |
Repositório Digital do Instituto Evandro Chagas (Patuá) |
repository_id_str |
|
spelling |
Viana, Giselle Maria RachidSiqueira, Nathália Nogueira ChammaBarbosa, Danielle Regina LimaCarmo, Ediclei Lima doPeres, José Mário VelosoNascimento, José Maria de SouzaPóvoa, Marinete Marins2018-02-23T14:07:01Z2018-02-23T14:07:01Z2016VIANA, Giselle Maria Rachid et al. Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films. Revista Pan-Amazônica de Saúde, v. 7, n. esp., p. 107-114, dez. 2016. Disponível em: <http://scielo.iec.pa.gov.br/pdf/rpas/v7nesp/2176-6223-rpas-7-esp-00107.pdf >. Acesso em: 23 fev. 2018.2176-6223https://patua.iec.gov.br/handle/iec/300810.5123/S2176-62232016000500012The study aimed to evaluate a protocol of nested PCR using archival Giemsa-stained thick blood smears (GTS)as source of Plasmodium DNA. A total of 138 GTS from patients of five municipalities from Pará State (Amazon Region, Brazil) was included in this survey. These samples were classified in three groups (group 1: 85 Plasmodium positive and negative GTS stored in plastic box during five years; group 2: 28 Plasmodium positive and negative GTS stored in wooden box during 10 years; and group 3: 25 Trypanosoma cruzi GTS negative for Plasmodium stored in plastic box during a month) and were submitted to DNA extraction with Chelex-100. Subsequently, extracted DNA samples were quantified and the integrity was verified by electrophoresis. Nested PCR protocol was performed to detect Plasmodium species. The results of nested PCR were compared to microscopy and statistic parameters were calculated by screening test. DNA samples from all groups had acceptable quantity and purity level, but the evaluation of integrity showed 19 degraded samples from group 2. By nested PCR, this group showed very low sensitivity (29.63%) and accuracy (32.14%), while nested PCR for samples from group 1 showed 100% of sensitivity and 97.65% of accuracy. The results of this research showed that samples stored until five years can be useful as Plasmodium DNA source for nested PCR to identify Plasmodium species, being an important alternative to support retrospective studies.O estudo teve como objetivo avaliar um protocolo de nested PCR, utilizando o teste de gota espessa corada por Giemsa (GTS) como fonte de DNA de Plasmodium. Um total de 138 amostras de GTS de pacientes de cinco municípios do Estado do Pará (Região Amazônica, Brasil) foi incluído neste estudo. Essas amostras foram classificadas em três grupos (grupo 1: 85 Plasmodium GTS positivos e negativos armazenados em uma caixa de plástico durante cinco anos; grupo 2: 28 Plasmodium GTS positivos e negativos armazenados na caixa de madeira durante 10 anos; e grupo 3: 25 Trypanosoma cruzi GTS negativos para Plasmodium armazenados em caixa de plástico durante um mês) e foram submetidas à extração de DNA com Chelex-100. Subsequentemente, as amostras de DNA extraídas foram quantificadas e sua integridade foi verificada pela eletroforese. O protocolo de nested PCR foi realizado para detectar resultados das espécies de Plasmodium. Os resultados do nested PCR foram comparados com os de microscopia e os parâmetros estatísticos foram calculados pelo teste de triagem. As amostras de DNA de todos os grupos obtiveram nível, quantidade e pureza aceitáveis, mas a avaliação da integridade mostrou 19 amostras degradadas do grupo 2. Pelo nested PCR, esse grupo mostrou baixa sensibilidade (29,63%) e precisão (32,14%), enquanto que as amostras do grupo 1 apresentaram 100% de sensibilidade e 97,65% de precisão. Os resultados desta pesquisa apontam que as amostras armazenadas até cinco anos podem ser úteis como fonte de DNA de Plasmodium para que o nested PCR identifique as espécies de Plasmodium, sendo uma alternativa importante para apoiar estudos retrospectivos.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.engInstituto Evandro ChagasNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood filmsNested PCR utilizando a gota espessa como fonte do DNA de Plasmodium: uma alternativa para esfregaços de sangue arquivadosinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleMalária / diagnósticoReação em Cadeia da Polimerase / métodosTécnicas e Procedimentos DiagnósticosSensibilidade e EspecificidadeParasitemiaProtocolosEcossistema Amazônicoinfo:eu-repo/semantics/openAccessreponame:Repositório Digital do Instituto Evandro Chagas (Patuá)instname:Instituto Evandro Chagas (IEC)instacron:IECORIGINALNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdfNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdfapplication/pdf498004https://patua.iec.gov.br/bitstreams/1db41ded-1314-45fd-9e2e-29b829bec00f/download261a47d1b37113e7088b80fdf76cb46aMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-871https://patua.iec.gov.br/bitstreams/6b9e266f-c2e5-483c-aa7f-94f44064026c/download52f1732ea66fbd1123abe39f5373b797MD52TEXTNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdf.txtNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdf.txtExtracted texttext/plain33679https://patua.iec.gov.br/bitstreams/65aa9dc4-3909-4bd4-bb5c-450a8eb85016/download6619b9588c960e04865c384eeca24368MD55THUMBNAILNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdf.jpgNested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films.pdf.jpgGenerated Thumbnailimage/jpeg6092https://patua.iec.gov.br/bitstreams/54ef1360-e6e3-4d43-ae47-f4f29ad4a508/download067a7952e388930b43c8f67ab953662cMD56iec/30082022-10-20 21:18:55.211oai:patua.iec.gov.br:iec/3008https://patua.iec.gov.brRepositório InstitucionalPUBhttps://patua.iec.gov.br/oai/requestclariceneta@iec.gov.br || Biblioteca@iec.gov.bropendoar:2022-10-20T21:18:55Repositório Digital do Instituto Evandro Chagas (Patuá) - Instituto Evandro Chagas (IEC)falseVG9kb3Mgb3MgZG9jdW1lbnRvcyBkZXNzYSBjb2xlw6fDo28gc2VndWVtIGEgTGljZW7Dp2EgQ3JlYXRpdmUgY29tbW9ucy4= |
dc.title.pt_BR.fl_str_mv |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
dc.title.alternative.pt_BR.fl_str_mv |
Nested PCR utilizando a gota espessa como fonte do DNA de Plasmodium: uma alternativa para esfregaços de sangue arquivados |
title |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
spellingShingle |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films Viana, Giselle Maria Rachid Malária / diagnóstico Reação em Cadeia da Polimerase / métodos Técnicas e Procedimentos Diagnósticos Sensibilidade e Especificidade Parasitemia Protocolos Ecossistema Amazônico |
title_short |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
title_full |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
title_fullStr |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
title_full_unstemmed |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
title_sort |
Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films |
author |
Viana, Giselle Maria Rachid |
author_facet |
Viana, Giselle Maria Rachid Siqueira, Nathália Nogueira Chamma Barbosa, Danielle Regina Lima Carmo, Ediclei Lima do Peres, José Mário Veloso Nascimento, José Maria de Souza Póvoa, Marinete Marins |
author_role |
author |
author2 |
Siqueira, Nathália Nogueira Chamma Barbosa, Danielle Regina Lima Carmo, Ediclei Lima do Peres, José Mário Veloso Nascimento, José Maria de Souza Póvoa, Marinete Marins |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Viana, Giselle Maria Rachid Siqueira, Nathália Nogueira Chamma Barbosa, Danielle Regina Lima Carmo, Ediclei Lima do Peres, José Mário Veloso Nascimento, José Maria de Souza Póvoa, Marinete Marins |
dc.subject.decsPrimary.pt_BR.fl_str_mv |
Malária / diagnóstico Reação em Cadeia da Polimerase / métodos Técnicas e Procedimentos Diagnósticos Sensibilidade e Especificidade Parasitemia Protocolos Ecossistema Amazônico |
topic |
Malária / diagnóstico Reação em Cadeia da Polimerase / métodos Técnicas e Procedimentos Diagnósticos Sensibilidade e Especificidade Parasitemia Protocolos Ecossistema Amazônico |
description |
The study aimed to evaluate a protocol of nested PCR using archival Giemsa-stained thick blood smears (GTS)as source of Plasmodium DNA. A total of 138 GTS from patients of five municipalities from Pará State (Amazon Region, Brazil) was included in this survey. These samples were classified in three groups (group 1: 85 Plasmodium positive and negative GTS stored in plastic box during five years; group 2: 28 Plasmodium positive and negative GTS stored in wooden box during 10 years; and group 3: 25 Trypanosoma cruzi GTS negative for Plasmodium stored in plastic box during a month) and were submitted to DNA extraction with Chelex-100. Subsequently, extracted DNA samples were quantified and the integrity was verified by electrophoresis. Nested PCR protocol was performed to detect Plasmodium species. The results of nested PCR were compared to microscopy and statistic parameters were calculated by screening test. DNA samples from all groups had acceptable quantity and purity level, but the evaluation of integrity showed 19 degraded samples from group 2. By nested PCR, this group showed very low sensitivity (29.63%) and accuracy (32.14%), while nested PCR for samples from group 1 showed 100% of sensitivity and 97.65% of accuracy. The results of this research showed that samples stored until five years can be useful as Plasmodium DNA source for nested PCR to identify Plasmodium species, being an important alternative to support retrospective studies. |
publishDate |
2016 |
dc.date.issued.fl_str_mv |
2016 |
dc.date.accessioned.fl_str_mv |
2018-02-23T14:07:01Z |
dc.date.available.fl_str_mv |
2018-02-23T14:07:01Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
VIANA, Giselle Maria Rachid et al. Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films. Revista Pan-Amazônica de Saúde, v. 7, n. esp., p. 107-114, dez. 2016. Disponível em: <http://scielo.iec.pa.gov.br/pdf/rpas/v7nesp/2176-6223-rpas-7-esp-00107.pdf >. Acesso em: 23 fev. 2018. |
dc.identifier.uri.fl_str_mv |
https://patua.iec.gov.br/handle/iec/3008 |
dc.identifier.issn.-.fl_str_mv |
2176-6223 |
dc.identifier.doi.-.fl_str_mv |
10.5123/S2176-62232016000500012 |
identifier_str_mv |
VIANA, Giselle Maria Rachid et al. Nested PCR using thick blood smears as source of Plasmodium DNA: an alternative to study archival blood films. Revista Pan-Amazônica de Saúde, v. 7, n. esp., p. 107-114, dez. 2016. Disponível em: <http://scielo.iec.pa.gov.br/pdf/rpas/v7nesp/2176-6223-rpas-7-esp-00107.pdf >. Acesso em: 23 fev. 2018. 2176-6223 10.5123/S2176-62232016000500012 |
url |
https://patua.iec.gov.br/handle/iec/3008 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Instituto Evandro Chagas |
publisher.none.fl_str_mv |
Instituto Evandro Chagas |
dc.source.none.fl_str_mv |
reponame:Repositório Digital do Instituto Evandro Chagas (Patuá) instname:Instituto Evandro Chagas (IEC) instacron:IEC |
instname_str |
Instituto Evandro Chagas (IEC) |
instacron_str |
IEC |
institution |
IEC |
reponame_str |
Repositório Digital do Instituto Evandro Chagas (Patuá) |
collection |
Repositório Digital do Instituto Evandro Chagas (Patuá) |
bitstream.url.fl_str_mv |
https://patua.iec.gov.br/bitstreams/1db41ded-1314-45fd-9e2e-29b829bec00f/download https://patua.iec.gov.br/bitstreams/6b9e266f-c2e5-483c-aa7f-94f44064026c/download https://patua.iec.gov.br/bitstreams/65aa9dc4-3909-4bd4-bb5c-450a8eb85016/download https://patua.iec.gov.br/bitstreams/54ef1360-e6e3-4d43-ae47-f4f29ad4a508/download |
bitstream.checksum.fl_str_mv |
261a47d1b37113e7088b80fdf76cb46a 52f1732ea66fbd1123abe39f5373b797 6619b9588c960e04865c384eeca24368 067a7952e388930b43c8f67ab953662c |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Digital do Instituto Evandro Chagas (Patuá) - Instituto Evandro Chagas (IEC) |
repository.mail.fl_str_mv |
clariceneta@iec.gov.br || Biblioteca@iec.gov.br |
_version_ |
1809190037347106816 |