Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola
Abstract Background Malaria is the parasitic disease with the highest morbidity and mortality worldwide. Angola is one of the five sub-Saharan African countries with the highest malaria burden. Real-time PCR diagnosis in endemic areas has not been implemented due to its high cost and the need for ad...
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BMC
2025-02-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-025-06685-3 |
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| author | Alejandro Mediavilla Begoña Febrer-Sendra Aroa Silgado Patricia Martínez-Vallejo Beatriz Crego-Vicente Arlette Nindia Carles Rubio Maturana Lidia Goterris Joan Martínez-Campreciós Sandra Aixut Pedro Fernández-Soto María Luisa Aznar Antonio Muro Inés Oliveira-Souto Israel Molina Elena Sulleiro |
| author_facet | Alejandro Mediavilla Begoña Febrer-Sendra Aroa Silgado Patricia Martínez-Vallejo Beatriz Crego-Vicente Arlette Nindia Carles Rubio Maturana Lidia Goterris Joan Martínez-Campreciós Sandra Aixut Pedro Fernández-Soto María Luisa Aznar Antonio Muro Inés Oliveira-Souto Israel Molina Elena Sulleiro |
| author_sort | Alejandro Mediavilla |
| collection | DOAJ |
| description | Abstract Background Malaria is the parasitic disease with the highest morbidity and mortality worldwide. Angola is one of the five sub-Saharan African countries with the highest malaria burden. Real-time PCR diagnosis in endemic areas has not been implemented due to its high cost and the need for adequate infrastructure. Dried blood spots (DBSs) are an alternative for collecting, preserving, and transporting blood samples to reference laboratories. The objective of the study was to assess the efficacy of DBS as a sampling method for malaria research studies employing real-time PCR. Methods The study was divided into two phases: (i) prospective study at the Hospital Universitario Vall d'Hebron (HUVH) to compare real-time PCR from whole blood or DBS, including 12 venous blood samples from patients with positive real-time PCR for Plasmodium spp. and 10 quality control samples (nine infected samples and one negative control). Samples were collected as DBSs (10, 20, 50 µl/circle). Samples from both phases of the study were analyzed by generic real-time PCR (Plasmodium spp.) and the subsequent positive samples underwent species-specific real-time PCR (Plasmodium species) and (ii) cross-sectional study conducted at the Hospital Nossa Senhora da Paz, Cubal (Angola), including 200 participants with fever. For each patient, a fresh capillary blood specimen [for thin and thick blood films and rapid diagnostic test (RDT)] and venous blood, collected as DBSs (two 10-µl circles were combined for a total volume of 20 µl of DBS), were obtained. DBSs were sent to HUVH, Barcelona, Spain. Results (i) Real-time PCR from whole blood collection was positive for 100% of the 21 Plasmodium spp.-infected samples, whereas real-time PCR from DBSs detected Plasmodium spp. infection at lower proportions: 76.19% (16/21) for 10 µl, 85.71% (18/21) for 20 µl, 88.24% (15/17) for 50 µl and 85.71% (18/21) for 100 µl DBSs. (ii) Field diagnosis (microscopy and/or RDT) showed a 51.5% (103/200) positivity rate, while 50% (100/200) of the DBS samples tested positive by real-time PCR. Using field diagnosis as the reference method, the sensitivity of real-time PCR in DBS samples was 77.67% with a specificity of 79.38%. Plasmodium species were identified in 86 samples by real-time PCR: 81.40% (16/86) were caused by Plasmodium falciparum, 11.63% (10/86) were coinfections of P. falciparum + P. malariae, 4.65% (4/86) were P. falciparum + P. ovale, and 2.33% (2/86) were triple coinfections. Conclusions The DBS volume used for DNA extraction is a determining factor in the performance of real-time PCR for Plasmodium DNA detection. A DBS volume of 50–100 µl appears to be optimal for malaria diagnosis and Plasmodium species determination by real-time PCR. DBS is a suitable method for sample collection in Cubal followed by real-time PCR analysis in a reference laboratory. Graphical Abstract |
| format | Article |
| id | doaj-art-337a14ca875e4d3e815ed75a70c1e10d |
| institution | Kabale University |
| issn | 1756-3305 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMC |
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| series | Parasites & Vectors |
| spelling | doaj-art-337a14ca875e4d3e815ed75a70c1e10d2025-02-09T12:15:11ZengBMCParasites & Vectors1756-33052025-02-0118111110.1186/s13071-025-06685-3Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in AngolaAlejandro Mediavilla0Begoña Febrer-Sendra1Aroa Silgado2Patricia Martínez-Vallejo3Beatriz Crego-Vicente4Arlette Nindia5Carles Rubio Maturana6Lidia Goterris7Joan Martínez-Campreciós8Sandra Aixut9Pedro Fernández-Soto10María Luisa Aznar11Antonio Muro12Inés Oliveira-Souto13Israel Molina14Elena Sulleiro15Microbiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaInfectious and Tropical Diseases Research Group (E-INTRO), Biomedical Research Institute of Salamanca-Center for Research in Tropical Diseases of the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of SalamancaMicrobiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaMicrobiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaInfectious and Tropical Diseases Research Group (E-INTRO), Biomedical Research Institute of Salamanca-Center for Research in Tropical Diseases of the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of SalamancaHospital Nossa Senhora da PazMicrobiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaMicrobiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaCentro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos IIIInternational Health Unit Vall d’Hebron-Drassanes, Infectious Diseases Department, Vall d’Hebron University Hospital, PROSICS BarcelonaInfectious and Tropical Diseases Research Group (E-INTRO), Biomedical Research Institute of Salamanca-Center for Research in Tropical Diseases of the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of SalamancaCentro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos IIIInfectious and Tropical Diseases Research Group (E-INTRO), Biomedical Research Institute of Salamanca-Center for Research in Tropical Diseases of the University of Salamanca (IBSAL-CIETUS), Faculty of Pharmacy, University of SalamancaCentro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos IIICentro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos IIIMicrobiology Department, Vall d’Hebron University Hospital, Autonomous University of Barcelona, PROSICS BarcelonaAbstract Background Malaria is the parasitic disease with the highest morbidity and mortality worldwide. Angola is one of the five sub-Saharan African countries with the highest malaria burden. Real-time PCR diagnosis in endemic areas has not been implemented due to its high cost and the need for adequate infrastructure. Dried blood spots (DBSs) are an alternative for collecting, preserving, and transporting blood samples to reference laboratories. The objective of the study was to assess the efficacy of DBS as a sampling method for malaria research studies employing real-time PCR. Methods The study was divided into two phases: (i) prospective study at the Hospital Universitario Vall d'Hebron (HUVH) to compare real-time PCR from whole blood or DBS, including 12 venous blood samples from patients with positive real-time PCR for Plasmodium spp. and 10 quality control samples (nine infected samples and one negative control). Samples were collected as DBSs (10, 20, 50 µl/circle). Samples from both phases of the study were analyzed by generic real-time PCR (Plasmodium spp.) and the subsequent positive samples underwent species-specific real-time PCR (Plasmodium species) and (ii) cross-sectional study conducted at the Hospital Nossa Senhora da Paz, Cubal (Angola), including 200 participants with fever. For each patient, a fresh capillary blood specimen [for thin and thick blood films and rapid diagnostic test (RDT)] and venous blood, collected as DBSs (two 10-µl circles were combined for a total volume of 20 µl of DBS), were obtained. DBSs were sent to HUVH, Barcelona, Spain. Results (i) Real-time PCR from whole blood collection was positive for 100% of the 21 Plasmodium spp.-infected samples, whereas real-time PCR from DBSs detected Plasmodium spp. infection at lower proportions: 76.19% (16/21) for 10 µl, 85.71% (18/21) for 20 µl, 88.24% (15/17) for 50 µl and 85.71% (18/21) for 100 µl DBSs. (ii) Field diagnosis (microscopy and/or RDT) showed a 51.5% (103/200) positivity rate, while 50% (100/200) of the DBS samples tested positive by real-time PCR. Using field diagnosis as the reference method, the sensitivity of real-time PCR in DBS samples was 77.67% with a specificity of 79.38%. Plasmodium species were identified in 86 samples by real-time PCR: 81.40% (16/86) were caused by Plasmodium falciparum, 11.63% (10/86) were coinfections of P. falciparum + P. malariae, 4.65% (4/86) were P. falciparum + P. ovale, and 2.33% (2/86) were triple coinfections. Conclusions The DBS volume used for DNA extraction is a determining factor in the performance of real-time PCR for Plasmodium DNA detection. A DBS volume of 50–100 µl appears to be optimal for malaria diagnosis and Plasmodium species determination by real-time PCR. DBS is a suitable method for sample collection in Cubal followed by real-time PCR analysis in a reference laboratory. Graphical Abstracthttps://doi.org/10.1186/s13071-025-06685-3MalariaDried blood spotsSamplingReal-time PCRDiagnosisWhole blood |
| spellingShingle | Alejandro Mediavilla Begoña Febrer-Sendra Aroa Silgado Patricia Martínez-Vallejo Beatriz Crego-Vicente Arlette Nindia Carles Rubio Maturana Lidia Goterris Joan Martínez-Campreciós Sandra Aixut Pedro Fernández-Soto María Luisa Aznar Antonio Muro Inés Oliveira-Souto Israel Molina Elena Sulleiro Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola Parasites & Vectors Malaria Dried blood spots Sampling Real-time PCR Diagnosis Whole blood |
| title | Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola |
| title_full | Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola |
| title_fullStr | Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola |
| title_full_unstemmed | Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola |
| title_short | Evaluation of dried blood spot sampling for real-time PCR malaria diagnostics in a rural setting in Angola |
| title_sort | evaluation of dried blood spot sampling for real time pcr malaria diagnostics in a rural setting in angola |
| topic | Malaria Dried blood spots Sampling Real-time PCR Diagnosis Whole blood |
| url | https://doi.org/10.1186/s13071-025-06685-3 |
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