Implementation and validation of a new qPCR assay to detect imported human Plasmodium species
ABSTRACT Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex (Plasmodium spp. and Pla...
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American Society for Microbiology
2025-01-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.01622-24 |
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| author | Camille Cordier Samia Hamane Théo Ghelfenstein-Ferreira Sarah Dellière Élodie Da Silva Blandine Denis Sandrine Houzé Valentin Joste Alexandre Alanio |
| author_facet | Camille Cordier Samia Hamane Théo Ghelfenstein-Ferreira Sarah Dellière Élodie Da Silva Blandine Denis Sandrine Houzé Valentin Joste Alexandre Alanio |
| author_sort | Camille Cordier |
| collection | DOAJ |
| description | ABSTRACT Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex (Plasmodium spp. and Plasmodium falciparum) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human Plasmodium species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving P. falciparum, P. vivax, P. ovale, and P. malariae were positive with duplex and species-specific qPCRs. P. knowlesi qPCR detected DNA from a P. knowlesi culture. Of eight cases of mixed Plasmodium species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log P. falciparum load decrease was observed by qPCR. These new Plasmodium qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of P. falciparum cases, malaria with low parasitaemia, and mixed Plasmodium species infection.IMPORTANCEMalaria is a disease transmitted by a Plasmodium parasite genus. Most cases are caused by Plasmodium falciparum. Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species (Plasmodium spp.) and P. falciparum. We have also developed specific qPCRs for each human plasmodial species. |
| format | Article |
| id | doaj-art-882fa7d541574c4bb7e518c5f7b98bbb |
| institution | Kabale University |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj-art-882fa7d541574c4bb7e518c5f7b98bbb2025-01-07T14:05:18ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-01-0113110.1128/spectrum.01622-24Implementation and validation of a new qPCR assay to detect imported human Plasmodium speciesCamille Cordier0Samia Hamane1Théo Ghelfenstein-Ferreira2Sarah Dellière3Élodie Da Silva4Blandine Denis5Sandrine Houzé6Valentin Joste7Alexandre Alanio8Laboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceLaboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceLaboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceLaboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceLaboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceService des maladies infectieuses, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceCentre National de Référence du Paludisme, Laboratoire de Parasitologie-Mycologie, Hôpital Bichat-Claude Bernard, Paris, FranceCentre National de Référence du Paludisme, Laboratoire de Parasitologie-Mycologie, Hôpital Bichat-Claude Bernard, Paris, FranceLaboratoire de Parasitologie-Mycologie, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, FranceABSTRACT Diagnosis of imported malaria is based on microscopic examination of blood smears (BS), detection of circulating plasmodial antigen by immunochromatography (ICT), or detection of Plasmodium spp. DNA by loop mediated isothermal amplification. We have developed duplex (Plasmodium spp. and Plasmodium falciparum) real-time PCR (qPCR) and species-specific qPCR assays for the identification and quantification of human Plasmodium species. Whole nucleic acids from 523 samples prospectively collected from 410 patients suspected of malaria between June 2016 and March 2021 were tested by qPCRs and compared to standard diagnostic procedures. All qPCRs were designed on 18S ribosomal ribonucleic acid. The limit of detection of duplex qPCR was 8 copies/reaction, and analytical specificity of species-specific qPCRs was 100%. Seventy-nine patients diagnosed for single species malaria involving P. falciparum, P. vivax, P. ovale, and P. malariae were positive with duplex and species-specific qPCRs. P. knowlesi qPCR detected DNA from a P. knowlesi culture. Of eight cases of mixed Plasmodium species infection, five were identified with our qPCR assays with better sensitivity as compared to BS and ICT. Eight out of 323 patients with negative BS were hospitalized for symptoms suggestive malaria after malaria-endemic area travel or known with history of malaria had a low positive duplex qPCR. Between day of diagnosis and post-treatment follow-up at D2-D4 of malaria, a 3.1-log P. falciparum load decrease was observed by qPCR. These new Plasmodium qPCRs allowing detection of human plasmodial species have excellent species specificity and allow rapid detection of P. falciparum cases, malaria with low parasitaemia, and mixed Plasmodium species infection.IMPORTANCEMalaria is a disease transmitted by a Plasmodium parasite genus. Most cases are caused by Plasmodium falciparum. Despite a significant drop of incidence and mortality since 2000, 249million cases and 608,000 deaths have been estimated in 2022, mainly in Africa. Due to the increasing number of travels to endemic areas, incidence of imported malaria is rising in Europe. Various techniques are used in European laboratories, such as microscopic examination of thin and thick smears and rapid diagnostic tests. However, these techniques require skilled operators to differentiate plasmodial species and have limited sensitivity. Actually, molecular diagnosis is carried out using point-of-care test for rapid results with excellent sensitivity but is unabled to determine involved species and assess parasitaemia. In this study, we developed a combined molecular tool based on both detection of all human plasmodial species (Plasmodium spp.) and P. falciparum. We have also developed specific qPCRs for each human plasmodial species.https://journals.asm.org/doi/10.1128/spectrum.01622-24malariaqPCRduplexPlasmodiumdiagnosis |
| spellingShingle | Camille Cordier Samia Hamane Théo Ghelfenstein-Ferreira Sarah Dellière Élodie Da Silva Blandine Denis Sandrine Houzé Valentin Joste Alexandre Alanio Implementation and validation of a new qPCR assay to detect imported human Plasmodium species Microbiology Spectrum malaria qPCR duplex Plasmodium diagnosis |
| title | Implementation and validation of a new qPCR assay to detect imported human Plasmodium species |
| title_full | Implementation and validation of a new qPCR assay to detect imported human Plasmodium species |
| title_fullStr | Implementation and validation of a new qPCR assay to detect imported human Plasmodium species |
| title_full_unstemmed | Implementation and validation of a new qPCR assay to detect imported human Plasmodium species |
| title_short | Implementation and validation of a new qPCR assay to detect imported human Plasmodium species |
| title_sort | implementation and validation of a new qpcr assay to detect imported human plasmodium species |
| topic | malaria qPCR duplex Plasmodium diagnosis |
| url | https://journals.asm.org/doi/10.1128/spectrum.01622-24 |
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