Simultaneous detection of <it>Plasmodium vivax</it> and <it>Plasmodium falciparum</it> gametocytes in clinical isolates by multiplex-nested RT-PCR

• Main Authors: Kuamsab Napaporn, Putaporntip Chaturong, Pattanawong Urassaya, Jongwutiwes Somchai
• Format: Article
• Language: English
• Published: BMC 2012-06-01
• Series: Malaria Journal
• Subjects: Malaria diagnosis; Gametocyte; Reverse transcription polymerase chain reaction; <it>Plasmodium falciparum</it>; <it>Plasmodium vivax</it>; <it>Pfs25</it>; <it>Pvs25</it>
• Online Access: http://www.malariajournal.com/content/11/1/190

<p>Abstract</p> <p>Background</p> <p>Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of <it>Plasmodium falciparum</it> gametocytes but very limited for that of <it>Plasmodium vivax</it>.</p> <p>Methods</p> <p>A multiplex-nested RT-PCR targeting <it>Pfs25</it> and <it>Pvs25</it> mRNA specific to mature gametocytes of <it>P. falciparum</it> and <it>P. vivax</it>, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and <it>18S rRNA</it> PCR.</p> <p>Results</p> <p>The multiplex-nested RT-PCR detected <it>Pfs25</it> mRNA in 75 of 86 (87.2%) <it>P. falciparum</it>-infected individuals and <it>Pvs25</it> mRNA in 82 of 90 (91.1%) <it>P. vivax</it> malaria patients diagnosed by <it>18S rRNA</it> PCR. Gametocytes were detected in 38 (eight <it>P. falciparum</it> and 30 <it>P. vivax</it>) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting <it>Pfs25</it> or <it>Pvs25</it> mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of <it>P. falciparum</it> and <it>P. vivax,</it> respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates.</p> <p>Conclusions</p> <p>The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both <it>P. falciparum</it> and <it>P. vivax</it> gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.</p>