Original article. A novel reverse transcription polymerase chain reaction reveals a high prevalence of Plasmodium vivax gametocyte carriage in an endemic area of Thailand

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Abstract

Background: Gametocytes are precursors of malaria sexual stages that are infective to mosquito vectors and play crucial roles in maintaining cycle of infection. Microscopy cannot determine the status of gametocyte carriage in those who had submicroscopic gametocytemia that may serve as infectious reservoirs in endemic areas. Meanwhile, gametocytes possess stage-specific mRNA that can be detected by molecular methods.

Objective: To develop a sensitive method for detection of Plasmodium vivax gametocytes using reverse transcription polymerase chain reaction (RT-PCR) and determine its diagnostic performance in clinical samples.

Materials and Methods: A nested RT-PCR was devised using primers targeting Pvs25, a mature gametocytespecific mRNA transcript of P. vivax (nested Pvs25 RT-PCR). Performance of the assay was evaluated using mRNA extracted from blood samples of 180 febrile patients attending a malaria clinic in Tak Province. Total RNA was extracted from blood samples that were preserved in RNAlater and from dried blood on filter papers. Malaria species was determined by microscopy from Giemsa stained blood smears and reaffirmed by nested PCR targeting mitochondrial cytochrome b (nested mtCytb PCR).

Results: Of 180 blood samples, malaria was diagnosed in 120 patients (69 P. vivax and 51 P. falciparum) by microscopy and 125 patients by nested mtCytb PCR (69 P. vivax, 51 P. falciparum and 5 coinfections with both these species). Microscopy detected gametocytes in 30 of all 74 (40.5%) P. vivax positives by nested mtCytb PCR. Meanwhile, 67 of 74 (90.5%) P. vivax-positive isolates that were preserved in RNAlater gave positive results by nested Pvs25 RT-PCR. Therefore, nested Pvs25 RT-PCR identified mature P. vivax gametocytes more than twice as frequently as microscopy. The minimum detection threshold for nested Pvs25 RT-PCR was 10 copies of template DNA whereas no cross-reactivity with other human malaria species was observed. Dried blood collected on filter papers offered comparable results for Pvs25 mRNA detection with blood stored in RNA preservative with only 2.7% difference in positive rates.

Conclusion: The nested RT-PCR targeting Pvs25 developed in this study is sensitive and specific for diagnosing mature P. vivax gametocytes and can be efficiently applied to both blood samples kept in RNA preservative and dried blood on filter paper.

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