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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Napaporn Kuamsab, Chaturong Putaporntip, Urassaya Pattanawong, Somchai Jongwutiwes
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-190) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SJ and CP involved in study design, supervised and managed the project. NK, CP, UP and SJ carried out field work. NK, SJ and CP carried out molecular assay. NK and UP performed microscopy-based detection and estimation of parasite density. NK, CP and SJ analysed data. SJ and CP wrote the manuscript. All authors read and approved the final manuscript.

Abstract

Background

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 Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax.

Methods

A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, 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 18S rRNA PCR.

Results

The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) 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 Pfs25 or Pvs25 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 P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates.

Conclusions

The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.
Zusatzmaterial
Authors’ original file for figure 1
12936_2012_2198_MOESM1_ESM.pdf
Literatur
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