Métodos moleculares para detecção e quantificação de gametócitos de Plasmodium.

2012. 91 p. Masters thesis The microscopic detection of gametocytes may underestimate their prevalence, leading to an inaccurate assessment of the potential for malaria transmission in receptive areas and a poor estimate of the proportion of infected individuals who are infectious. We aimed to standardize molecular methods for detection and quantification of gametocytes of Plasmodium falciparum and Plasmodium vivax . Here, we describe a quantitative reverse transcriptase (RT) real-time PCR (qRT-PCR) that targets transcripts of the mature gametocyte-specific pvs25 gene. We found mature gametocytes in 53 of 55 (96.4%) P. vivax infections diagnosed during an ongoing cohort study in a farming settlement located in the southern of Amazonas state. SYBR green qRT-PCR was more sensitive than a conventional RT-PCR that targets the same gene. We also standardized a nested PCR, with the same target, for detecting P. vivax gametocytes in blood samples spotted onto FTA membranes and obtained positive results in 71.4% (23 of 35) samples tested. Most (61.9%) gametocyte carriers were either asymptomatic or had subpatent parasitemias, and would have been missed by routine malaria control strategies. However, potentially undiagnosed gametocyte carriers usually had low-density infections and contributed a small fraction (up to 4%) to the overall gametocyte burden in the community. Further studies are required to determine the relative contribution to malaria transmission of long-lasting but low-density gametocytemias in asymptomatic carriers that are left undiagnosed and untreated. " Molecular methods can detect gametocytes in nearly all Plasmodium vivax infections. " Most P. vivax gametocyte carriers would be missed by routine malaria control strategies. " Low-density (subpatent) gametocyte carriage may be a major contributor to malaria transmission. The proportion of Plasmodium vivax- infected subjects that carry mature gametocytes, and thus are poten- tially infectious, remains poorly characterized in endemic settings. Here, we describe a quantitative reverse transcriptase (RT) real-time PCR (qRT-PCR) that targets transcripts of the mature gametocyte- speciﬁc pvs25 gene. We found mature gametocytes in 42 of 44 (95.4%) P. vivax infections diagnosed during an ongoing cohort study in northwestern Brazil. SYBR green qRT-PCR was more sensitive than a con- ventional RT-PCR that targets the same gene. Molecular detection of gametocytes failed, however, when dried bloodspots were used for RNA isolation and complementary DNA synthesis. Estimating the number of pvs25 gene transcripts allowed for examining the potential infectiousness of gametocyte carriers in a quantitative way. We found that most (61.9%) gametocyte carriers were either asymptomatic or had sub- patent parasitemias and would have been missed by routine malaria control strategies. However, potentially undiagnosed gametocyte carriers usually had low-density infections and contributed a small fraction (up to 4%) to the overall gametocyte burden in the community. Further studies are required to determine the relative contribution to malaria transmission of long-lasting but low-density gametocyte- mias in asymptomatic carriers that are left undiagnosed and untreated.