Assessment of submicroscopic infections and gametocyte carriage of Plasmodium falciparum during peak malaria transmission season in a community-based cross-sectional survey in western Kenya, 2012

Abstract

Background: Although malaria control intervention has greatly decreased malaria morbidity and mortality in many African countries, further decline in parasite prevalence has stagnated in western Kenya. In order to assess if malaria transmission reservoir is associated with this stagnation, submicroscopic infection and gametocyte carriage was estimated. Risk factors and associations between malaria control interventions and gametocyte carriage were further investigated in this study.

Methods: A total of 996 dried blood spot samples were used from two strata, all smear-positives (516 samples) and randomly selected smear-negatives (480 samples), from a community cross-sectional survey conducted at peak transmission season in 2012 in Siaya County, western Kenya. Plasmodium falciparum parasite presence and density were determined by stained blood smear and by 18S mRNA transcripts using nucleic acid sequence-based amplification assay (NASBA), gametocyte presence and density were determined by blood smear and by Pfs25 mRNA-NASBA, and gametocyte diversity by Pfg377 mRNA RT-PCR and RT-qPCR.

Results: Of the randomly selected smear-negative samples, 69.6 % (334/480) were positive by 18S-NASBA while 18S-NASBA detected 99.6 % (514/516) smear positive samples. Overall, 80.2 % of the weighted population was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the weighted population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Children 5-15 years old were more likely to be parasitaemic and gametocytaemic by NASBA than individuals >15 years old or children <5 years old while gametocyte density decreased with age. Anaemia and self-reported fever within the past 24 h were associated with increased odds of gametocytaemia. Fever was also positively associated with parasite density, but not with gametocyte density. Anti-malarial use within the past 2 weeks decreased the odds of gametocytaemia, but not the odds of parasitaemia. In contrast, recent anti-malarial use was associated with lowered parasite density, but not the gametocyte density. Use of ITNs was associated with lower odds for parasitaemia in part of the study area with a longer history of ITN interventions. In the same part of study area, the odds of having multiple gametocyte alleles were also lower in individuals using ITNs than in those not using ITNs and parasite density was positively associated with gametocyte diversity.

Conclusion: A large proportion of submicroscopic parasites and gametocytes in western Kenya might contribute to the stagnation in malaria prevalence, suggesting that additional interventions targeting the infectious reservoir are needed. As school aged children and persons with anaemia and fever were major sources for gametocyte reservoir, these groups should be targeted for intervention and prevention to reduce malaria transmission. Anti-malarial use was associated with lower parasite density and odds of gametocytaemia, but not the gametocyte density, indicating a limitation of anti-malarial impact on the transmission reservoir. ITN use had a protective role against parasitaemia and gametocyte diversity in western Kenya.

Keywords: Antimalarials; Gametocytes; ITNs; Kenya; Plasmodium falciparum; Risk factors.

Fig. 1

Weighted prevalence of parasitaemia and gametocytaemia measured by 18S-NASBA and Pfs25-NASBA compared to blood smear method for residents of Asembo and Karemo, western Kenya, 2012. It shows weighted parasite prevalence of 80.2 % by 18S-NASBA (grey filled bars) vs 30.6 % by slide smear (white open bars) and weighted gametocyte prevalence of 44.0 % by Pfs25-NASBA (grey filled bars) vs 2.6 % by slide smear (white open bars)

Fig. 2

Gametocyte diversity in human blood samples from Asembo, western Kenya (n = 130), 2012. a shows the frequency of each single allele of gametocytes detected by Pfg377 RT-PCR. The predominant genotype (56.57 %) is allele E (size 357 bp). b shows frequency distribution of Pfg377 alleles per sample. c shows each single band on 4 % electrophoresis gel. d shows the single or multiple bands of Pfg377 region 3. The gels were run with TrackIt™ 50 bp DNA ladders (Thermo Fisher Scientific, MA)

Fig. 3

Density of parasites and gametocytes and association with risk factors. Mean densities (± 95 % CI) of parasitaemia by 18S (grey filled bars) or gametocytaemia by Pfs25 (white open bars) were estimated by GLM, and presented using observed marginals. Left Y axis is parasites/µL, while right Y axis is gametocytes/µL. The brackets indicates the columns compared. Asterisks indicates that the column is significantly different (p < 0.05) than the second column with same colour for all panels. a Density of parasite and gametocyte was lower in individuals >15 years old than young or old children by 18S and Pfs25 (p < 0.001 for all comparisons). b Gametocytaemia density did not differ between anaemic and non-anaemic (NoAnaemia) individuals. c Parasite density was higher in individuals with fever. d There was no associations of ITN use on density of parasitaemia or gametocytaemia. e Parasite density was lower among individuals who had received anti-malarials (AM) in the past 2 weeks, but gametocyte density was not significantly different between receiving AM and not receiving AM (NoAM)

Fig. 4

Association of gametocyte diversity with risk factors among Pfg377 positive individuals. Odds ratios for multiple alleles and 95 % confidence intervals. Grey bars are non- significant, while dark bars indicate significant parameters. Risk factors are on the Y axes and odds ratios on the X axis. A 10-fold increased parasite density as measured by 18S-NASBA was associated with higher odds of having multiple gametocyte alleles, while sleeping under ITNs was associated with lower odds of having multiple gametocyte alleles