Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis

Abstract

In Papua New Guinea (PNG), complex patterns of malaria commonly include single and mixed infections of Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Here, we assess recent epidemiologic characteristics of Plasmodium blood-stage infections in the Wosera region through four cross-sectional surveys (August 2001 to June 2003). Whereas previous studies performed here have relied on blood smear/light microscopy (LM) for diagnosing Plasmodium species infections, we introduce a newly developed, post-polymerase chain reaction (PCR), semi-quantitative, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). A direct comparison of the two methods for > 1,100 samples showed that diagnosis was concordant for > 80% of the analyses performed for P. falciparum (PF), P. vivax (PV), and P. malariae (PM). Greater sensitivity of the LDR-FMA accounted for 75% of the discordance between diagnoses. Based on LM, the prevalence of blood-stage PF, PV, and PM infections was found to be markedly reduced compared with an early 1990s survey. In addition, there were significant shifts in age distribution of infections, with PV becoming the most common parasite in children < 4 years of age. Consistent with previous studies, prevalence of all Plasmodium species infections increased significantly in samples analyzed by the PCR-based LDR-FMA. This increase was most pronounced for PM, PO, and mixed infections and in adolescent (10-19 years) and adult age groups, suggesting that LM may lead to under-reported prevalence of less common Plasmodium species, infection complexity, and a skewed distribution of infections towards younger age groups. This study shows that the application of LDR-FMA diagnosis in large epidemiologic studies or malaria control interventions is feasible and may contribute novel insights regarding the epidemiology of malaria.

Figure 1

Prevalence of Plasmodium species infections in different age groups. (A) 2001–2003 surveys (A–D combined). (B) 1990–1992 surveys. Any, infected with any Plasmodium species; PF, P. falciparum; PV, P. vivax; PM, P. malariae; Mixed, concurrent infection with more than one Plasmodium species.

Figure 2

Age-specific prevalence of malaria infection as detected by LM and LDR-FMA assay. Solid line, LM; dashed line, LDR-FMA assay; Any, infected with any Plasmodium species; Mixed, concurrent infection with more than one Plasmodium species.

Figure 3

Association between semi-quantitative blood-stage parasitemia and LDR-FMA fluorescent signal intensity. The association between species-specific Plasmodium fluorescent signal intensity and parasitemia was determined using blood smear LM for N = 1,182 paired samples. Quadrants delineated by the horizontal and vertical bars denote the limits of negative and positive values. Top left quadrant, LDR-FMA+:LM−; top right quadrant, LDR-FMA+:LM+; bottom left quadrant, LDR-FMA−:LM−; bottom right quadrant, LDR-FMA−:LM+. In the top left quadrant, LDR-FMA+:LM− samples designated as + showed evidence of one or more Plasmodium species in the blood smear other than the species detected by LDR-FMA; samples designated as – showed evidence of no other Plasmodium species. In the bottom right quadrant, LDR-FMA−:LM+ positive samples designated as + showed evidence of one or more Plasmodium species by LDR-FMA other than the species detected by microscopy; samples designated as – showed evidence of no other Plasmodium species. Panel A reports results for PF, panel B for PV, and panel C for PM.

Figure 4

Age distribution of geometric mean blood-stage parasitemia determined by semi-quantitative blood smear microscopy and LDR-FMA. Blood smear parasitemia (infected red blood cells [IRBCs]/µL of blood) and median fluorescent signal intensity (MFI) for individuals who were positive for Plasmodium species infection by either LDR-FMA and/or LM. (A) P. falciparum. (B) P. vivax. (C) P. malariae. Black circles, LDR-FMA MFI values; gray circles, no. of IRBCs/µL; black solid line, geom. mean LDR-FMA MFI; gray dashed line, geom. mean parasitaemia/µL. Mean values estimated using a symmetric local regression (Loess) smoother with 1 degree of freedom.