Ligase Detection Reaction-fluorescent Microsphere Research Articles

Peripheral and Placental Prevalence of Sulfadoxine-Pyrimethamine Resistance Markers in Plasmodium falciparum among Pregnant Women in Southern Province, Rwanda.

Intermittent preventive therapy during pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended in areas of moderate to high malaria transmission intensity. As a result of the increasing prevalence of SP resistance markers, IPTp-SP was withdrawn from Rwanda in 2008. Nonetheless, more recent findings suggest that SP may improve birthweight even in the face of parasite resistance, through alternative mechanisms that are independent of antimalarial effects. The prevalence of single nucleotide polymorphisms in Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes associated with SP resistance among 148 pregnant women from 2016 to 2018 within Rwanda's Southern Province (Huye and Kamonyi districts) was measured using a ligase detection reaction-fluorescent microsphere assay. The frequency of pfdhps K540E, A581G, and the quintuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E) and sextuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E + A581G) mutant genotypes was 90%, 38%, 75%, and 28%, respectively. No significant genotype difference was seen between the two districts, which are approximately 50 km apart. Observed agreements for matched peripheral to placental blood were reported and found to be 207 of 208 (99%) for pfdhfr and 239 of 260 (92%) for pfdhps. The peripheral blood sample did not miss any pfdhfr drug-resistant mutants or pfdhps except at the S436 loci. At this level of the sextuple mutant, the antimalarial efficacy of SP for preventing low birthweight is reduced, although overall SP still exerts a nonmalarial benefit during pregnancy. This study further reveals the need to intensify preventive measures to sustain malaria control in Rwanda to keep the overall incidence of malaria during pregnancy low.

Creating and Validating Ligase Primers to Detect Single Nucleotide Polymorphisms Associated with Atovaquone Resistance in Plasmodium falciparum.

Atovaquone-proguanil is one of the most commonly prescribed malaria prophylactic drugs. However, sporadic mutations conferring resistance to atovaquone have been detected in recent years associated with single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum cytochrome b ( pfcytb) gene. Monitoring polymorphisms linked with resistance is essential in assessing the prevalence of drug resistance and may help in designing strategies for malaria control. Several approaches have been used to study genetic polymorphisms associated with antimalarial drug resistance. However, they either lack high throughput capacity or are expensive in time or money. Ligase detection reaction fluorescent microsphere assay (LDR-FMA) provides a high-throughput method to detect genetic polymorphisms in P. falciparum. In this study, we have created primers to detect SNPs associated with clinically relevant atovaquone resistance using LDR-FMA and validated them in clinical samples. Four SNPs from pfcytb gene were analyzed using LDR-FMA. The results were 100% consistent with DNA sequence data, indicating that this method has potential as a tool to detect genetic polymorphisms associated with atovaquone resistance in P. falciparum.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Plasmodium falciparum Parasitemia and Band Sensitivity of the SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test in Madagascar.

Current malaria rapid diagnostic tests (RDTs) contain antibodies against Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2), Plasmodium lactate dehydrogenase (pLDH), and aldolase in various combinations. Low or high parasite densities/target antigen concentrations may influence the accuracy and sensitivity of PfHRP2-detecting RDTs. We analyzed the SD Bioline Malaria Ag P.f/Pan RDT performance in relation to P. falciparum parasitemia in Madagascar, where clinical Plasmodium vivax malaria exists alongside P. falciparum. Nine hundred sixty-three samples from patients seeking care for suspected malaria infection were analyzed by RDT, microscopy, and Plasmodium species-specific, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Plasmodium infection positivity by these diagnostics was 47.9%, 46.9%, and 58%, respectively. Plasmodium falciparum-only infections were predominant (microscopy, 45.7%; LDR-FMA, 52.3%). In all, 16.3% of P. falciparum, 70% of P. vivax, and all of Plasmodium malariae, Plasmodium ovale, and mixed-species infections were submicroscopic. In 423 P. falciparum mono-infections, confirmed by microscopy and LDR-FMA, the parasitemia in those who were positive for both the PfHRP2 and pan-pLDH test bands was significantly higher than that in those who were positive only for the PfHRP2 band (P < 0.0001). Plasmodium falciparum parasitemia in those that were detected as P. falciparum-only infections by microscopy but P. falciparum mixed infections by LDR-FMA also showed similar outcome by the RDT band positivity. In addition, we used varying parasitemia (3-0.0001%) of the laboratory-maintained 3D7 strain to validate this observation. A positive pLDH band in high P. falciparum-parasitemic individuals may complicate diagnosis and treatment, particularly when the microscopy is inconclusive for P. vivax, and the two infections require different treatments.

Comparative Prevalence of Plasmodium falciparum Resistance-Associated Genetic Polymorphisms in Parasites Infecting Humans and Mosquitoes in Uganda.

Controlling malaria in high transmission areas, such as much of sub-Saharan Africa, will require concerted efforts to slow the spread of drug resistance and to impede malaria transmission. Understanding the fitness costs associated with the development of drug resistance, particularly within the context of transmission, can help guide policy decisions to accomplish these goals, as fitness constraints might lead to decreased transmission of drug-resistant strains. To determine if Plasmodium falciparum resistance-mediating polymorphisms impact on development at different parasite stages, we compared the genotypes of parasites infecting humans and mosquitoes from households in Uganda. Genotypes at 14 polymorphic loci in genes encoding putative transporters (pfcrt and pfmdr1) and folate pathway enzymes (pfdhfr and pfdhps) were characterized using ligase detection reaction-fluorescent microsphere assays. In paired analysis using the Wilcoxon signed-rank test, prevalences of mutations at 12 loci did not differ significantly between parasites infecting humans and mosquitoes. However, compared with parasites infecting humans, those infecting mosquitoes were enriched for the pfmdr1 86Y mutant allele (P = 0.0001) and those infecting Anopheles gambiae s.s. were enriched for the pfmdr1 86Y (P = 0.0001) and pfcrt 76T (P = 0.0412) mutant alleles. Our results suggest modest directional selection resulting from varied fitness costs during the P. falciparum life cycle. Better appreciation of the fitness implications of drug resistance mediating mutations can inform optimal malaria treatment and prevention strategies.

Drug resistance mediating Plasmodium falciparum polymorphisms and clinical presentations of parasitaemic children in Uganda

BackgroundPlasmodium falciparum genetic polymorphisms that mediate altered drug sensitivity may impact upon virulence. In a cross-sectional study, Ugandan children with infections mutant at pfcrt K76T, pfmdr1 N86Y, or pfmdr1 D1246Y had about one-fourth the odds of symptomatic malaria compared to those with infections with wild type (WT) sequences. However, results may have been confounded by greater likelihood in those with symptomatic disease of higher density mixed infections and/or recent prior treatment that selected for WT alleles.MethodsPolymorphisms in samples from paired episodes of asymptomatic and symptomatic parasitaemia in 114 subjects aged 4–11 years were followed longitudinally in Tororo District, Uganda. Paired episodes occurred within 3–12 months of each other and had no treatment for malaria in the prior 60 days. The prevalence of WT, mixed, and mutant alleles was determined using multiplex ligase detection reaction-fluorescent microsphere assays.ResultsConsidering paired episodes in the same subject, the odds of symptomatic malaria were lower for infections with mutant compared to WT or mixed sequence at N86Y (OR 0.26, 95% CI 0.09–0.79, p = 0.018), but not K76T or D1246Y. However, symptomatic episodes (which had higher densities) were more likely than asymptomatic to be mixed (for N86Y OR 2.0, 95% CI 1.04–4.0, p = 0.036). Excluding mixed infections, the odds of symptomatic malaria were lower for infections with mutant compared to WT sequence at N86Y (OR 0.33, 95% CI 0.11–0.98, p = 0.046), but not the other alleles. However, if mixed genotypes were grouped with mutants in this analysis or assuming that mixed infections consisted of 50% WT and 50% mutant genotypes, the odds of symptomatic infection did not differ between infections that were mutant or WT at the studied alleles.ConclusionsAlthough infections with only the mutant pfmdr1 86Y genotype were associated with symptomatic infection, this association could primarily be explained by greater parasite densities and therefore greater prevalence of mixed infections in symptomatic children. These results indicate limited association between the tested polymorphisms and risk of symptomatic disease and highlight the value of longitudinal studies for assessing associations between parasite factors and clinical outcomes.

Molecular surveillance of Plasmodium falciparum drug resistance markers reveals partial recovery of chloroquine susceptibility but sustained sulfadoxine-pyrimethamine resistance at two sites of different malaria transmission intensities in Rwanda

Faced with intense levels of chloroquine (CQ) resistance in Plasmodium falciparum malaria, Rwanda replaced CQ with amodiaquine (AQ)+sulfadoxine-pyrimethamine (SP) in 2001, and subsequently with artemether–lumefantrine (AL) in 2006, as first-line treatments for uncomplicated malaria. Following years of discontinuation of CQ use, re-emergence of CQ-susceptible parasites has been reported in countries including Malawi, Kenya and Tanzania. In contrast, high levels of SP resistant mutant parasites continue to be reported even in countries of presumed reduced SP drug selection pressure. The prevalence and distributions of genetic polymorphisms linked with CQ and SP resistance at two sites of different malaria transmission intensities are described here to better understand drug-related genomic adaptations over time and exposure to varying drug pressures in Rwanda. Using filter paper blood isolates collected from P. falciparum infected patients, DNA was extracted and a nested PCR performed to identify resistance-mediating polymorphisms in the pfcrt, pfmdr1, pfdhps and pfdhfr genes. Amplicons from a total of 399 genotyped samples were analysed by ligase detection reaction fluorescent microsphere assay. CQ susceptible pfcrt 76K and pfmdr1 86N wild-type parasites were found in about 50% and 81% of isolates, respectively. Concurrently, SP susceptible pfdhps double (437G-540E), pfdhfr triple (108N-51I-59R), quintuple pfdhps 437G-540E/pfdhfr 51I-59R-108N and sextuple haplotypes were found in about 84%, 85%, 74% and 18% of isolates, respectively. High-level SP resistance associated pfdhfr 164L and pfdhps 581G mutant prevalences were noted to decline. Mutations pfcrt 76T, pfdhfr 59R and pfdhfr 164L were found differentially distributed between the two study sites with the pfdhfr 164L mutants found only at Ruhuha site, eastern Rwanda. Overall, sustained intense levels of SP resistance mutations and a recovery of CQ susceptible parasites were found in this study following 7 years and 14 years of drug withdrawal from use, respectively. Most likely, the sustained high prevalence of resistant parasites is due to the use of DHFR/DHPS inhibitors like trimethoprim-sulfamethoxazole (TS) for the treatment of and prophylaxis against bacterial infections among HIV infected individuals as well as the continued use of IPTp-SP within the East and Central African regions for malaria prevention among pregnant women. With regard to CQ, the slow recovery of CQ susceptible parasites may have been caused partly by the continued use of CQ and/or CQ mimicking antimalarial drugs like AQ in spite of policies to withdraw it from Rwanda and the neighbouring countries of Uganda and Tanzania. Continued surveillance of P. falciparum CQ and SP associated polymorphisms is recommended for guiding future rational drug policy-making and mitigation of future risk of anti-malaria drug resistance development.

Intermittent Preventive Treatment with Dihydroartemisinin-Piperaquine in Ugandan Schoolchildren Selects for Plasmodium falciparum Transporter Polymorphisms That Modify Drug Sensitivity

Dihydroartemisinin-piperaquine (DP) offers prolonged protection against malaria, but its impact on Plasmodium falciparum drug sensitivity is uncertain. In a trial of intermittent preventive treatment in schoolchildren in Tororo, Uganda, in 2011 to 2012, monthly DP for 1 year decreased the incidence of malaria by 96% compared to placebo; DP once per school term offered protection primarily during the first month after therapy. To assess the impact of DP on selection of drug resistance, we compared the prevalence of key polymorphisms in isolates that emerged at different intervals after treatment with DP. Blood obtained monthly and at each episode of fever was assessed for P. falciparum parasitemia by microscopy. Samples from 160 symptomatic and 650 asymptomatic episodes of parasitemia were assessed at 4 loci (N86Y, Y184F, and D1246Y in pfmdr1 and K76T in pfcrt) that modulate sensitivity to aminoquinoline antimalarials, utilizing a ligase detection reaction-fluorescent microsphere assay. For pfmdr1 N86Y and pfcrt K76T, but not the other studied polymorphisms, the prevalences of mutant genotypes were significantly greater in children who had received DP within the past 30 days than in those not treated within 60 days (86Y, 18.0% versus 8.3% [P = 0.03]; 76T, 96.0% versus 86.1% [P = 0.05]), suggesting selective pressure of DP. Full sequencing of pfcrt in a subset of samples did not identify additional polymorphisms selected by DP. In summary, parasites that emerged soon after treatment with DP were more likely than parasites not under drug pressure to harbor pfmdr1 and pfcrt polymorphisms associated with decreased sensitivity to aminoquinoline antimalarials. (This study has been registered at ClinicalTrials.gov under no. NCT01231880.).

Significant geographical differences in prevalence of mutations associated with Plasmodium falciparum and Plasmodium vivax drug resistance in two regions from Papua New Guinea.

BackgroundDrug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries.MethodsAn improved multiplexed post-PCR ligase detection reaction—fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010.ResultsDespite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p < 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from <5 % to >30 % (p < 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012).ConclusionsThis LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0879-9) contains supplementary material, which is available to authorized users.

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea

BackgroundIn northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013.MethodsA validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay.ResultsCQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005–2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71–107) vs 167 (141–197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted.ConclusionsReflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12610000913077.

Validation of the ligase detection reaction fluorescent microsphere assay for the detection of Plasmodium falciparum resistance mediating polymorphisms in Uganda

BackgroundMalaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting.MethodsFor 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared.ResultsSNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K = 0.77) for pfcrt K76T, 79% (K = 0.55) for pfmdr1 N86Y, 83% (K = 0.65) for pfmdr1 Y184F, and 91% (K = 0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes.ConclusionThe LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.

Optimization of a Ligase Detection Reaction-Fluorescent Microsphere Assay for Characterization of Resistance-Mediating Polymorphisms in African Samples of Plasmodium falciparum

Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

Molecular Assessment of Plasmodium falciparum Resistance to Antimalarial Drugs in Papua New Guinea Using an Extended Ligase Detection Reaction Fluorescent Microsphere Assay

Surveillance for Plasmodium falciparum drug resistance mutations is becoming an established tool for assessing antimalarial treatment effectiveness. We used an extended version of a high-throughput post-PCR multiplexed ligase detection reaction fluorescent microsphere assay (LDR-FMA) to detect single-nucleotide P. falciparum drug resistance polymorphisms in 402 isolates from children in Papua New Guinea (PNG) participating in an antimalarial treatment trial. There was a fixation of P. falciparum crt (pfcrt) K76T, pfdhfr C59R and S108N, and pfmdr1 mutations (92%, 93%, 95%, and 91%, respectively). Multiple mutations were frequent. Eighty-eight percent of isolates possessed a quintuple mutation (underlined), SVMNT, NRNI, KAA, and YYSND, in codons 72 to 76 for pfcrt; 51, 59, 108, and 164 for pfdhfr; 540, 581, and 613 for pfdhps; and 86, 184, 1034, 1042, and 1246 for pfmdr1, and four of these carried the K540E pfdhps allele. The pfmdr1 D1246Y mutation was associated with PCR-corrected day 42 in vivo treatment failure in children allocated piperaquine-dihydroartemisinin (P = 0.004). Although the pfmdr1 NFSDD haplotype was found in only four isolates, it has been associated with artemether-lumefantrine treatment failure in Africa. LDR-FMA allows the large-scale assessment of resistance-associated single-nucleotide polymorphisms (SNPs). Our findings reflect previous heavy 4-aminoquinoline/sulfadoxine-pyrimethamine use in PNG. Since artemether-lumefantrine and piperaquine-dihydroartemisinin will become first- and second-line treatments, respectively, the monitoring of pfmdr1 SNPs appears to be a high priority.

High sensitivity detection of Plasmodium species reveals positive correlations between infections of different species, shifts in age distribution and reduced local variation in Papua New Guinea

BackgroundWhen diagnosed by standard light microscopy (LM), malaria prevalence can vary significantly between sites, even at local scale, and mixed species infections are consistently less common than expect in areas co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. The development of a high-throughput molecular species diagnostic assay now enables routine PCR-based surveillance of malaria infections in large field and intervention studies, and improves resolution of species distribution within and between communities.MethodsThis study reports differences in the prevalence of infections with all four human malarial species and of mixed infections as diagnosed by LM and post-PCR ligase detection reaction – fluorescent microsphere (LDR-FMA) assay in 15 villages in the central Sepik area of Papua New Guinea.ResultsSignificantly higher rates of infection by P. falciparum, P. vivax, P. malariae and Plasmodium ovale were observed in LDR-FMA compared to LM diagnosis (p < 0.001). Increases were particularly pronounced for P. malariae (3.9% vs 13.4%) and P. ovale (0.0% vs 4.8%). In contrast to LM diagnosis, which suggested a significant deficit of mixed species infections, a significant excess of mixed infections over expectation was detected by LDR-FMA (p < 0.001). Age of peak prevalence shifted to older age groups in LDR-FMA diagnosed infections for P. falciparum (LM: 7–9 yrs 47.5%, LDR-FMA: 10–19 yrs 74.2%) and P. vivax (LM: 4–6 yrs 24.2%, LDR-FMA: 7–9 yrs 50.9%) but not P. malariae infections (10–19 yrs, LM: 7.7% LDR-FMA: 21.6%). Significant geographical variation in prevalence was found for all species (except for LM-diagnosed P. falciparum), with the extent of this variation greater in LDR-FMA than LM diagnosed infections (overall, 84.4% vs. 37.6%). Insecticide-treated bednet (ITN) coverage was also the dominant factor linked to geographical differences in Plasmodium species infection prevalence explaining between 60.6% – 74.5% of this variation for LDR-FMA and 81.8% – 90.0% for LM (except P. falciparum), respectively.ConclusionThe present study demonstrates that application of molecular diagnosis reveals patterns of malaria risk that are significantly different from those obtained by standard LM. Results provide insight relevant to design of malaria control and eradication strategies.

Microscopy Underestimates the Frequency of Plasmodium Falciparum Infection in Symptomatic Individuals in a Low Transmission Highland Area

In an area with unstable malaria transmission, detection of Plasmodium falciparum infection in 379 symptomatic individuals was assessed by microscopy and three polymerase chain reaction (PCR) methodologies. P. falciparum infection was detected in 25% of patients by microscopy, 37% by nested PCR, 41% by merozoite surface protein-2 (MSP-2) PCR, and 45% by a ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Of the 64 individuals who were LDR-FMA positive, microscopy negative and did not receive treatment, 8 (12.5%) had persistent symptoms and returned for treatment. Malaria attributable fraction (MAF) in symptomatic individuals was 14.6% by microscopy (95% confidence interval [CI] = 6.6-21.8%) and 28.2% by nested PCR (95% CI = 17.9-37.2%). In this highland area, P. falciparum infection in symptomatic individuals is detected more frequently by PCR than microscopy, and most frequently by LDR-FMA. P. falciparum infection appears to resolve without treatment in most LDR-FMA-positive, microscopy-negative individuals, but is persistent in a subset of these individuals and requires treatment.

A Polymerase Chain Reaction/Ligase Detection Reaction–Fluorescent Microsphere Assay to Determine Plasmodium falciparum MSP-119 Haplotypes

The merozoite surface protein-1 (MSP-1) is a blood stage antigen currently being tested as a vaccine against Plasmodium falciparum malaria. Determining the MSP-1(19) haplotype(s) present during infection is essential for assessments of MSP-1 vaccine efficacy and studies of protective immunity in human populations. The C-terminal fragment (MSP-1(19)) has four predominant haplotypes based on point mutations resulting in non-synonymous amino acid changes: E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type). Current techniques using direct DNA sequencing are laborious and expensive. We present an MSP-1(19) allele-specific polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA) that allows simultaneous detection of the four predominant MSP-1(19) haplotypes with a sensitivity and specificity comparable with other molecular methods and a semi-quantitative determination of haplotype contribution in mixed infections. Application of this method is an inexpensive, accurate, and high-throughput alternative to distinguish the predominant MSP-1(19) haplotypes in epidemiologic studies.

CHANGING PATTERNS OF PLASMODIUM BLOOD-STAGE INFECTIONS IN THE WOSERA REGION OF PAPUA NEW GUINEA MONITORED BY LIGHT MICROSCOPY AND HIGH THROUGHPUT PCR DIAGNOSIS

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.

DIAGNOSING INFECTION LEVELS OF FOUR HUMAN MALARIA PARASITE SPECIES BY A POLYMERASE CHAIN REACTION/LIGASE DETECTION REACTION FLUORESCENT MICROSPHERE-BASED ASSAY

Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings.