Ligase Detection Reaction-fluorescent Microsphere Assay 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 As a result of the increasing prevalence of SP resistance markers, IPTp-SP was withdrawn from Rwanda in 2008.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.

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.

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.

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.

A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes

BackgroundReports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates.MethodsPvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples.ResultsGenotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele.ConclusionsThe LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.

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.

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.