Post-polymerase Chain Reaction Research Articles

Label-Free Citrate-Stabilized Silver Nanoparticles-Based, Highly Sensitive, Cost-Effective, and Rapid Visual Method for the Differential Detection of Mycobacterium tuberculosis and Mycobacterium bovis.

Tuberculosis poses a global health challenge, and it demands improved diagnostics and therapies. Distinguishing between Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis) infections holds critical "One Health" significance due to the zoonotic nature of these infections and inherent resistance of M. bovis to pyrazinamide, a key part of the directly observed treatment, short-course (DOTS) regimen. Furthermore, most of the currently used molecular detection methods fail to distinguish between the two species. To address this, our study presents an innovative molecular-biosensing strategy. We developed a label-free citrate-stabilized silver nanoparticle aggregation assay that offers sensitive, cost-effective, and swift detection. For molecular detection, genomic markers unique to M. tb and M. bovis were targeted using species-specific primers. In addition to amplifying species-specific regions, these primers also aid the detection of characteristic deletions in each of the mycobacterial species. Post polymerase chain reaction (PCR), we compared two highly sensitive visual detection methods with respect to the traditional agarose gel electrophoresis. The paramagnetic bead-based bridging flocculation assay successfully discriminates M. tb from M. bovis with a sensitivity of ∼40 bacilli. The second strategy exploits citrate-stabilized silver nanoparticles, which aggregate in the absence of amplified dsDNA on the addition of sodium chloride (NaCl). This technique enables the precise, sensitive, and differential detection of as few as ∼4 bacilli. Our study hence advances tuberculosis detection, overcoming the challenges of M. tb and M. bovis differentiation and offering a quicker alternative to time-consuming methods.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Application of CRISPR/Cas9-based mutant enrichment technique to improve the clinical sensitivity of plasma EGFR testing in patients with non-small cell lung cancer

BackgroundApproximately 50%–60% of secondary resistance to primary EGFR- tyrosine kinase inhibitors (TKI) therapy is caused by acquired p.Thr790Met (T790M) mutation; however, highly fragmented, low-quantity circulating tumor DNA is an obstacle for detecting mutations. Therefore, more sensitive mutation detection techniques are required. Here, we report a new mutant enrichment technology, the CRISPR system combined with post-polymerase chain reaction (PCR) cell-free DNA (cfDNA) (CRISPR-CPPC) to detect the T790M mutation using droplet digital PCR (ddPCR) from cfDNA.MethodsThe CRISPR-CPPC process comprises the following three steps: (1) cfDNA PCR, (2) assembly of post-PCR cfDNA and CRISPR/CRISPR associated protein 9 complex, and (3) enrichment of the target DNA template. After CRISPR-CPPC, the target DNA was detected using ddPCR. We optimized and validated CRISPR-CPPC using reference cfDNA standards and cfDNA from patients with non-small cell lung cancer who underwent TKI therapy. We then compared the detection sensitivity of CRISPR-CPPC assay with the results of real-time PCR and those of ddPCR.ResultsCRISPR-CPPC aided detection of T790M with 93.9% sensitivity and 100% specificity. T790M mutant copies were sensitively detected achieving an approximately 13-fold increase in the detected allele frequency. Furthermore, positive rate of detecting a low T790M copy number (< 10 copies/mL) were 93.8% (15/16) and 43.8% (7/16) for CRISPR-CPPC assay and ddPCR, respectively.ConclusionsCRISPR-CPPC is a useful mutant enrichment tool for the sensitive detection of target mutation. When tested in patients with progressive disease, the diagnostic performance of CRISPR-CPPC assay is exceptionally better than that of any other currently available methods.

Development of antibody response to SARS‐CoV‐2 following asymptomatic infection in patients with plasma cell disorders on immunomodulatory therapy

Development of antibody response to SARS‐CoV‐2 following asymptomatic infection in patients with plasma cell disorders on immunomodulatory therapy

Exposure to aflatoxin B1 and associated risk factors in hepatitis C patients in cosmopolitan city of Pakistan: facility-based study.

Introductionpopulation-based follow-up study has been designed to investigate the contributing factors to high exposure to Aflatoxin B1 (AFB1) and the subsequent associated risk factors among hepatitis C-infected patients at a referral centre, Karachi. Pakistan. Hepatitis C infection affects millions of individuals worldwide and confers high morbidity and mortality, especially in lower middle-income countries (LMICs) including Pakistan. A literature review of recent studies has revealed that a number of hepatocellular carcinomas (HCC) cases are markedly increased in Pakistan, where one of the potential causes of HCC is hepatitis C virus. The objectives of this study were to determine frequency of Aflatoxin B1 (AFB1) exposure and other associated characteristics among hepatitis C patients at a referral centre, Karachi, Pakistan.Methodsa semi-structured pre-coded pro forma designed to collect socio-demographic, Pharmacological, biochemical and clinical information from patients and hospital records. Patient´s pre and post polymerase chain reaction (PCR), serum alanine aminotransferase (ALT) levels and other blood parameters were analysed. AFB1 exposure was determined using an ELISA kit and validated through high-performance liquid chromatography (HPLC).ResultsAFB1 exposure was found in 30 (34%) patients. Post treatment responders were 49 (55.6%). More than 37% of study participants had a family history of hepatitis C. About 74% had a history of surgical procedure, and around 36% of study participants had a blood transfusion history. Up to 36% participants were fond of spicy food and around 25% study participants were eating roadside food on daily basis.Conclusionhigh frequency of AFB1 exposure due to risky dietary habits, low level of formal education and awareness are contributing factors may be responsible for high exposure of AFB1. Effective and multidimensional strategies are needed to prevent advance stage progression of disease and associated complications.

Impaired anti-SARS-CoV-2 antibody response in non-severe COVID-19 patients with diabetes mellitus: A preliminary report

Background and aimsPatients with diabetes mellitus (DM) often demonstrate impaired antibody response to influenza/hepatitis B vaccines. Hence, we compared anti-SARS-CoV-2 antibody response in non-severe COVID-19 patients with and without type 2 diabetes mellitus (T2DM). MethodsRecords of non-severe COVID-19 patients admitted at our institution between April 10, 2020 and May 20, 2020 were retrieved. Qualitative detection of total (IgG + IgM) anti-SARS-CoV-2 antibody was performed using electrochemiluminescence immunoassay in plasma samples collected at least 14 days post-polymerase chain reaction (PCR) confirmation of diagnosis. ResultsThirty-one non-severe COVID-19 patients were included. Nine patients (29%) had T2DM with mean HbA1c at admission of 8.3 ± 1.0%. Anti-SARS-CoV-2 antibody was estimated at a median of 16 (14–17) days post-PCR confirmation of COVID-19 diagnosis. Only three patients (10%) were seronegative, and all had T2DM. Patients with T2DM were more likely to have non-detectable anti-SARS-CoV-2 antibodies than those without DM (p = 0.019). ConclusionsCOVID-19 patients with T2DM may not undergo seroconversion even after two weeks of diagnosis. Impaired seroconversion could theoretically increase the risk of reinfections in patients with DM. However, the finding requires validation in large-scale studies involving serial estimations of anti-SARS-CoV-2 antibodies in patients with and without DM.

Authentication of olive oil based on DNA analysis

Olive oil, which has been produced mainly in the Mediterranean area since the ancient times, has a high nutritional value linked to many health benefits. Extra virgin, which is the purest form of olive oil, has excellent quality and premium prices. Many cases of adulteration and fraud necessitate the development of reliable and accurate methods for olive oil authentication. DNA-based methods analyze the residual DNA extracted from olive oil and use molecular markers for genetic identification of different species, subspecies or cultivars because these markers act as signs which reflect distinct genetic profiles. This study reviews the process by which DNA from olive oil is extracted and analyzed by the most recently used markers in the authentication of olive oil, such as Simple Sequence Repeats (SSR) or microsatellites and the single nucleotide polymorphisms (SNPs). Methods of analysis such as qPCR and digital PCR are also discussed with a special emphasis placed on the method of High-Resolution Melting (HRM), a post-polymerase chain reaction method, which enables rapid, high performing identification of genetic variants in the DNA regions of interest without sequencing, and may differentiate very similar cultivars which differ in only one nucleotide in a specific locus.

One-Step Differential Detection of OXA-48-Like Variants Using High-Resolution Melting (HRM) Analysis.

OXA-48-like carbapenemase gene remains a hidden threat, as different OXA-48 variants have varying presentations of susceptibility to antibiotics that might affect the treatment decisions. Rapid detection and differentiation of OXA-48-like carbapenemase genes are critical for targeted treatment and infection control. In this study, we aimed to develop high-resolution melting (HRM) analysis for the differentiation of OXA-48 variants. HRM analysis is a post-polymerase chain reaction (post-PCR) method for identification of small variations in nucleic acid sequences based on the PCR dissociation curve. A total of 82 bacterial strains, which consisted of Enterobacteriaceae and non-Enterobacteriaceae, were collected from a tertiary teaching hospital. The sensitivity and specificity of the assay were determined, and the developed assay was evaluated using the collected isolates against conventional-sequencing method. Overall, the developed assay was able to detect isolates that harboured OXA-48 and OXA232/OXA-181 by showing two distinct peaks at 81.1 ± 0.2 °C and 82.1 ± 0.2 °C, respectively. The detection limit of the assay was 1.6 x 10−6 ng/µL for OXA-48 and 1.8 × 10−7 ng/µL for OXA-232/OXA-181. This assay showed 100% specificity when evaluated on a panel of 37 isolates comprised of different species of bacteria and yeasts. When the assay with isolates collected in the year 2016 was first evaluated, the assay showed comparable results with conventional PCR-sequencing method where 34 OXA-48 and OXA-232/OXA-181 were detected. By using HRM analysis, the presence of OXA-48-like variants could be easily identified within 3 h from the pure culture.

Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

In the chaotic market of fur goods, genetic distinction is increasingly important for identifying species. A vast diversity of species identification methods has been proposed, while little is developed, particularly those easy, fast, and cost-effective ones. In this study, a simple and reliable novel loop-mediated isothermal amplification method for identifying cytochrome c oxidase I of felis and vulpes was established. It saves laborious post–polymerase chain reaction procedures and shortens the time for high-fidelity gene amplification. The sensitivity of this method for felis and vulpes identification, which is well matched to quantitative polymerase chain reaction, could be 10 or 1.0 pg, respectively. Predominantly, the sensitivity of loop-mediated isothermal amplification is more tolerant to those polymerase chain reaction inhibitors such as pigments, dyes, or other fur ingredients, compared to quantitative polymerase chain reaction. Even without costly specialized equipment, a water bath is sufficient for genetic distinction. Our approach is a new technique with broad application perspective, such as on-site species identity tests, commercial fraud, and wildlife crimes.

High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction.

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T0 transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T0 transgenic plants, which will be widely used in the area of plant gene editing.

Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis.

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.

Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

Nanotechnology: An Applied and Robust Approach for Forensic Investigation

Nanotechnology, the manipulation of matter at the atomic and molecular level to form novel materials with unusually diverse and unique properties, is a quickly expanding area of research with immense potential in several fields, ranging from health care to production and physical science. In forensic science it promises to revolutionize the applicability of micro-fluidic systems in post-polymerase chain reaction (PCR) quantification, DNA extraction media, fingerprint visualization using nano-powder. In general, Forensic Nanotechnology will transform the bulky instruments into small chip-based platforms, and shorten analysis methods to make investigations, sensitive, timely, and applicable.

High resolution melt analysis to confirm the establishment ofLactobacillus plantarumandEnterococcus faeciumfrom silage inoculants during ensiling of wheat

High resolution melt (HRM) analysis is a sensitive post-polymerase chain reaction (PCR) method that enables to distinguish between similar DNA sequences, because of its high resolution capability. Thus, we aimed to use the HRM analysis in order to identify species of lactic acid bacteria (LAB) used as inoculants for silage and to confirm their establishment during the ensiling of wheat. Four silage inoculants including Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus faecium and Lactobacillus buchneri were used as reference strains for HRM. The 16S rDNA gene of the four strains was amplified by real-time PCR and the amplicons were used for subsequent HRM analysis. The different LAB species tested generated distinctive HRM profiles, allowing the discrimination and differentiation of each species. Two ensiling experiments were performed using wheat silages prepared in anaerobic jars. Treatments included control (no additives), L. plantarum and E. faecium each added at 106 colony forming unit per 1 g of fresh wheat. Three jars per treatment were sampled for analysis on different days of the experiment. Melting curves were obtained from DNA extracted from LAB colonies generated on de Man, Rogosa and Sharpe agar media. In Experiment 1, DNA was obtained from random colonies, whereas, in Experiment 2, in addition to random colonies, the rest of the colonies from a given plate were analyzed. In all experimental setups, when random colonies were used, the results indicated that different LAB species are present in the silage, which changes along with the decrease in pH during the ensiling of wheat. In Experiment 2, when all colonies were sampled from given plates, the dominance of the L. plantarum showed up during ensiling of wheat, especially in wheat treated with L. plantarum. In summary, a simple molecular approach based on HRM analysis used in the present study, enables us to confirm establishment of L. plantarum throughout ensiling of wheat.

Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner.DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19.The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process.In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI.

Rapid differentiation of Alectoris rufa L., 1758 and Alectoris chukar (Gray, 1830) (Galliformes: Phasianidae) by melting curve analysis of a parathyroid hormone gene SNP

Current DNA-based methods for identifying introgression levels of closely related species imply post-polymerase chain reaction manipulations that requires additional time and expenses for sample processing. Melting curves analysis is a technique that uses species-specific primers. Here we show this method to be highly accurate for the rapid differentiation of pure individual partridges of Alectoris chukar and A. rufa species, using a SNP of the parathyroid hormone gene in a simple, rapid and efficient way. Seventy eight individuals were analyzed, (A. chukar, n=15; A. rufa, n=63), and no hybrids were detected. This method should find wide use for the management polices based on strict genetic controls for partridge stocks reproduced in farms and used in restocking or other hunting activities.

Nanotechnology - The future armour of forensics: A short review

Nanotechnology is the study of the control of matter of an atomic and molecular scale. At present the most widespread forensic application of micro fluidic systems is post-polymerase chain reaction (PCR) quantization. These systems are currently being used in several forensic laboratories to perform post-PCR quantification of mitochondrial DNA. Another innovation relates to assisting in solving gun crime. Using a nanoscale developer and an X-ray source, it is possible to image the etched fingerprints even if the casing has been wiped or washed. This technology is going to revolutionize the fields of virtopsy, crime scene investigation, identification, forensic ballistics, and toxicology.

HLA‐B*5701 typing: evaluation of an allele‐specific polymerase chain reaction melting assay

Inheritance of HLA-B*5701 is a strong predictor of a hypersensitivity reaction to the anti-HIV drug abacavir. The identification of susceptible individuals prior to the institution of abacavir therapy is therefore of clinical importance and has generated demand for a simple and rapid diagnostic test for carriage of HLA-B*5701. In this study, we describe the development of such a method based on allele-specific polymerase chain reaction (AS-PCR) and melting curve analysis. Ninety-six patient samples including 36 HLA-B*5701-positive samples and 60 HLA-B*5701-negative samples were analysed. Compared with sequence-based typing, this method had 100% sensitivity and specificity for the HLA-B*5701 allele. In conclusion, the AS-PCR/melting curve approach minimises post-polymerase chain reaction handling processing and provides an attractive alternative to currently described AS-PCR methods.

Gel‐free species identification using melt‐curve analysis

AbstractDNA‐based identification of organisms is an important tool in biosecurity, ecological monitoring and wildlife forensics. Current methods usually involve post‐polymerase chain reaction (PCR) manipulations (e.g. restriction digest, gel electrophoresis), which add to the expense and time required for processing samples, and may introduce error. We developed a method of species identification that uses species‐specific primers and melt‐curve analysis, and avoids post‐PCR manipulation of samples. The method was highly accurate when trialled on DNA from six large carnivore species from Tasmania, Australia. Because of its flexibility and cost‐effectiveness, this method should find wide use in many areas of applied biological science.

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.