Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria (original) (raw)
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Cellular, Molecular and Biomedical Reports
Malaria control and its elimination heavenly depend on successful and reliable diagnosis using recommended diagnostic techniques. These available techniques often have certain peculiarities and mode applications, thus making them have different levels of performance and accuracy. Therefore the aim of this study was to evaluate the performance of PCR in relation to Rapid Diagnostic Test Kit (SD Bio line Malaria Ag P.f (05fk50)) in malaria diagnosis. A total of 200 blood samples were collected from the consented study subjects using the vein puncture technique and analysed using PCR and RDTs. Plasmodium falcifarum's DNA was extracted using Quick-DNA™ Miniprep Plus Kit with catalog number D4069. 18SrRNA gene of Plasmodium falciparum from chromosome 13 was amplified using the two primers. For the RDTs technique, the SD Bio line Malaria Ag P.f (05fk50) test kit was used. Malaria prevalence of 106(53.0%) and 132(66.0%) were recorded using PCR and RDTs respectively. The PCR demonstrates an overall accuracy of 0.53 with sensitivity and specificity values of 56.06 and 52.94% respectively. The negative and positive predictive values were 69.81 and 38.30% respectively. PCR demonstrated a good level of performance and is therefore recommended as an effective diagnostic tool for malaria, especially in patients where the parasite density/parasitaemia level is very low.
Malaria journal, 2013
The use of malaria-specific quantitative real-time PCR (qPCR) is increasing due to its high sensitivity, speciation and quantification of malaria parasites. However, due to the lack of consensus or standardized methods in performing qPCR, it is difficult to evaluate and/or compare the quality of work reported by different authors for a cross-study and/or cross-platform assay analysis. The performances of seven published qPCR assays that detect Plasmodium spp or Plasmodium falciparum were compared using standard DNA and samples from a clinical trial. Amplification and qPCR measurements were performed using the Applied Biosystems 7500 Fast Real-Time PCR System. All the analyses were automatically established using the default settings. For the TaqMan probe format, the assays were performed in the background of QuantiFast Probe Master Mix whereas in SYBR Green format, the assays were performed in the background of QuantiFast SYBR Green Master Mix and QuantiTect SYBR Green Master Mix ba...
Sample-ready multiplex qPCR assay for detection of malaria
Malaria Journal, 2014
Background: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. Methods: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The C T values and the standard deviations (SD) were used in the analysis of the assay performance.
Short Report: Malaria Diagnosis by a Polymerase Chain Reaction-Based Assay Using a Pooling Strategy
The American Journal of Tropical Medicine and Hygiene, 2009
Pooling clinical specimens reduces the number of assays needed when screening for infectious diseases. Polymerase chain reaction (PCR)-based assays are the most sensitive tests to diagnose malaria, but its high cost limits its use. We adapted a pooling platform that could reduce the number of assays needed to detect malaria infection. To evaluate this platform, two sets of 100 serum samples, with 1% and 5% malaria prevalence, were tested. DNA, extracted from pooled samples, was amplified by malaria-specific PCR. Additional validation was performed by determining the level of PCR detection based on 1:10 and 1:100 dilution. The platform correctly detected all malaria samples in the two test matrices. The use of stored serum samples also has important implications for studies investigating malaria prevalence rates retrospectively. Field studies, using serum and whole blood specimens, are needed to validate this technique for the adaptation of these methods for clinical utility.
2001
Polymerase chain reaction (PCR)-based assays targeting the small-subunit rRNA were developed and evaluated, allowing for the simultaneous diagnosis of Plasmodium falciparum and Plasmodium vivax DNA in human blood samples. The PCR methods and quantitative buffy coat (QBC) were compared in 402 patients. The heminested PCR method showed a sensitivity of 97.4%, which was superior to the sensitivity of the QBC method (91.7%, P Ͻ 0.05), to simple PCR (84.6%, P Ͻ 0.001), and to PCR with digoxigenin labeling (PCR-DIG) (88.5%, P Ͻ 0.001). The PCR-DIG and QBC analyses were more sensitive than simple PCR (P Ͻ 0.003 and P Ͻ 0.05, respectively). There was no significant difference between the sensitivities of the QBC assay and the PCR-DIG assay. The specificity for the 3 PCR-based methods was 100%, superior to the specificity calculated for the QBC assay (88.95%, P Ͻ 0.009). The frequency of a positive result in groups from endemic areas but without detectable parasitemia increased, in order, from simple PCR, QBC test, PCR-DIG, to heminested PCR. An association between a positive PCR result and a history of malaria was also found. Taken together, these data suggest that this technology could be further developed to screen people with oligoparasitemia and to monitor malaria treatment.
Infectious Disease Reports, 2020
Background: The diagnostic test for malaria is mostly based on Rapid Diagnostic Test (RDT) and detection by microscopy. Polymerase Chain Reaction (PCR) is also a sensitive detection method that can be considered as a diagnostic tool. The outcome of malaria microscopy detection depends on the examiner's ability and experience. Some RDT has been distributed in Indonesia, which needs to be evaluated for their results. Objective: This study aimed to compare the performance of RightSign RDT and ScreenPlus RDT for detection of Plasmodium in human blood. We used specific real-time polymerase chain reaction abTESTMMalaria qPCRII) and gold standard of microscopy detection method to measure diagnostic efficiency. Methods: Blood specimens were evaluated using RightSign RDT, ScreenPlus RDT, Microscopy detection, and RT-PCR as the protocol described. The differences on specificity (Sp), sensitivity (Sn), positive predictive value (PPV), and negative predictive value (NPV) were analyzed using...
Annals of Tropical Medicine & Parasitology, 2007
Malaria is a common, life-threatening infection in endemic tropical areas and one that presents a diagnostic challenge to laboratories in most non-endemic countries. A rapid and accurate diagnosis is a prerequisite for effective treatment, especially for the potentially fatal cases of Plasmodium falciparum infection. In the present, multi-centre study, the performances of a rapid diagnostic test (NOWH Malaria) and several, commercial, PCRbased assays (AMS61H, AMS42H, AMS43H, AMS44H and AMS45H) were compared against the results of microscopical examination of bloodsmears (the current 'gold standard'). The subjects were either non-European immigrants (N5135) or international travellers (N5171). There was good concordance between the results of all the detection methods, with kappa values of .0.8. Although the NOW Malaria rapid test was both sensitive (100%) and specific (100%) in detecting P. falciparum infections, it was less specific (93.1%) and sensitive (90.7%) in identifying the other Plasmodium species. The results from the AMS61 assay, designed to detect any malarial infection, generally parallelled those of the microscopy (kappa50.89), giving a specificity of 98.2% and a sensitivity of 91.0%. Although the use of speciesspecific molecular primers to identify pure infections with P. falciparum and P. vivax gave results that were in good
Malaria Diagnosis by a Polymerase Chain Reaction-Based Assay Using a Pooling Strategy
American Journal of Tropical Medicine and Hygiene, 2009
Pooling clinical specimens reduces the number of assays needed when screening for infectious diseases. Polymerase chain reaction (PCR)-based assays are the most sensitive tests to diagnose malaria, but its high cost limits its use. We adapted a pooling platform that could reduce the number of assays needed to detect malaria infection. To evaluate this platform, two sets of 100 serum samples, with 1% and 5% malaria prevalence, were tested. DNA, extracted from pooled samples, was amplified by malaria-specific PCR. Additional validation was performed by determining the level of PCR detection based on 1:10 and 1:100 dilution. The platform correctly detected all malaria samples in the two test matrices. The use of stored serum samples also has important implications for studies investigating malaria prevalence rates retrospectively. Field studies, using serum and whole blood specimens, are needed to validate this technique for the adaptation of these methods for clinical utility.
Malaria Journal, 2010
Background: Malaria presents a diagnostic challenge in most tropical countries. Microscopy remains the gold standard for diagnosing malaria infections in clinical practice and research. However, microscopy is labour intensive, requires significant skills and time, which causes therapeutic delays. The objective of obtaining result quickly from the examination of blood samples from patients with suspected malaria is now made possible with the introduction of rapid malaria diagnostic tests (RDTs). Several RDTs are available, which are fast, reliable and simple to use and can detect Plasmodium falciparum and non-falciparum infections or both. A study was conducted in tribal areas of central India to measure the overall performance of several RDTs for diagnosis of P. falciparum and non-falciparum infections in comparison with traditional and molecular techniques. Such data will be used to guide procurement decisions of policy makers and programme managers.
Journal of the Egyptian Society of Parasitology, 2008
A real-time PCR assay with conventional microscopy by Giemsa-stained blood films was used. PCR was completed in an hour and identified the Plasmodium species in a single reaction. Blood was collected, and DNA was extracted. A genus-specific primer set corresponding to 18S ribosomal RNA was used to amplify target sequence. Fluorescence resonance energy technology hybridization probes were designed for P. falciparum over a region containing base pair mismatches allowed Plasmodium species differentiation. Microscopically positive patients (n = 60) were positive with real-time assay (100% sensitivity). 58 were single-species infections caused by P. falciparum; mixed infections (P. falciparum & P. vivax) were shown by real-time assay. Six out of 30 negative microscopy specimens were positive by real-time PCR (80% specificity). The discrepant results could be due to the subjective nature of microscopy and analytical objectivity of PCR, and high analytical sensitivity of real-time assay (1...