Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand
TL;DR: PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand, and data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather thanpoor performance of PCR.
Abstract: The main objective of this study was to compare the performance of nested PCR with expert microscopy as a means of detecting Plasmodium parasites during active malaria surveillance in western Thailand. The study was performed from May 2000 to April 2002 in the village of Kong Mong Tha, located in western Thailand. Plasmodium vivax (PV) and Plasmodium falciparum (PF) are the predominant parasite species in this village, followed by Plasmodium malariae (PM) and Plasmodium ovale (PO). Each month, fingerprick blood samples were taken from each participating individual and used to prepare thick and thin blood films and for PCR analysis. PCR was sensitive (96%) and specific (98%) for malaria at parasite densities ≥ 500/μl; however, only 18% (47/269) of P. falciparum- and 5% (20/390) of P. vivax-positive films had parasite densities this high. Performance of PCR decreased markedly at parasite densities <500/μl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/μl. Although PCR performance appeared poor when compared to microscopy, data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather than poor performance of PCR. These data are not unusual when the diagnostic method being evaluated is more sensitive than the reference method. PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand.
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TL;DR: RDTs are highlighted, including challenges in assessing their performance, internationally available RDTs, their effectiveness in various health care settings, and the selection of R DTs for different health care systems.
Abstract: To help mitigate the expanding global impact of malaria, with its associated increasing drug resistance, implementation of prompt and accurate diagnosis is needed. Malaria is diagnosed predominantly by using clinical criteria, with microscopy as the current gold standard for detecting parasitemia, even though it is clearly inadequate in many health care settings. Rapid diagnostic tests (RDTs) have been recognized as an ideal method for diagnosing infectious diseases, including malaria, in recent years. There have been a number of RDTs developed and evaluated widely for malaria diagnosis, but a number of issues related to these products have arisen. This review highlights RDTs, including challenges in assessing their performance, internationally available RDTs, their effectiveness in various health care settings, and the selection of RDTs for different health care systems.
489 citations
Cites background from "Comparison of PCR and microscopy fo..."
...Despite these strengths, microscopy possesses a number of limitations (4, 19, 25, 42, 43, 45, 56, 72, 97, 113, 114)....
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...While MRDTs would reduce logistical challenges in these studies, their parasitemia threshold of detection does not appear to be sufficiently low to be useful for asymptomatic screening (19, 57, 85)....
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TL;DR: A systematic review of endemic population surveys in which P. falciparum prevalence had been measured by both microscopy and a more-sensitive polymerase chain reaction (PCR)-based technique found that microscopy can miss a substantial proportion of P. Falconerum infections in surveys of endemic populations, especially in areas with low transmission of infection.
Abstract: Introduction. Light microscopy examination of blood slides is the main method of detecting malaria infection; however, it has limited sensitivity. Low-density infections are most likely to be missed, but they contribute to the infectious reservoir. Quantifying these submicroscopic infections is therefore key to understanding transmission dynamics and successfully reducing parasite transmission. Methods. We conducted a systematic review of endemic population surveys in which P. falciparum prevalence had been measured by both microscopy and a more-sensitive polymerase chain reaction (PCR)-based technique. The combined microscopy: PCR prevalence ratio was estimated by random-effects meta-analysis, and the effect of covariates was determined by meta-regression. Results. Seventy-two pairs of prevalence measurements were included in the study. The prevalence of infection measured by microscopy was, on average, 50.8% (95% confidence interval [CI], 45.2%-57.1%) of that measured by PCR. For gametocyte-specific detection, the microscopy prevalence was, on average, 8.7% (95% CI, 2.8%-26.6%) of the prevalence measured by PCR. A significantly higher percentage of total infections was detected by microscopy in areas of high, compared with low, transmission (74.5% when the prevalence determined by PCR was >75% versus 12.0% when the prevalence determined by PCR was <10%). Discussion. Microscopy can miss a substantial proportion of P. falciparum infections in surveys of endemic populations, especially in areas with low transmission of infection. The extent of the submicroscopic reservoir needs to be taken into account for effective surveillance and control.
487 citations
TL;DR: Without strategies accounting for P. vivax-specific characteristics, progress toward elimination of endemic malaria transmission will be substantially impeded.
Abstract: Plasmodium vivax is the most widespread human malaria, putting 2.5 billion people at risk of infection. Its unique biological and epidemiological characteristics pose challenges to control strategies that have been principally targeted against Plasmodium falciparum Unlike P. falciparum, P. vivax infections have typically low blood-stage parasitemia with gametocytes emerging before illness manifests, and dormant liver stages causing relapses. These traits affect both its geographic distribution and transmission patterns. Asymptomatic infections, high-risk groups, and resulting case burdens are described in this review. Despite relatively low prevalence measurements and parasitemia levels, along with high proportions of asymptomatic cases, this parasite is not benign. Plasmodium vivax can be associated with severe and even fatal illness. Spreading resistance to chloroquine against the acute attack, and the operational inadequacy of primaquine against the multiple attacks of relapse, exacerbates the risk of poor outcomes among the tens of millions suffering from infection each year. Without strategies accounting for these P. vivax-specific characteristics, progress toward elimination of endemic malaria transmission will be substantially impeded.
300 citations
TL;DR: Loop-mediated isothermal amplification, a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites and may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.
Abstract: Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.
282 citations
Cites background from "Comparison of PCR and microscopy fo..."
...Therefore, this situation can lead to false-negative results or unreliable species determinations (1)....
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TL;DR: A general pattern recognition framework to perform diagnosis, which includes image acquisition, pre-processing, segmentation, and pattern classification components, is described and the open problems are addressed.
Abstract: This paper reviews computer vision and image analysis studies aiming at automated diagnosis or screening of malaria infection in microscope images of thin blood film smears. Existing works interpret the diagnosis problem differently or propose partial solutions to the problem. A critique of these works is furnished. In addition, a general pattern recognition framework to perform diagnosis, which includes image acquisition, pre-processing, segmentation, and pattern classification components, is described. The open problems are addressed and a perspective of the future work for realization of automated microscopy diagnosis of malaria is provided.
168 citations
Cites background from "Comparison of PCR and microscopy fo..."
...Polymerase chain reaction (PCR) methods are known to be more sensitive and more specific than (manual) microscopy [19-21]....
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References
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TL;DR: This poster presents a probabilistic procedure to quantify the numbers of Gram-positive and Gram-negative parasites found in the blood of Malaria patients infected with E.coli.
1,409 citations
"Comparison of PCR and microscopy fo..." refers background in this paper
...In general, PCR was more sensitive and specific than examination of thick or thin blood smears, particularly in cases with low parasite rates or mixed infections [2,8,10,13-16]....
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TL;DR: Most new technology for malaria diagnosis incorporates immunochromatographic capture procedures, with conjugated monoclonal antibodies providing the indicator of infection, and clinical studies allow effective comparisons between different formats.
Abstract: Malaria presents a diagnostic challenge to laboratories in most countries. Endemic malaria, population movements, and travelers all contribute to presenting the laboratory with diagnostic problems for which it may have little expertise available. Drug resistance and genetic variation has altered many accepted morphological appearances of malaria species, and new technology has given an opportunity to review available procedures. Concurrently the World Health Organization has opened a dialogue with scientists, clinicians, and manufacturers on the realistic possibilities for developing accurate, sensitive, and cost-effective rapid diagnostic tests for malaria, capable of detecting 100 parasites/microl from all species and with a semiquantitative measurement for monitoring successful drug treatment. New technology has to be compared with an accepted "gold standard" that makes comparisons of sensitivity and specificity between different methods. The majority of malaria is found in countries where cost-effectiveness is an important factor and ease of performance and training is a major consideration. Most new technology for malaria diagnosis incorporates immunochromatographic capture procedures, with conjugated monoclonal antibodies providing the indicator of infection. Preferred targeted antigens are those which are abundant in all asexual and sexual stages of the parasite and are currently centered on detection of HRP-2 from Plasmodium falciparum and parasite-specific lactate dehydrogenase or Plasmodium aldolase from the parasite glycolytic pathway found in all species. Clinical studies allow effective comparisons between different formats, and the reality of nonmicroscopic diagnoses of malaria is considered.
1,290 citations
"Comparison of PCR and microscopy fo..." refers background or methods in this paper
...The limitations and shortcomings of microscopy are welldocumented [1,2], with significant problems existing even in fairly sophisticated laboratories....
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...Background The detection of asexual parasites by light microscopy of Giemsa-stained thick and thin films remains the standard laboratory method for the diagnosis of malaria [1,2]....
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TL;DR: Genus- and species-specific sequences are present within the small subunit ribosomal RNA genes of the four human malaria parasites that have proven to be more sensitive and accurate than by routine diagnostic microscopy.
836 citations
"Comparison of PCR and microscopy fo..." refers background or methods in this paper
...In general, PCR was more sensitive and specific than examination of thick or thin blood smears, particularly in cases with low parasite rates or mixed infections [2,8,10,13-16]....
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...Several experimental assays using various primers and extraction and detection techniques have been reported [8-12]....
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01 Jan 1996
764 citations
"Comparison of PCR and microscopy fo..." refers methods in this paper
...Epi-Info version 6 [26] was used to calculate test performance and acceptability evaluation indices of PCR, with microscopy used as the reference standard....
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TL;DR: Methods for collecting fingerstick blood onto filter paper strips that are air-dried, then stored and transported at room temperature and a nested PCR technique that has improved sensitivity and specificity are reported.
Abstract: As chloroquine resistance spreads across Africa, the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and proguanil are being used as alternative first-line drugs for the treatment and prevention of Plasmodium falciparum malaria. Resistance to these drugs is conferred by point mutations in parasite DHFR. These point mutations can be detected by polymerase chain reaction (PCR) assays, but better methods for sample collection, DNA extraction, and a diagnostic PCR are needed to make these assays useful in malaria-endemic areas. Here we report methods for collecting fingerstick blood onto filter paper strips that are air-dried, then stored and transported at room temperature. Cell lysis and DNA extraction are accomplished by boiling in Chelex-100. We also report a nested PCR technique that has improved sensitivity and specificity. These procedures readily detect mixed infections of parasites with both sensitive and resistant genotypes (confirmed by direct sequencing) and are reliable at parasite densities less than 250/mm3 in field surveys.
640 citations