PCR-based methods to the diagnosis of imported malaria

Service de Parasitologie-Mycologie, Hôpital Rangueil, Centre Hospitalier Universitaire de Toulouse, TSA 50032, 31059 Toulouse 9, France.
Parasite (Impact Factor: 1.09). 10/2008; 15(3):484-8. DOI: 10.1051/parasite/2008153484
Source: PubMed


Rapid and precise diagnosis of malaria is needed to take care febrile patient returning from endemic areas. Since the first description of the diagnosis of Plasmodium infection by polymerase-chain-reaction (PCR), the role of this kind of molecular method in the laboratory diagnosis of imported malaria is still a topical question. PCR-based assays were found to be more sensitive and more specific than all conventional methods. The highest contribution of the molecular diagnosis is that a PCR negative result would ascertain the lack of any malaria infection, thus quickly orienting the investigations toward other aetiology. This technique should be now considered as the gold standard for the diagnosis of imported malaria.

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Available from: Antoine Berry, May 04, 2015
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    • "Assays such as real-time PCR (qPCR) are more sensitive and specific than microscopy and mRDTs [8,9], and have allowed for explicit identification and quantification of malaria parasites [8-13]. Most of the malaria qPCR assays target the multicopy 18S ribosomal RNA (rRNA) genes [14] with detection limits ranging from 0.002 to 30 parasites/μL [10,14]. "
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    ABSTRACT: 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. A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-ReadyTM 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[degree sign]C, room temperature (RT), 37[degree sign]C and 42[degree sign]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 CT values and the standard deviations (SD) were used in the analysis of the assay performance. The limit of detection for the MMSR assay was 0.244 parasites/muL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/muL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37[degree sign]C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. The MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.
    Full-text · Article · Apr 2014 · Malaria Journal
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    • "Indeed, it has been shown that, in areas with low or very low intensity of malaria transmission, sub-microscopic infections were commonly seen [35,36]. It would be important to improve this detection using more sensitive methods such as gene amplification by Polymerase Chain Reaction (PCR) [37-39]. Nevertheless, the presence of P. falciparum infection has been revealed with microscopic detection, suggesting that malaria transmission occurs during the dry season in this area. "
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    ABSTRACT: The Northern part of Senegal is characterized by a low and seasonal transmission of malaria. However, some Plasmodium falciparum infections and malaria clinical cases are reported during the dry season. This study aims to assess the relationship between IgG antibody (Ab) responses to gSG6-P1 mosquito salivary peptide and the prevalence of P. falciparum infection in children during the dry season in the Senegal River Valley. The positive association of the Ab response to gSG6-P1, as biomarker of human exposure to Anopheles vector bite, and P. falciparum infectious status (uninfected, infected-asymptomatic or infected-symptomatic) will allow considering this biomarker as a potential indicator of P. falciparum infection risk during the dry season. Microscopic examination of thick blood smears was performed in 371 and 310 children at the start (January) and at the end (June) of the dry season, respectively, in order to assess the prevalence of P. falciparum infection. Collected sera were used to evaluate IgG response to gSG6-P1 by ELISA. Association between parasitological and clinical data (infected-asymptomatic or infected-symptomatic) and the anti-gSG6-P1 IgG levels were evaluated during this period. The prevalence of P. falciparum infection was very low to moderate according to the studied period and was higher in January (23.5%) compared to June (3.5%). Specific IgG response was also different between uninfected children and asymptomatic carriers of the parasite. Children with P. falciparum infection in the dry season showed higher IgG Ab levels to gSG6-P1 than uninfected children. The results strengthen the hypothesis that malaria transmission is maintained during the dry season in an area of low and seasonal transmission. The measurement of IgG responses to gSG6-P1 salivary peptide could be a pertinent indicator of human malaria reservoir or infection risk in this particular epidemiological context. This promising immunological marker could be useful for the evaluation of the risk of P. falciparum exposure observed during dry season and, by consequences, could be used for the survey of potential pre-elimination situation.
    Full-text · Article · Aug 2013 · Malaria Journal
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    • "PCR offers several advantages over microscopy in diagnosis of malaria in several regards. First, PCR is both highly sensitive and highly specific allowing explicit identification of malarial species [9], [10]. PCR can also be used for precise parasite quantification through qPCR methods. "
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    ABSTRACT: We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.
    Full-text · Article · Aug 2013 · PLoS ONE
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