An innovative tool for moving malaria PCR detection of parasite reservoir into the field

Abstract

BackgroundTo achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field.MethodsDNA extraction and real-time PCR assays were implemented in an “in-house” designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831).ResultsThe real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24–48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%.ConclusionsThe operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture.

Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots Versus Venous Blood Samples

“…The 5 μL DBS samples were lysed overnight with hepes buffered saline (HBS) 1 + /Saponin 0.5% and DNAs were extracted using the Bio-Rad Instagene matrix (Bio-Rad, Singapore) as previously described. 15 The DNAs from the 50 and 200 μL aliquots were extracted using the QiaAmp DNA blood mini kit (Qiagen, Courtaboeuf, France) and 1 mL aliquots were extracted using the QiaAmp DNA blood midi kit (Qiagen) following manufacturer's recommendations. All DNA samples were eluted with 200 μL buffer AE.…”

“…Details on the PCR program, mix composition, and primer sequences have been described in Canier and others. 15 Real-time PCR assays were followed by a melt curve analysis.…”

“…Here, the detection limit of malaria polymerase chain reaction (PCR) assays, and then the prevalence of parasite carriers in a set of field samples, were evaluated by using different volumes of blood and DNA extraction methods, by comparing the following conditions: 1) DNA extracted from dried blood spots (DBS) (5 μL) with a fast and inexpensive extraction method, commonly performed in large-scale epidemiological studies in malaria-endemic countries 15 ; 2 and 3) DNA extracted from 50 μL and 200 μL venous blood using the Qiagen mini kit based extraction method (Courtaboeuf, France), commonly used in research studies; 4) DNA extracted from 1 mL venous blood using the Qiagen midi kit based extraction method, representing here the gold standard. To this end, 521 samples were collected in Preah Vihear province, a low malaria transmission area (north eastern Cambodia) and aliquoted into four volumes: 5 μL capillary blood spot onto filter paper, 50 μL, 200 μL, and 1 mL venous blood.…”

Reduced Polymorphism in the Kelch Propeller Domain in Plasmodium vivax Isolates from Cambodia

“…Briefly, parasite DNA was extracted from whole blood using the QIAamp DNA blood minikit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. The parasite species was confirmed by real-time PCR, as described by Canier et al (20). The first round of PCR amplification was performed with a 20-l reaction mixture containing 5 l DNA, 0.25 M each primer, 2.5 mM MgCl 2 , and 0.25 l HOT FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia) under the following conditions: 95°C for 15 min, followed by 20 cycles at 95°C for 30 s, 58°C for 60 s, and 72°C for 130 s, and a final extension at 72°C for 10 min.…”

A Review of Plasmodium knowlesi among Macaques in Malaysia

“…Other PCR-based amplification assays for identification of malaria include multiplex PCR (Kho et al, 2003;Patsoula et al, 2003), real-time or quantitative PCR (qPCR) (Rougemont et al, 2004), and loop-mediated isothermal amplification (LAMP) (Han et al, 2007). These methods are simpler, less time consuming, less labour intensive and better quantitative diagnostic tools in comparison to nested PCR (Canier et al, 2013). These techniques are more reliable for accurate species detection (Erdman and Kain, 2008) and also can be a helpful tool in identifying drug-resistant parasites and follow-up therapeutic response (Berry et al, 2008;Tavares et al, 2011).…”