Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

Safeukui, Innocent; Millet, Pascal; Boucher, Sébastien; Melinard, Laurence; Fregeville, Frédéric; Receveur, Marie‐Catherine; Pistone, Thierry; Fialon, P.; Vincendeau, Philippe; Fleury, Hervé; Malvy, Denis

doi:10.1186/1475-2875-7-70

Cited by 38 publications

(39 citation statements)

References 34 publications

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“…Since its establishment in 1997 (19), this method has been particularly useful in detecting known mutations causing human diseases such as cancer (27) and other disorders (30). The melting curve analysis genotyping technique is also used in other fields, such as molecular haplotyping (21), bacteriology (22), parasitology (26), and virology (25). Using this method allowed us to determine the genotype of human lice at each mutation site.…”

Section: Discussionmentioning

confidence: 99%

Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

et al. 2012

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Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) ␣ subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the ␣ subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies.T he human body louse Pediculus humanus corporis (P. h. corporis) is a hematophagous ectoparasite that lives and multiplies in clothing. Body lice are vectors of 3 major infectious diseases: epidemic typhus caused by Rickettsia prowazekii, relapsing fever caused by Borrelia recurrentis, and trench fever caused by Bartonella quintana (24). The poor living conditions and crowded situations in homeless, war refugee, or natural disaster victim populations provide ideal conditions for the spread of lice (23). Body louse infestation has been observed in 22% of the sheltered homeless population in Marseille, France, causing trench fever, epidemic typhus, and relapsing fever (4), and coinfestation with head lice is often noted. Consequently, all measures that can be used to decrease the burden of these ectoparasites in homeless people are warranted to avoid the spread and/or outbreak of these diseases. Clothing change as well as the use of ivermectin to eradicate the body lice in a cohort of homeless individuals in Marseille was unsuccessful (10), suggesting that the complete eradication of this ectoparasite is a true challenge (1, 5). It was therefore essential for us to investigate other strategies to reach this goal.During the 1980s, a study conducted by the U.S. Army demonstrated that the use of uniforms impregnated with 0.125 mg/ cm 2 permethrin effectively eradicated body lice (28). The resistance to permethrin, ...

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Section: Discussionmentioning

confidence: 99%

Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

et al. 2012

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“…Low parasitemia samples can be difficult to detect using single-step DNA PCR approaches, 14 although reports vary. 8 Some RNA assays have improved sensitivities at low parasite densities because of the increased copy number relative to coding DNA. 22 Third, many assays do not include a multiplexed internal control, which is critical for ruling out PCR inhibitors in every sample.…”

Section: Discussionmentioning

confidence: 99%

“…42 There are many factors to consider when choosing from the diverse molecular methods reported for malaria. [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] First, pre-analytical handling steps (such as leukocyte filtering reported in some assays) should be minimized or highly controlled to reduce the risk of contamination. Second, as the sample volume decreases, the limit of detection increases.…”

Section: Discussionmentioning

confidence: 99%

“…4 A variety of molecular methods of malaria parasite detection were developed in the past 20 years and include singlestep and nested polymerase chain reaction (PCR) with gel detection, RNA-specific nucleic acid-based sequence amplification (NASBA), and probe-based real-time PCR and reverse transcription (RT)-PCR (using various combinations of genus-or species-specific primers and TaqMan probes or molecular beacons). [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Species identification can be achieved by SYBR Green melting curve analysis, 20 by real-time PCR with species-specific probes, 5,6 or most sensitively using laborintensive species-specific nested PCR. 24,25 In addition to diagnosing clinical cases with low parasite densities, some species-specific methods can detect pre-patent parasitemia in samples from field studies and from malaria human challenge trials.…”

Section: Introductionmentioning

confidence: 99%

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Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

Murphy¹,

Prentice²,

Williamson³

et al. 2012

The American Society of Tropical Medicine and Hygiene

104

133

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Abstract. To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified 98.7% of parasite-containing samples to ±0.5 log 10 parasites/mL of the nominal value without false positives. The analytical sensitivity was 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1 10 4 copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 µL) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.

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“…Over the past 20 years, many molecular assays for malaria have been developed. [72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91] Among published methods, 65 primer sets have been reported with at least five molecular targets used to test for as many as five human malaria species. Methods include single-step PCR with electrophoresis gel-based detection or DNA probe-based real-time detection, nested PCR with gel-based or real-time SYBR Green dye-based detection, nucleic acid-based sequence amplification (NASBA) and real-time RT-PCR with probe-based detection.…”

Section: Molecular Assaysmentioning

confidence: 99%

Malaria Diagnostics in Clinical Trials

Murphy¹,

Shott²,

Parikh³

et al. 2013

The American Society of Tropical Medicine and Hygiene

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Abstract. Malaria diagnostics are widely used in epidemiologic studies to investigate natural history of disease and in drug and vaccine clinical trials to exclude participants or evaluate efficacy. The Malaria Laboratory Network (MLN), managed by the Office of HIV/AIDS Network Coordination, is an international working group with mutual interests in malaria disease and diagnosis and in human immunodeficiency virus/acquired immunodeficiency syndrome clinical trials. The MLN considered and studied the wide array of available malaria diagnostic tests for their suitability for screening trial participants and/or obtaining study endpoints for malaria clinical trials, including studies of HIV/ malaria co-infection and other malaria natural history studies. The MLN provides recommendations on microscopy, rapid diagnostic tests, serologic tests, and molecular assays to guide selection of the most appropriate test(s) for specific research objectives. In addition, this report provides recommendations regarding quality management to ensure reproducibility across sites in clinical trials. Performance evaluation, quality control, and external quality assessment are critical processes that must be implemented in all clinical trials using malaria tests.

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Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

Cited by 38 publications

References 34 publications

Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

Malaria Diagnostics in Clinical Trials

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