Dataset Information

Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping.

ABSTRACT: 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.

SUBMITTER: Drali R

PROVIDER: S-EPMC3405629 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping.

Drali Rezak R Benkouiten Samir S Badiaga Sékéné S Bitam Idir I Rolain Jean Marc JM Brouqui Philippe P

Journal of clinical microbiology 20120509 7

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 parao ...[more]

PMID: 22573588

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Project description:Human head lice are blood-sucking insects causing an infestation in humans called pediculosis capitis. The infestation is more prevalent in the school-aged population. Scalp itching, a common presenting symptom, results in scratching and sleep disturbance. The condition can lead to social stigmatization which can lead to loss of self-esteem. Currently, the mainstay of treatment for pediculosis is chemical insecticides such as permethrin. The extended use of permethrin worldwide leads to growing pediculicide resistance. The aim of this study is to demonstrate the presence of the knockdown resistance (kdr) mutation in head lice populations from six different localities of Thailand. A total of 260 head lice samples in this study were collected from 15 provinces in the 6 regions of Thailand. Polymerase chain reaction (PCR) was used to amplify the α subunit of voltage-sensitive sodium channel (VSSC) gene, kdr mutation (C→T substitution). Restriction fragment length polymorphism (RFLP) patterns and sequencing were used to identify the kdr T917I mutation and demonstrated three genotypic forms including homozygous susceptible (SS), heterozygous genotype (RS), and homozygous resistant (RR). Of 260 samples from this study, 156 (60.00%) were SS, 58 (22.31%) were RS, and 46 (17.69%) were RR. The overall frequency of the kdr T917I mutation was 0.31. Genotypes frequencies determination using the exact test of Hardy-Weinberg equilibrium found that northern, central, northeastern, southern, and western region of Thailand differed from expectation. The five aforementioned localities had positive inbreeding coefficient value (Fis > 0) which indicated an excess of homozygotes. The nucleotide and amino acid sequences of RS and RR showed T917I and L920F point mutations. In conclusion, this is the first study detecting permethrin resistance among human head lice from Thailand. PCR-RFLP is an easy technique to demonstrate the kdr mutation in head louse. The data obtained from this study would increase awareness of increasing of the kdr mutation in head louse in Thailand.

Project description:BackgroundHead louse females secrete liquid glue during oviposition, which is solidified to form the nit sheath over the egg. Recently, two homologous proteins, named louse nit sheath protein (LNSP) 1 and LNSP 2, were identified as adhesive proteins but the precise mechanism of nit sheath solidification is unknown.MethodsWe determined the temporal transcriptome profiles of the head louse accessory glands plus oviduct, from which putative major structural proteins and those with functional importance were deduced. A series of RNA interference (RNAi) experiments and treatment of an inhibitor were conducted to elucidate the function and action mechanism of each component.ResultsBy transcriptome profiling of genes expressed in the louse accessory glands plus uterus, the LNSP1 and LNSP2 along with two hypothetical proteins were confirmed to be the major structural proteins. In addition, several proteins with functional importance, including transglutaminase (TG), defensin 1 and defensin 2, were identified. When LNSP1 was knocked down via RNA interference, most eggs became nonviable via desiccation, suggesting its role in desiccation resistance. Knockdown of LNSP2, however, resulted in oviposition failure, which suggests that LNSP2 may serve as the basic platform to form the nit sheath and may have an additional function of lubrication. Knockdown of TG also impaired egg hatching, demonstrating its role in the cross-linking of nit sheath proteins. The role of TG in cross-linking was further confirmed by injecting or hair coating of GGsTop, a TG inhibitor.ConclusionsBoth LNSP1 and LNSP2 are essential for maintaining egg viability besides their function as glue. The TG-mediated cross-linking plays critical roles in water preservation that are essential for ensuring normal embryogenesis. TG-mediated cross-linking mechanism can be employed as a therapeutic target to control human louse eggs, and any topically applied TG inhibitors can be exploited as potential ovicidal agents.

Dataset Information

Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping.

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.

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