Determination, mechanism and monitoring of knockdown resistance in permethrin-resistant human head lice, Pediculus humanus capitis

Abstract

Permethrin resistance has been reported worldwide and clinical failures to commercial pediculicides containing permethrin have likewise occurred. Permethrin resistance in head lice populations from the U.S. is widespread but is not yet uniform and the level of resistance is relatively low (~4-8 fold). Permethrin-resistant lice are cross-resistant to pyrethrins, PBO-synergized pyrethrins and to DDT. Nix((R)), when applied to human hair tufts following manufacture's instructions, did not provide 100% control when assessed by the hair tuft bioassay in conjunction with the in vitro rearing system. Resistance to permethrin is due to knockdown resistance (kdr), which is the result of three point mutations within the alpha-subunit gene of the voltage-gated sodium channel that causes amino acid substitutions, leading to nerve insensitivity.A three-tiered resistance monitoring system has been established based on molecular resistance detection techniques. Quantitative sequencing (QS) has been developed to predict the kdr allele frequency in head lice at a population level. The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance monitoring tool to screen a large number of louse populations as an alternative to conventional bioassay. As a secondary monitoring method, real-time PASA (rtPASA) has been devised for a more precise determination of low resistance allele frequencies. To obtain more detailed information on resistance allele zygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) has been developed as an individual genotyping method. Our approach of using three tiers of molecular resistance detection should facilitate large-scale routine resistance monitoring of permethrin resistance in head lice using field-collected samples.

Fig. 1

Comparative sodium current traces from Xenopus oocytes obtained before and after exposure to increasing concentrations of permethrin. Reproduced with permission from Yoon et al, 2008. Copyright 2008 Elsevier.

Fig. 2

Exon-intron structure of the 908-bp head louse VSSC genomic region that contains the M815I, T917I and L920F mutations. Shaded boxes and solid lines indicate exons and introns, respectively. Locations of three resistance mutations are marked with black circles. Vertical arrows indicate the approximate locations of intron polymorphisms. Horizontal arrows indicate the locations of the QS primers.

Fig. 3

Sequencing chromatograms of the standard template DNA mixtures with different ratios of resistant and susceptible alleles at the T917I mutation site. The numbers on the top of each column indicate the resistance allele ratios at each mutation site. The relative intensities of the resistance allele signals are indicated with arrows.

Fig. 4

Resistance sequence signal ratios obtained from the sense (A)- and antisense (B)-directional sequencing were plotted with corresponding resistance allele frequencies at the T917F mutation site. Quadratic regression lines are indicated by solid black lines with the upper and lower 95% prediction lines indicated by dotted red lines. Nucleotide signal ratio (x-axis) was calculated as [resistant nucleotide signal/(resistant nucleotide signal + susceptible nucleotide signal)].

Fig. 5

Diagram of the exon 3 fragment in the VSSC genomic DNA region that encompasses the T917I and L920F mutation sites. Locations of the two mutations are marked with black circles. Horizontal arrows indicate the locations of the rtPASA primers.

Fig. 6

Typical amplification patterns of rtPASA using the DNA templates containing 0~16% resistant alleles (A) and the regression line generated from the plot of normalized Ct value versus the log of resistance allele frequency (B). The regression line is indicated by a solid line with the upper and lower 95% prediction lines indicated by dashed lines in Panel B.

Fig. 7

Comparisons of the genotypic make up of head louse populations by SISAR. Red bars are homozygotes susceptible (SS), grey bars are heterozygotes (RS) and black bars are homozygote resistant (RR).