Introduction

In 2020, infestations with all types of human lice (head, body, and pubic lice) were considered a global public health problem. Notably, 19% of schoolchildren worldwide are estimated to be infected with Pediculus humanus capitis1. Several factors contribute to the persistence of lice infestations, including a lack of patient compliance with treatment instructions and poor drug efficacy against some stages of lice, which frequently lead to re-infestation2.

Pyrethroids are the most common over-the-counter insecticides prescribed for the treatment of head lice. They affect the nervous system of lice by keeping voltage-gated sodium channels open for long periods. Consequently, spastic paralysis occurs, leading to the death of lice2.

Permethrin is the most widely used over-the-counter pesticide for the treatment of human head lice. It is most often used as a 1% cream, while pyrethrin is used as a 0.33% shampoo or mousse formulation. Both agents interfere with sodium channel currents, leading to a delay in nerve depolarization. The treatment is usually applied to the hair for 10 min and repeated a week later to kill previously unhatched lice. However, a major concern is that, because of their widespread use, head lice have become resistant to both pyrethrin and permethrin3,4,5. There is significant evidence of drug resistance, including reports of treatment failures2, though the extent of resistance in lice varies between countries6.

Head lice resistant to pyrethroids contain knockdown resistance (KDR) point mutations that involve amino acid substitutions at M815I, T917I, and L920F on the α subunit of the voltage-sensitive sodium channel (VSSC) gene7,8. The M815I and L920F mutations alone reduce the susceptibility of lice to permethrin compounds by 2- to 3-fold, whereas the T917I mutation, either alone or in combination with others, totally mitigates the effect of permethrin9,10.

Several studies have monitored the presence of KDR mutations in head lice isolated from different regions (Table 1). In this study, we aimed to determine pyrethroid resistance genes in lice samples isolated from schoolchildren in Riyadh, Saudi Arabia.

Table 1 Summary of previous studies investigating KDR mutations in head lice.
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Results

The objective of this research was to investigate the presence of mutations in the VSSC gene in head lice, to determine if our collected samples were susceptible to pyrethroid insecticides or developed resistance against them.

After applying the Sanger sequencing, four mutations were detected in the genetic sequences of the samples (T917I, L920F, V966F, and F967L). Analysis of the mutations revealed significant differences in the distribution ratio of genotypes in the studied lice samples, which were explained in (Table 2).

Table 2 Distribution of KDR-like alleles in head lice populations in Riyadh.
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Substitution of KDR T917I was detected in 99% of the samples. Sixty six were homozygous resistant (RR) genotypes, 33% were heterozygotes resistant (RS), and 1% were homozygous susceptible (SS). Three new mutations were detected in L920H, V966F, and F967L. L920H showed no RS individuals. RR and SS were found in proportions of (75%, 80%, 70%) and (25%, 0%, 0%), respectively. The RR genotype was significantly higher in V966F and F967L mutations, with a percentage roughly centered around 70–80%, then RS 21 and 31 for successive mutations with no SS genotype detected for either mutation.

The statistical examination showed negative values ranging from − 0.15 to 1 in the Fis values, indicating an excess of heterozygosity for T917I, V966F, and F967L. The excess indicates that the observed heterozygosity is greater than the expected heterozygosity under Hardy–Weinberg equilibrium, also, there is a value of Fis in L920H greater than 0, which indicates that there is an excess of homozygotes for this mutation (Table 2).

For the three mutations T917I, V966F, and F967L, the chi-square values did not exceed the critical value of 3.841, indicating that the observed genotype frequencies for these mutations do not differ significantly from what would be expected under Hardy–Weinberg equilibrium. The remaining mutation L920H had chi-square values that exceeded the critical value, and thus the observed genotype frequencies differ from what would be expected.

Using the MEGA software, the nucleotide sequencing of the VSSC gene in the studied samples was aligned and compared to the wild-type sequence of Pediculus human capitis from the GeneBank database.

According to the chromogram result, the first mutation T917I involves the substitution ACA > ATA, thus leading to the change of threonine (Thr) to isoleucine (Ile) amino acids at position 917 in the gene sequencing. The mutation L920H occurs by substitution at position 920 from CTT > CAT instead of leucine (Leu) to histidine (His). The mutation, V966F involves a change in GTT > TTT, after that, a change in valine (Val) to Phe. Finally, the mutation F967L has substitution nucleotides in TTT > TTA then changes at amino acids Phe to Leu. Notably, all mutations were associated with homozygous resistance (Fig. 1).

Fig. 1
figure 1

The nucleotide substitution mutations within VSSC gene of head lice were collected from Riyadh. MEGA software was used to find and visualize the position of KDR gene mutations in T917I, L920H, and F967L.

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Discussion

Head lice are the most common infection caused by ectoparasites and infect humans worldwide. Infestations are particularly prevalent among schoolchildren aged 3–11 years, causing skin irritation, anxiety, and an increased risk of infection for those around them20. Many insects use similar resistance mechanisms that some specialists fail to determine, when treatment fails. They attribute the failure to re-infestation or lack of commitment to using the treatment. While this may be possible, we cannot ignore the effect of resistance on treatment failure21.

To the best of our knowledge, this is the first study aiming to discover the resistance of pyrethroids in samples collected from schoolchildren in Riyadh. Sanger sequencing was used to detect pyrethroid resistance by targeting the VSSC gene.

Four mutations were found in this study. T917I showed a high percentage of resistant mutations of the two genotypes RR (66%) and RS (33%), with a greater prevalence in the genotype RR, and a low percentage of SS genotype (1%). T917I is a molecular biomarker of the presence of a pyrethroid resistance mutation, either if it is found alone or with other mutations9,10.

The frequency of resistance alleles in T917I is 0.83 in the present study, which is notably higher than the frequency recorded in Jeddah of 0.6211. Of the resistant genotypes, we found 33% heterogenous alleles, compared to the absence of heterozygosity in Jeddah. This may be attributed to the larger sample size collected in Riyadh than in Jeddah. According to the Center of Education Statistics and Decision Support on the Ministry of Education website 2023, the number of students in elementary schools in the Riyadh region is nearly double (636,634 students) that in Jeddah (340,722)22. This means that Riyadh has a high population in the age group targeted by head lice.

The differences in resistance frequencies between the present study and other studies may be due to several genetic, social, and environmental factors. High frequency of resistance was reported in the United States and Canada (88–98%;16), France (57%;14), Mexico (62%;15), Argentina (88%;19) beside Saudi Arabia (62.2–83%;11) and (83%, in the current study), may be attributed to the high levels of urbanization in these countries. This may impede lice control efforts, by reducing the effectiveness of pyrethroid-based treatments.

The difference between our study and the study in Thailand may be attributed to a cultural difference18, which is that the Thai population relies heavily on traditional treatments, especially in rural areas of Thailand. In areas that contain a lower frequency of the resistance allele, lice may still be susceptible to pyrethroids compared to Riyadh. The high rate of resistance in Riyadh city is an indication of the need to produce alternative treatments to control the infection.

Most studies in KDR mutations reveal different levels of resistant alleles in T917I. This may be affected by the amount and duration of exposure to the insecticide. In addition, contact between infected people carrying resistant lice with non-carriers increases the spread and reproduction of resistant lice10,11,13,14,15,16,17,18,19.

Previously, a mutation of L920F was identified for its involvement in increasing pyrethroid resistance10,19,23. This mutation is similar to L920H, which is detected in this study. This suggests that this L920H may have the same role as L920F.

The results of the chi-square analysis show us important information about the genetic mutations, for the three mutations (T917I, V966F, F967L) and their values below the critical value, this is evidence that they agree with Hardy–Weinberg expectations. That is, the population is experiencing random mating, not controlled by any pressures affecting allele frequencies.

In contrast, the L920H mutation showed chi-square values above the critical threshold, indicating a large deviation from Hardy–Weinberg equilibrium. This suggests the possible influence of factors causing pressure on allele frequencies, leading to an overrepresentation of homozygotes.

Two other novel mutations were identified in this study (V966F and F967L), which have not been reported previously and are expected to also have an effect on pyrethroid resistance. Although the role of the mutations has not yet been determined, they are located in the gene containing the resistance-causing mutations, which increases the possibility that they are also involved in resistance.

It is concluded that the widespread use of insecticides, rather than traditional treatments, has led to the development of insecticide resistance, which causes treatment failure. In addition, the diverse population in these countries, due to the cultural openness, has contributed to the spread of various species of head lice among schoolchildren. A treatment alternative is strongly needed to control head lice infestations in urban countries.

Materials and methods

Study samples

One hundred head lice were collected from six to 12-year-old schoolchildren at the Spotless Hair Center in Riyadh City, Saudi Arabia. Samples were obtained from children who visited the center for treatment, using a metal comb specialized for removing head lice. Most visitors to the center were children, though some adults were infected by their children. This study was approved by the Biomedical Ethics Committee of King Abdulaziz University Faculty of Medicine (reference no: 130-2). All methods were performed in accordance with relevant guidelines and regulations. The guardians of all children provided informed consent for their participation in this study.

DNA extraction

DNA was extracted from dead stage III larva and the adult stages of head lice using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol.

PCR amplification of KDR gene mutation

The amplification of a 332-bp portion of the VSSC α-subunit was done by using the Dream Taq Hot Green Start Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Twenty microliters of the reaction mixture for each sample contained 10 µL of Master Mix, 1 µL of the primer (5′-AAATCGTG GCCAACGTTAAA-3′ (sense) and 5′-TGAATCCATTCACCGCATAA-3′ (antisense)), and variable amounts of deionized water and the DNA extract, depending on the concentration of DNA in the sample. PCR amplification was conducted in a 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA) with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and the final extension at 72 °C for 7 min14. The PCR products were analyzed using agarose gel electrophoresis at 135 V for 30 min using the Mupid-exU system, 2 g of agarose powder, and 100 mL of Tris–borate-EDTA buffer, with 4 µL of the sample in each well.

Sanger sequencing for the samples

During Sanger sequencing, dideoxynucleoside triphosphate is modified with fluorescent dyes, allowing the detection and identification of terminal fragments in the sequencing process24. The resulting sequences were aligned and visualized using Molecular Evolutionary Genetics Analysis version 11. All sequences were compared with AY191156.1 in Gene Bank.

Statistical analysis

To determine the frequency of each genotype (RR, RS, and SS) in the collected samples, all lice belonging to each genotype were counted and divided by the total number of analyzed lice and examined with Hardy–Weinberg expectations of Wright’s inbreeding coefficient using the following equation:

$$F_{is} = 1 - \frac{{H_{O} }}{{H_{E} }}$$

(\({H}_{O}\): number of observed heterozygote genotypes; \({H}_{E}\): Number of expected heterozygote genotypes). Deviations were detected by using Hardy–Weinberg ratios. The chi-square (χ2) test was used to evaluate the distribution of genotypes in the samples compared to the values obtained using the Hardy–Weinberg test.