Distribution patterns of three sodium channel mutations associated with pyrethroid resistance in Rhipicephalus (Boophilus) microplus populations from North and South America, South Africa and Australia

AbstractResistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with target site pyrethroid resistance. We developed a multiplex PCR using allele-specific primers to amplify wild type or mutated genotypes of the three mutations simultaneously. This assay was used to screen tick samples originating from Brazil, Argentina, Mexico, South Africa and Australia whose phenotype to flumethrin and cypermethrin had been determined by the use of the Larval Tarsal test (LTT) or the Larval Packet Test (LPT). These mutations were found to have distinct geographical distributions and result in different resistance phenotypes. The L64I Domain II mutation conferring resistance to several SP compounds was found in all the Brazilian, Argentinean and Australian populations and in one South African population, with frequencies between 38% and 100% in flumethrin and cypermethrin resistant populations. In contrast, this mutation was not found in samples from Mexico, while the Domain III mutation was found exclusively in this country. The G72V Domain II flumethrin-specific mutation was found in a single Australian population, with a very low resistant allele frequency (3%). The homozygous resistant RR genotype of the L64I Domain II mutation correlated significantly with the survival rates at the discriminating doses of flumethrin and cypermethrin. This survey shows the widespread distribution of the L64I Domain II mutation and provides evidence of its geographic separation from the Domain III mutation

PCR Diagnosis of Opisthorchis viverrini and Haplorchis taichui Infections in a Lao Community in an Area of Endemicity and Comparison of Diagnostic Methods for Parasitological Field Surveys▿

Opisthorchiasis is a major public health problem in Southeast Asia. Affected individuals often have mixed infections with the liver fluke Opisthorchis viverrini and minute intestinal flukes such as Haplorchis taichui. The usual methods of diagnosing these infections involve the demonstration of fluke eggs in stool samples under light microscopy, but sensitivity and specificity are low. We developed two PCR tests that detect and discriminate between O. viverrini and H. taichui infections. PCR tests were validated by stool samples from purged individuals. We then applied the PCR tests to estimate the prevalence of O. viverrini and H. taichui infections from a random sample of individuals selected from a community in an area of endemicity in Khong District, Laos. PCR results were compared with those from the Kato-Katz (KK) method and the formalin-ether concentration technique (FECT). When validated with purge results, PCR tests of O. viverrini and H. taichui had sensitivities of 93.7% (95% confidence interval [CI], 85.8 to 97.9%) and 73.3% (95% CI, 60.3 to 83.9%) and could detect as little as 0.75 pg DNA and 1.32 ng DNA, respectively. The PCR-determined community prevalences of O. viverrini and H. taichui infections were 63.9% (95% CI, 54.1 to 72.9%) and 30.6% (95% CI, 22.1 to 40.2%), respectively. Using PCR as the gold standard to detect O. viverrini, three KK thick smears performed comparably well, whereas one KK smear and FECT were poorer (sensitivities of 91.4% [95% CI, 81.0 to 97.1%,], 62.3% [95% CI, 49.8 to 73.7%], and 49.3% [95% CI, 37.0 to 61.6%], respectively). PCR may be a valuable and sensitive diagnostic tool, particularly for low-intensity O. viverrini and H. taichui infections

Geometric mean fecal egg counts according to the sampling effort.

<p>Geometric mean fecal egg counts before and after PZQ treatment, by the number of days of stool specimen collection (x-axis), based on children diagnosed “infected” following maximum Kato-Katz thick smear sampling effort. (a) <i>S. mekongi</i> infected at baseline (day 0), <i>n</i> = 79; days 28–30 after treatment, <i>n</i> = 14; and (b) <i>O. viverrini</i> infected at baseline (day 0), <i>n</i> = 89; days 28–30 after treatment, <i>n</i> = 11. Each point on a curve represents the geometric mean fecal egg count for each sampling effort (number of Kato-Katz thick smears examined per stool specimen).</p

Solicited adverse events reported 24 hours following PZQ administration (<i>n</i> = 93).

*<p>according to exact χ² test.</p><p>The two study groups were 40 mg/kg <i>vs.</i> 75 mg/kg divided into 2 doses of 50 mg/kg+25 mg/kg, 4 hours apart.</p

Cumulative prevalence according to the sampling effort.

<p>Cumulative infection prevalences for (a) <i>S. mekongi</i> and (b) <i>O. viverrini</i> by the number over consecutive days of stool specimen collection (x-axis). Each point on a curve represents a cumulative prevalence value for each sampling effort (number of Kato-Katz thick smears per stool specimen). At baseline (day 0), <i>n</i> = 90; after treatment (days 28–30), <i>n</i> = 66.</p

Sensitivity of different sampling efforts to detect <i>S. mekongi</i> and <i>O. viverrini</i> infections.

<p>Study was carried out among 93 children in primary and secondary schools on Don Long Island, Khong district, Champasack province, Lao PDR in February and March 2007. Sensitivity is compared before (<i>n</i> = 90) and after PZQ administration (<i>n</i> = 66), using the maximum sampling effort as the diagnostic ‘gold’ standard for the following sampling efforts: 1×1 sampling effort examines the first Kato-Katz thick smear only; 1×3 examines the first stool specimen by triplicate Kato-Katz thick smears; 3×1 examines 3 stool specimens by a single Kato-Katz thick smear for each specimen.</p

<i>S. mekongi</i> infection intensity before (D0) and posttreatment (D28) and egg reduction rate for maximum and minimum diagnostic effort.

<p>EPG, eggs per gram of stool; ERR, egg reduction rate; GM, geometric mean; na, not applicable.</p

Flowchart of subjects with cure and egg reduction rates.

<p>Cure and egg reduction rates are presented for <i>O. viverrini</i> and <i>S. mekongi</i> infections following 40 mg/kg and 75 mg/kg (50 mg/kg+25 mg/kg 4 hours apart) PZQ treatment considering (a) the maximum sampling effort (3×3, 3 stool specimens with triplicate Kato-Katz thick smears per specimen); (b) the minimum sampling effort (1×1, single Kato-Katz thick smear from the first stool specimen).</p

<i>O. viverrini</i> infection intensity before (D0) and posttreatment (D28) and egg reduction rate for maximum and minimum diagnostic effort.

<p>EPG, eggs per gram of stool; ERR, egg reduction rate; GM, geometric mean; na, not applicable.</p