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Periodicity, the cyclical rise and fall in microfilaria (mff) numbers in the peripheral blood over time, is observed in many filarial infections. It is correlated with the necessity for these larval stages to be ingested by the blood feeding vector before they can be transmitted to a new vertebrate host. Microfilariae of the dog heartworm Dirofilaria immitis have been described to show periodicity, but the circadian pattern does not seem to be consistent. Most publications describe the lowest mff-concentrations in the peripheral blood in the early morning, while the highest counts occurred either in the afternoon, in the late evening or shortly after midnight. Sixteen dogs were experimentally infected with D. immitis isolates originating from Italy (one isolate, 14 dogs), and the USA (two isolates, one dog each). The dogs were housed indoors with a natural light source (windows) and heating that prevented temperature-drops below 20 °C during winter. When patency was reached, blood samples were collected at weekly and monthly intervals over a period of up to 3 years, and at given hours of the day (morning, noon, evening) for the duration of one year in order to determine seasonal, as well as daily variations of microfilaremia. Despite the fact that the dogs were kept indoors, there was an apparent seasonality of the D. immitis-microfilaremia, with peaks in summer and 5–49-times lower counts in winter. This difference was statistically significant and the ratio remained constant over the years, regardless of the fact that the mff-counts increased from the first to the second year of patency. Since the temperature was kept constantly in a range between 20 to 26 °C (with some single outliners in both directions) the climatic conditions may not explain this observation. Therefore, day length may be the most obvious reason for the seasonality in the given study set-up. Interestingly, the Italian D. immitis-isolate lost seasonality after three passages of experimental infections in dogs. The circadian cycle of mff in the peripheral blood varied considerably between dogs and season. There was no consistent or apparent pattern, which led to the conclusion that many individual factors seem to influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.

Infestations with ticks have an important economic impact on the cattle industry worldwide and resistance to acaricides has become a widespread phenomenon. To optimize their treatment strategy, farmers need to know if and against which classes potential acaricide-resistance does occur. Bioassays are used to assess the resistance level and pattern of Rhipicephalus (Boophilus) microplus populations. The objective of the current study was to assess the susceptibility of field populations originating from Argentina (8), South Africa (3), and Australia (2) using the Larval Tarsal Test. Nine acaricidal compounds from five major classes were tested: organosphosphates, synthetic pyrethroids (SP), macrocyclic lactones, phenylpyrazols, and amidines. The resistance ratios at concentrations inducing 50 and 90% mortality were used to detect established and emerging resistance. This study confirmed the newly reported presence of amitraz resistance in populations from Argentina. In addition, resistance to SP appeared to be widespread (88%) in the Argentinean farms, which had been selected based on the observation of lack of treatment efficacy by farmers. In South Africa one of the three populations was found to be resistant to SP and to a phenylpyrazol compound (pyriprol). Furthermore, resistance to organosphosphates and SP was observed in Australia. Finally, the Larval Tarsal Test proved to be a suitable test to evaluate the susceptibility of R. microplus field populations to the most relevant acaricidal classes.

In Argentina, the cattle tick Rhipicephalus microplus has already developed resistance to organophospates and synthetic pyrethroids. However, no cases of amitraz resistance have ever been recorded in this country despite its heavy use. A recent failure of amitraz to control ticks in a farm located in Santo Tome, province of Corrientes, resulted in the collection of samples for acaricide resistance diagnosis. The modified Drummond adult immersion test (AIT) and the larval tarsal test (LTT) were performed separately in Argentina and Switzerland to evaluate efficacy of amitraz and other acaricides. The AIT showed that oviposition in the Santo Tome field isolate was not inhibited when it was challenged to 250 and 500 ppm amitraz, and 50 ppm deltamethrin. However, oviposition was reduced by 90.6% when this field isolate was challenged to a combination of 400 ppm ethion and 100 ppm cypermethrin. To confirm the results obtained with the AIT, 2 additional tick samples were collected and shipped to Switzerland for resistance diagnosis of amitraz, cypermethrin and flumethrin, using the LTT. With this bioassay, the resistance ratios of the 2 field isolates were 32.5 and 57.0 for amitraz and between 5.9 and 27.2 for the synthetic pyrethroids. Both in vitro bioassays confirmed amitraz and synthetic pyrethroid resistance in the Santo Tome samples. These results account for the first evidence of amitraz resistance in R. microplus in Argentina.

Acaricide resistance of the cattle tick Rhipicephalus (Boophilus) microplus is widespread in most of the countries where this parasite is present. Bioassays are used to diagnose the level and pattern of resistance in tick populations. In the present study, we describe a detailed protocol of the Larval Tarsal Test (LTT) using simplified equipment and data on the resistance of 17 tick field populations originating from 5 Brazilian states. Nine acaricidal compounds from 5 major classes were tested: organophosphates (OP), synthetic pyrethroids (SP), macrocyclic lactones (ML), phenylpyrazols (PYZ) and amidines. For comparison, four of the tick populations were also tested with the Larval Packet Test (LPT) with one compound per class. The most common resistances were to SP, amitraz and OP, with frequencies of 94%, 88% and 82%, respectively. Resistance to PYZ was also found to be widespread (65%), suggesting a rapid development of fipronil resistance in Brazil. One case of ML resistance and 2 cases of suspected ML resistance were identified with the LTT. The LTT led to higher resistance ratios to all compounds than the LPT, reflecting its high sensitivity to detect resistance. Finally, the LTT allowed testing a larger number of compounds and doses with reduced labour in comparison to the LPT and turned out to be a reliable bioassay to detect resistance in field populations.

Resistance 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.

Acaricide resistance has become widespread in countries where cattle ticks, Rhipicephalus (Boophilus) microplus, are a problem. Resistance arises through genetic changes in a cattle tick population that causes modifications to the target site, increased metabolism or sequestration of the acaricide, or reduced ability of the acaricide to penetrate through the outer protective layers of the tick’s body. We review the molecular and biochemical mechanisms of acaricide resistance that have been shown to be functional in R. (B.) microplus. From a mechanistic point of view, resistance to pyrethroids has been characterized to a greater degree than any other acaricide class. Although a great deal of research has gone into discovery of the mechanisms that cause organophosphate resistance, very little is defined at the molecular level and organophosphate resistance seems to be maintained through a complex and multifactorial process. The resistance mechanisms for other acaricides are less well understood. The target sites of fipronil and the macrocyclic lactones are known and resistance mechanism studies are in the early stages. The target site of amitraz has not been definitively identified and this is hampering mechanistic studies on this acaricide. A resistência aos acaricidas tornou-se amplamente difundida nos países onde os carrapatos bovinos, Rhipicephalus .Boophilus. microplus, são um problema. A resistência surge por meio de alterações genéticas em umapopulação de carrapatos que causam modificações no local de ação, aumento do metabolismo ou sequestro do acaricida, ou ainda redução na capacidade do acaricida em penetrar através das camadas protetoras do corpo do carrapato. Neste artigo, foram revisados os mecanismos moleculares e bioquímicos da resistência aos acaricidas que ocorrem em R. (B.) microplus. A partir de um ponto de vista dos mecanismos envolvidos, a resistência aos piretróides tem sido caracterizada em maior grau do que em qualquer outra classe de acaricida. Embora uma grande quantidade de pesquisas têm sido direcionada para a descoberta de mecanismos que causam resistência aos organofosforados, muito pouco é conhecido ao nível molecular, e essa resistência parece ser mantida por intermédio de um processo multifatorial e complexo. Os mecanismos de resistência para os demais acaricidas são bem menos compreendidos. Os alvos de ação do fipronil e das lactonas macrocíclicas são conhecidos, e os estudos dos mecanismos de ação envolvidos estão ainda em estágios iniciais. O alvo de ação do amitraz ainda não foi definitivamente identificado, e isso é limitante aos estudos dos mecanismos envolvidos na resistência a esse acaricida.

Background Schistosomiasis and opisthorchiasis are of public health importance in Southeast Asia. Praziquantel (PZQ) is the drug of choice for morbidity control but few dose comparisons have been made. Methodology Ninety-three schoolchildren were enrolled in an area of Lao PDR where Schistosoma mekongi and Opisthorchis viverrini coexist for a PZQ dose-comparison trial. Prevalence and intensity of infections were determined by a rigorous diagnostic effort (3 stool specimens, each examined with triplicate Kato-Katz) before and 28–30 days after treatment. Ninety children with full baseline data were randomized to receive PZQ: the 40 mg/kg standard single dose (n = 45) or a 75 mg/kg total dose (50 mg/kg+25 mg/kg, 4 hours apart; n = 45). Adverse events were assessed at 3 and 24 hours posttreatment. Principal Findings Baseline infection prevalence of S. mekongi and O. viverrini were 87.8% and 98.9%, respectively. S. mekongi cure rates were 75.0% (95% confidence interval (CI): 56.6–88.5%) and 80.8% (95% CI: 60.6–93.4%) for 40 mg/kg and 75 mg/kg PZQ, respectively (P = 0.60). O. viverrini cure rates were significantly different at 71.4% (95% CI: 53.4–84.4%) and 96.6% (95% CI: not defined), respectively (P = 0.009). Egg reduction rates (ERRs) against O. viverrini were very high for both doses (>99%), but slightly lower for S. mekongi at 40 mg/kg (96.4% vs. 98.1%) and not influenced by increasing diagnostic effort. O. viverrini cure rates would have been overestimated and no statistical difference between doses found if efficacy was based on a minimum sampling effort (single Kato-Katz before and after treatment). Adverse events were common (96%), mainly mild with no significant differences between the two treatment groups. Conclusions/Significance Cure rate from the 75 mg/kg PZQ dose was more efficacious than 40 mg/kg against O. viverrini but not against S. mekongi infections, while ERRs were similar for both doses. Trial Registration Controlled-Trials.com ISRCTN57714676, Author Summary Parasitic worm infections are of public health importance in Southeast Asia. Particularly, the blood-dwelling Schistosoma mekongi worm, which is acquired by skin contact with the infectious cercariae in freshwater, can lead to liver enlargement. An infection with Opisthorchis viverrini is obtained by consumption of undercooked freshwater fish, and this infection increases the risk of developing cholangiocarcinoma. A single oral dose of 40 mg/kg praziquantel is recommended for mass treatment of schistosomiasis and opisthorchiasis, while at the individual level, a total dose of 75 mg/kg divided into three doses, is currently common practice to treat O. viverrini infection. Diagnosis is based on stool examination under a microscope for detection of worm eggs, but is limited by the low sensitivity of the widely used Kato-Katz technique. In this study, we showed that a 75 mg/kg total dose of praziquantel (50 mg/kg+25 mg/kg given 4 hours apart) cleared significantly more O. viverrini infections than a single 40 mg/kg dose, but no difference was observed for S. mekongi. Solicited adverse event profiles were mainly mild and similar in both groups. Repeated stool examination before and after treatment was essential for an accurate assessment of drug efficacy in terms of cure rate, but showed no effect on assessing egg reduction rates.

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus ( Boophilus ) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC 50 ) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo . The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.

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.