Molecular diagnosis of scabies using a novel probe-based polymerase chain reaction assay targeting high-copy number repetitive sequences in the Sarcoptes scabiei genome

Background The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. Methodology/Principal findings High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. Conclusions/Significance This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests

Optimizing dissection, sample collection and cell isolation protocols for frugivorous bats

Bats are becoming increasingly important as an experimental model due to their unique biological features. These include the ability of powered flight, minimal consequences from a heightened metabolic state, extended longevity in most species and minimal inflammation in response to most otherwise pathogenic viruses. To date there has been limited work done on the optimal procedures for necropsy, extraction of tissues or preparation of cell suspensions for downstream experimental work. Here we use Pteropus alecto black flying fox as an example model of the fruit bat to develop optimal procedures for anaesthetizing, necropsy methods, safety, sequence and protocols for cell/tissue extraction and isolation protocols. These methods were successfully used to yield high-quality RNA, DNA and protein samples from tissues along with viable cells for various molecular and immunological studies. Procedures utilized are suitable for comparative biology studies with most protocols being directly modified from those used in mice and humans. While mainly optimized for the larger fruit bats (flying foxes) in this study, the majority of protocols can readily be adapted to all species of bats. This study provides the framework for greater consistency with in vivo bat experiments, application for comparative biology studies and greater engagement of the bat community for suitable protocols to be harmoniously adopted

Treatment of pigs with endectocides as a complementary tool for combating malaria transmission by Anopheles farauti (s.s.) in Papua New Guinea

Abstract Background Outdoor, early-biting, zoophagic behaviours by Anopheles farauti (s.s.) can compromise the effectiveness of bed nets for malaria control. In the Western Pacific region, pigs and dogs represent significant alternative blood sources for mosquitoes. Treating these animals with endectocides may impact mosquito survival and complement control measures. This hypothesis was explored using membrane feeding assays (MFAs), direct feeds on treated pigs, pharmacokinetic analyses and a transmission model. Results Ivermectin was 375-fold more mosquitocidal than moxidectin (24 h LC50 = 17.8 ng/ml vs 6.7 µg/ml) in MFAs, and reduced mosquito fecundity by > 50% at ≥ 5 ng/ml. Treatment of pigs with subcutaneous doses of 0.6 mg/kg ivermectin caused 100% mosquito mortality 8 days after administration. Lethal effects persisted for up to 15 days after administration (75% death within 10 days). Conclusion The application of these empirical data to a unique malaria transmission model that used a three-host system (humans, pigs and dogs) predicts that the application of ivermectin will cause a significant reduction in the entomological inoculation rate (EIR = 100 to 0.35). However, this is contingent on local malaria vectors sourcing a significant proportion of their blood meals from pigs. This provides significant insights on the benefits of deploying endectocides alongside long-lasting insecticide-treated nets (LLINs) to address residual malaria transmission