ABSTRACT
BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.
Subject(s)
Dirofilaria immitis , Dirofilariasis , Microfilariae , Animals , Dirofilaria immitis/genetics , Dirofilaria immitis/metabolism , Dogs , Microfilariae/genetics , Dirofilariasis/parasitology , Dirofilariasis/blood , Dirofilariasis/diagnosis , Dog Diseases/parasitology , Dog Diseases/blood , Ivermectin/pharmacology , Doxycycline/pharmacology , Drug Resistance/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/geneticsABSTRACT
This Guidance provides information for clinicians providing women with copper-bearing intrauterine devices as long-term contraception. A key to the grades of recommendations, based on levels of evidence, is given at the end of this document. Details of the methods used by the Clinical Effectiveness Unit (CEU) in developing this Guidance and evidence tables summarising the research basis of the recommendations are available on the Faculty website (www.ffprhc.org.uk). Abbreviations (in alphabetical order) used include: acquired immune deficiency syndrome (AIDS); actinomyces-like organisms (ALOs); automated external defibrillator (AED); blood pressure (BP); British National Formulary (BNF); confidence interval (CI); copper-bearing intrauterine contraceptive device (IUD); emergency contraception (EC); Faculty Aid to Continuing Professional Development Topic (FACT); levonorgestrel-releasing intrauterine system (IUS); human immunodeficiency virus (HIV); Medicines and Healthcare products Regulatory Agency (MHRA); non-steroidal antiinflammatory drugs (NSAIDs); odds ratio (OR); pelvic inflammatory disease (PID); relative risk (RR); Royal College of Obstetricians and Gynaecologists (RCOG); Scottish Intercollegiate Guidelines Network (SIGN); sexually transmitted infection (STI); termination of pregnancy (TOP); World Health Organization (WHO); WHO Medical Eligibility Criteria (WHOMEC); WHO Selected Practice Recommendations (WHOSPR).
