Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

<div><p>A gp63PCR method was evaluated for the detection and characterization of <i>Leishmania (Leishmania)</i> (<i>L.</i>) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a <i>L. infantum</i> specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different <i>L. infantum</i> endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to <i>L. infantum</i> species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of <i>L. infantum</i> infections in dogs in Tunisia.</p></div

Results of parasitology, serology and PCR investigations on Tunisian and control dogs.

<p>Results of parasitology, serology and PCR investigations on Tunisian and control dogs.</p

Gp63PCR-RFLP patterns of <i>Leishmania</i> parasites obtained from dog biopsies.

<p>A: digestion with <i>Msc</i>I restriction enzyme; B: digestion with <i>Sal</i>I restriction enzyme. 1: <i>L. donovani</i>, 2: <i>L. infantum</i>, 3: LN112, 4: LN129, 5: LN26, 6: LN11, 7: LN80, 8: LN2, 9: LN39, 10: LN77, 11: LN102, 12: LN110, 13: J1, 14: J3, 15: J5, 16: J6, 17: J7. All sizes are indicated in bp.</p

PCR primers sequences, reaction and cycling conditions used.

<p>PCR primers sequences, reaction and cycling conditions used.</p

Pair wise concordance values calculated using the kappa coefficient for parasitology, serology and PCR investigations.

<p>Pair wise concordance values calculated using the kappa coefficient for parasitology, serology and PCR investigations.</p

Kitch dendrogram constructed using Nei-modified distances calculated from gp63PCR-RFLP results obtained with lymph node biopsies of dogs from Tunisia.

<p>Branches corresponding to Old World representative <i>Leishmania</i> strains are indicated.</p

Connaissance des temps ou des mouvements célestes à l'usage des astronomes et des navigateurs... / publiée par le Bureau des longitudes

19711971

Additional file 2: of South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on α4β7 for replication

IECs and IMCs show similar binding to and dependence on α4β7 for replication. IECs and IMCs of CAP210 T/F and CAP239 T/F bound to α4β7 at similar levels as defined by (A) p24 binding assay where the dashed line represents IMC binding and the solid line represents IEC binding and (B) the competition assay. SF162 IEC and IMC also bound similarly see Figure 1A. (C) All three viruses showed no differences between their IECs and respective IMCs in dependence on α4β7 for replication. All results are representative of three experiments with error bars representing SEM. No differences were significant by the paired t test

Additional file 6: of South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on ι4β7 for replication

Characteristics of IECs used in this study