Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis.

Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR-LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.

Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species.

BACKGROUND: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species. METHODOLOGY: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples. FINDINGS: Sequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex. A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples. CONCLUSION: The study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.

HLA class I polymorphisms in Tunisian patients with otosclerosis.

BACKGROUND: Otosclerosis is a common form of hearing impairment among Western-Eurasian adults. The cause of otosclerosis remains unknown. Autoimmune reaction against the otic capsule has been suggested as a possible aetiologic factor in otosclerosis. AIM: The present study is the first report to evaluate the relationship between class I major histocompatibility complex (MHC) genes (HLA-A, HLA-B and HLA-Cw) and genetic susceptibility to otosclerosis in Tunisian patients. SUBJECTS AND METHODS: Fifty unrelated Tunisian patients exhibiting clinical otosclerosis were typed for HLA-A, HLA-B and HLA-Cw antigens and compared with 100 ethnically-matched healthy controls. RESULTS: Increased frequencies of HLA-A*03 (OR = 4.16, Pc < 0.043), HLA-B*35 (OR = 2.76, Pc < 0.043) and HLA-Cw*03 (OR = 4.57, Pc < 0.043) antigens were found in the patients with otosclerosis compared with healthy controls. Individuals with HLA-A*30 (OR=0.25, Pc < 0.043), HLA-B*51 (OR = 0.11, Pc < 0.043), HLA-Cw*16 (OR = 0.08, Pc < 0.043) and Cw*06 (OR=0.32, Pc < 0.043) antigens have a protective effect against otosclerosis. CONCLUSIONS: In conclusion, the data suggest that a variation in class I HLA antigens could be a genetic factor involved in susceptibility to otosclerosis in the Tunisian population.

A new locus for otosclerosis, OTSC8, maps to the pericentromeric region of chromosome 9.

Otosclerosis is a common disorder of the otic capsule resulting in hearing impairment in 0.3-0.4% of the Caucasian population. The aetiology of the disease remains unclear. In most cases, otosclerosis can be considered as a complex disease. In some cases, the disease is inherited as an autosomal dominant trait, sometimes with reduced penetrance. To date, seven autosomal dominant loci have been reported, but none of the disease-causing genes has been identified. In this study, we present the results of a genome-wide linkage analysis in a large Tunisian family segregating autosomal dominant otosclerosis. Linkage analysis localised the responsible gene to chromosome 9p13.1-9q21.11 with a maximal LOD score of 4.13, and this locus was named OTSC8. Using newly generated short tandem repeat polymorphism markers, we mapped this new otosclerosis locus to a 34.16 Mb interval between the markers D9S970 and D9S1799. This region comprises the pericentromeric region on both arms of chromosome 9, a highly complex region containing many duplicated sequences.