Consistence of miniexon polymerase chain reaction-restriction fragment length polymorphism and single-copy gene sequence analyses in discriminating Leishmania genotypes.

Recently, a new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay had been developed using the miniexon sequences for genotyping Leishmania isolates. We had used this method for rapid diagnosis and genotyping of visceral and cutaneous leishmaniasis with the combination of microcapillary cultivation. In this study, we have evaluated this approach by examining genomic DNAs from 47 independent isolates, which were grouped into 19 genotypes of Leishmania subgenus complexes by sequence polymorphism of single-copy genes. Results obtained provide miniexon RFLP configurations specific to Leishmania enriettii, Leishmania tarentolae, and Leishmania gerbilli for the first time. Altogether, 92% of the results from miniexon PCR-RFLP are in agreement with those based on the sequence database of single-copy genes from the same isolates. The miniexon PCR-RFLP method is simple, sensitive, and specific method useful for routine diagnosis of different Leishmania.

[Investigation of SEN virus genotypes D and H among blood donors in Mersin University Medical School Hospital]. / Mersin Universitesi Tip Fakültesi Hastanesi kan donörlerinde SEN virusu genotip D ve H varliginin arastirilmasi.

SEN virus is a recently discovered DNA virus, and eight genotypes (A to H) were detected by phylogenetic analysis. Genotype D (SENV-D) and H (SENV-H) are thought to be associated in the etiology of post-transfusion hepatitis. Although no strong association was revealed between liver pathology and SEN virus, the viral replication in hepatocytes and transmission by blood transfusions were well characterized. The aim of this study was to investigate the prevalence of SENV-D and SENV-H in blood donors. One hundred serum samples were included in the study which were obtained from blood donors comprised of 98 male and 2 female with a mean age 36.4 years who were enrolled at Mersin University Faculty of Medicine blood center. The DNAs of SENV-D and SENV-H were detected with polymerase chain reaction (PCR), by using D10S/L2AS and C5S/L2AS primers, respectively. SEN virus DNA positivity was detected in 25 of 100 (25%) sera, of which 10 (10%) were positive for SENV-D and 15 (15%) were positive for SENV-H DNA. Although, the number of samples were not sufficient to reflect the general prevalence, our study relatively reveals that asymptomatic carriage rate of these viruses were 25% in our province.

SEN virus prevalence among non-B and non-C hepatitis patients with high liver function tests in the south of Turkey.

We investigated the characteristics and detection rates of SEN virus (SENV) infection among 100 Turkish patients who had with high alanine aminotransferase (ALT) and aspartate aminotransferase levels but were negative for HBV DNA and HCV RNA and had no history of transfusion. As a control group, we also analyzed 50 healthy individuals who had normal ALT levels, were negative for HBV DNA and HCV RNA, and had no history of transfusion. The serum samples of patient and controls were analyzed by PCR to detect the presence of SENV DNA and its two genotypes (SENV-H and SENV-D). We detected SENV DNA in 13 of 100 (13%) patients. Five of 13 (38.46%) patients were positive for SENV-D and 8 of 13 (61.53%) patients were positive for SENV-H DNA. We also detected SENV DNA in 5 of 50 (10%) patients in the control group. Two of 5 (40%) patients were positive for SENV-D and 3 of 5 (60%) patients were positive for SENV-H DNA in the control group. SENV was detected at almost the same frequency in the patient and control group. SENV did not seem to contribute to the pathogenesis of liver disease (P > 0.05) in this cohort. Our results also showed that SENV transmission was not only associated with blood transfusion but also with some other possible routes.

Rapid diagnosis and genotyping of Leishmania isolates from cutaneous and visceral leishmaniasis by microcapillary cultivation and polymerase chain reaction-restriction fragment length polymorphism of miniexon region.

We have performed a combination of microcapillary cultivation method and restriction fragment length polymorphism (RFLP) analysis of amplified products by 1 single PCR of miniexon region of Leishmania for molecular diagnosis and genotyping of different Leishmania species isolated from cutaneous leishmaniasis (CL) and visceral leishmaniasis. We have analyzed 10 microcapillary cultivated isolates from cutaneous cases and 5 microcapillary cultivated isolates from visceral cases (totally 15) by polymerase chain reaction-RFLP (PCR-RFLP). Of 10 isolates, 3 (30%) were genotyped as Leishmania infantum and 7 (70%) of 10 isolates were genotyped as Leishmania tropica from the microcapillary cultivated isolates of cutaneous cases. On the other hand, all 5 isolates (100%) were genotyped as L. infantum from microcapillary cultivated visceral cases. Our most interesting finding is the presence of 3 L. infantum isolates in CL cases without kala-azar history. Therefore, we suggest that further investigations must be done about this subject. On the other hand, we suggest the combination of microcapillary culture method and PCR-RFLP of miniexon region of leishmaniae can be used in routine laboratory experimentation because of their simple, cheap, and rapid benefits (within a week), whereas other different approaches offer a multitude of valid taxonomic characters for species identification.

Genotyping of hepatitis B virus isolated from chronic hepatitis B patients in the south of Turkey by DNA cycle-sequencing method.

The 8 genotypes of hepatitis B virus (HBV A-H) show a distinct geographic distribution and influence the course of disease and the prognosis of treatment. In this study, we have genotyped 50 HBV isolates circulating in the south of Turkey by DNA cycle sequencing, based on their compatibility with reference sequences of a part of S gene. In our cases, all 50 (100%) HBV sequences from the patients demonstrated full compatibility with the sequences of ayw subtype viruses in genotype D. However, we have found some nucleotide sequence variations within genotype D, 47 (94%) of which were related to HBVGEN1 (Z35716 genotype D) and 3 (6%) were related to HBVDNA (X68292, genotype D).