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Amaç: HIV tedavisinde Abakavir'in kullanımı için en büyük kontrendikasyonu aşırı duyarlılık reaksiyonudur. Abakavir'e karşı gelişen aşırı duyarlılık reaksiyonu HLA-B57:01 alleli varlıǧı ile yakın ilişkili olduǧu saptanmıştır. Bu çalışmanın yapıldıǧı tarihlerde ülkemizde bu alanda çok merkezli farklı bir çalışma yapılmamıştı. Bu çalışmada, HIV pozitif yeni tanı almış, henüz tedaviye başlamamış hasta grubundaki HLA-B57:01 allel sıklıǧının allel spesifik PCR ile taranması amaçlanmıştır. Gereç ve Yöntemler: Retrospektif olan çalışma için Ocak-Aralık 2021 tarihlerinde \"Halk Saǧlıǧı Genel Müdürlüǧü, Ulusal HIV-AIDS Doǧrulama ve Viral Hepatitler Referans Laboratuvarına\" yeni tanı almış 235 hastaya ait plazma örneklerinden izole edilen DNA örnekleri dâhil edilmiştir. HLA-B57:01 allel sıklıǧının allel spesifik PCR ile taranması amaçlanmış, pozitif bulunan örnekler için semi-nested PCR yapılmıştır. DNA örneklerinin izlenebilmesi için EDTA 0,5 M, 10XTBE solüsyonları hazırlanmıştır. Verilerin analizi SPSS 15 paket programında yapılmıştır. Bulgular: HLA-B57:01 allel varlıǧı yönünden test edilen toplam 235 örneǧin 5'inde (%2,12) allel pozitifliǧi saptandı. Pozitif bulunan 5 hastanın 35 yaş ve altındaki hasta grubunda olduǧu belirlendi. HLA-B57:01 pozitifliǧi ile cinsiyet arasındaki ilişkide; pozitif hastaların erkek olduǧu, tüm erkek hastaların %2,8'inin HLA-B57:01 alleli taşıdıǧı belirlendi (p=0,371). Coǧrafik daǧılım deǧerlendirildiǧinde; en sık allel pozitifliǧi Karadeniz Bölgesinde izlenmiştir. Sonuç: Test edilen örneklerin azlıǧı sebebiyle, bölgesel temsiliyet saǧlanamamıştır. İlerleyen dönemde, tüm bölgeleri temsil edecek düzeyde örneklerin dâhil edildiǧi, allel varlıǧı ile birlikte klinik yansımalarının da deǧerlendirilebileceǧi, HLA-B57:01 allel ile CD4 Th lenfosit sayısı arasındaki ilişkinin saptanabileceǧi, aşırı duyarlılık reaksiyonu veya benzeri semptomlarda deri yama testi gibi geniş kapsamlı çalışmaların yapılması gerektiǧi sonucuna varılmış olup bu çalışmamızın da sonraki çalışmalara ışık tutacaǧı kanaati oluşmuştur.

Background Fungal species are responsible for 40%–50% of all microbial keratitis cases. Due to the low amount of extracted DNA in ocular Formalin‐fixed Paraffin‐embedded (FFPE) samples, selecting a reliable molecular method is a substantial issue in this field. Methods Sixty‐six samples were collected via the penetrating keratoplasty (PK) technique. Histopathology assays were performed using hematoxylin–eosin (H&E) and periodic acid Schiff (PAS) staining methods. The ITS1/ITS4 and ITS1/ITS2 primer pairs were used in a semi‐nested polymerase chain reaction (PCR) to target the universal internal transcribed spacer (ITS) region. Some PCR results were validated through sequencing. Results Fungal DNA was detected in 44 of 66 samples (66.7%), and histopathology was positive for 41 of 66 samples (62.1%). Of 41 histopathologically proven fungal‐positive cases, 39 were PCR‐positive (95%). Moreover, of 44 PCR‐positive samples, 39 (88.6%) were histopathology‐positive, and 5 (11.3%) were histopathology‐negative. Totally in 39 cases (59%), both histopathology and PCR yielded positive results. The Kappa agreement rate between the two diagnostic methods, including histopathology and PCR, was 0.77. Sensitivity, specificity, positive predictive value, and false predictive value were reported as 88.64%, 90.9%, 95.12%, and 80%, respectively. Conclusion As we reached the acceptable Kappa agreement rate, we concluded that applying the semi‐nested PCR assay is a promising method for supporting the evidence by histopathology. Finally, we suggest targeting more specific gene regions using primer pairs that amplify smaller amplicon sizes and surveying novel molecular methods such as NGS to achieve higher sensitivity and Kappa agreement rates. Following the clinical suspicion for fungal keratitis (FK), the corneal samples were collected by penetrating keratoplasty (PK) from the patients. Specimens were prepared by microtome and were stained by hematoxylin and eosin (H&E), periodic acid schiff (PAS) stainings. For the molecular assay, samples were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8 S‐ITS2) to identify causative agents was performed on PCR products. The results were analyzed by researchers.

Background Fungal rhinosinusitis (FRS) encompasses a various spectrum of diseases. Histopathology is the “reference method” for diagnosing FRS, but it cannot determine the genus and species. Moreover, in more than 50% of the histopathologically proven cases, the culture elicited no reliable results. This study was an attempt to evaluate the diagnostic efficiency of semi‐nested polymerase chain reaction (PCR) from formalin‐fixed paraffin‐embedded (FFPE) functional endoscopic sinus surgery (FESS) in FRS patients. Methods One hundred ten specimens were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8S‐ITS2) to identify causative agents was performed on PCR products. Results Sixty‐four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi‐nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests. Conclusion Due to the acceptable sensitivity and specificity of the present method, it might be used to diagnose fungal sinusitis infections along with microscopic techniques. This method is recommended to confirm the diagnose of suspected fungal sinusitis with negative histopathology results. Following clinical susception, for the fungal rhinosinusitis (FRS), the samples were collected from the rhino‐nasal cavity of 110 patients and were stained by hematoxylin and eosin (H&E), periodic acid‐Schiff (PAS) stains. For the molecular assay, 110 specimens were subjected to DNA extraction and histopathology examination. The amplification of the β‐globin gene by conventional PCR was used to confirm the quality of extracted DNA. The semi‐nested PCR was performed using ITS1, ITS2, and ITS4 primers during two steps. Sequencing the internal transcribed spacer region (ITS1‐5.8S‐ITS2) to identify causative agents was performed on PCR products. Sixty‐four out of 110 samples were positive by histopathology evidence, of which 56 samples (87.5%) were positive by PCR. Out of 46 negative samples by histopathological methods, five samples (10.9%) yielded positive results by PCR. Sensitivity, specificity, positive predictive value, and negative predictive value of the semi‐nested PCR method were reported 87.5%, 89.2%, 92.7%, and 85.2%, respectively. The kappa factor between PCR and histopathological methods was 0.76, indicating substantial agreements between these two tests.

Background The orf virus (ORFV) is a viral pathogen that primarily causes contagious ecthyma in humans and different ruminants. The infection, which is common worldwide, causes large‐scale economic losses to animal breeders. Objective and Methods In this study, tissue samples collected from eight randomly selected goats with dermatological lesions on the teats were examined in different goat herds. B2L gene‐specific primer pairs (PP1, PP3 and PP4) were used to reveal the presence of ORFV by molecular methods and for phylogenetic analysis. Results Viral DNA was detected in four of eight tissues using the semi‐nested PCR method. In addition, the data obtained by performing sequence analyses of the amplicons with positive results were compared with the information of different ORFV isolates registered in the GenBank database. Based on the sequence analysis of the field isolates obtained in our study, it was found that the nucleotide similarities among these isolates and those from Asian countries were 100%. Furthermore, ORFV isolates collected from different species and produced in Türkiye over various periods exhibited homologous nucleotide sequences with similarities ranging from 98.1% to 98.8%. In the phylogenetic tree drawn based on the B2L genomic region, it was observed that our field isolates were classified in Group I together with other Turkish and Asian strains. Conclusion As a result, while other pathogenic agents are considered the cause of disease in goats with dermatological lesions on their mammary tissue, the ORFV should also be evaluated, and protection and control programs should be prepared accordingly. Viral DNA (orf virus) was detected in tissue samples using the semi‐nested PCR method. In addition, the data obtained by performing sequence analyses of the amplicons with positive results were compared with the information of different orf virus isolates registered in the GenBank database. Based on the sequence analysis of the field isolates obtained in our study, it was found that the nucleotide similarities among these isolates and those from Asian countries were 100%. Furthermore, orf virus isolates collected from different species and produced in Türkiye over various periods exhibited homologous nucleotide sequences with similarities ranging from 98.1% to 98.8%.

Background: Zoonotic cutaneous leishmaniasis caused by Leishmania major is endemic in 17 of 31 Iranian provinces. Various species of rodents have been introduced as the main reservoirs of the disease. This study was conducted to de­termine the natural infection of hedgehogs with Leishmania spp. in an endemic area of the disease, northern Iran. Methods: Fifteen long-eared hedgehogs were captured alive during 18 months study period, from Apr 2015 to Sep 2016, in Damghan City, Semnan Province, Iran. The animals were identified using apparent characteristics and to de­termine the Leishmania infection, impression smears were prepared from their ear lobes, hind feet, livers, and spleens. Micro­scopic examination and semi-nested PCR were applied to determine the infection and to identify the parasites species respectively. Results: All examined animals were identified as Hemiechinus auritus (Family: Erinaceidae). In microscopic examina­tion, 8 (53.3%) samples were shown to be infected with Leishmania parasites. The higher and lower rate of the infection was observed in the ears as well as the feet and in the liver specimens, 53.3%, and 33.3% respectively. Forty percent (6/ 15) of the samples were molecularly positive and all were identified as L. major parasites. All the examined animals in au­tumn and 50% of them in summer were shown to be infected with Leishmania parasites. Conclusion: This study demonstrated the natural infection of H. auritus with L. major for the first time in Damghan City and introduced these mammals as new potential reservoirs of ZCL in the study area.

Babesia is an intraerythrocytic protozoan parasite which is transmitted by hard ticks of the Ixodidae family. One of the problems associated with protozoan infection is the determination and characterization of the vectors. The aim of the present study was to detect Babesia ovis in the salivary gland of Dermacentor spp. A total of 200 adult Dermacentor ticks (139 D. niveus and 61 D. marginatus) were collected from sheep suspected to be infected with babesiosis in the Ardabil region of Iran from April to September 2015 (active season of ticks); and were identified using standard taxonomic keys. Deoxyribonucleic acid (DNA) was isolated from the salivary glands of ticks and analyzed with the primers derived from the hyper variable V4 region of 18S ribosomal ribonucleic acid (18S rRNA) of the Babesia species using polymerase chain reaction (PCR). Babesia ovis was detected in 5.8% of the D. niveus and 3.3% of the D. marginatus positive samples in the second round of semi-nested PCR. Based on the results obtained from this study, it is concluded that D. niveus and D. marginatus, which are distributed in Ardabil region of Iran, might play a major role in the transmission of infection as a natural vector of B. ovis.

Besides group A rotaviruses, group B and C rotaviruses have been detected as the cause of diarrheal diseases in pigs. Out of a set of 329 faecal samples from pigs, 16 samples were selected in which rotavirus was detected by electron microscopy and at the same time group A rotavirus was excluded by the ELISA method. Rotaviruses were assayed using specific primers for detection of group B and C rotaviruses, and RT-PCR and semi-nested PCR methods. In one sample, no rotavirus of group B or C was detected; in the remaining 15 samples rotavirus group C was detected, in two samples together with group B rotavirus. Sequencing of the obtained PCR products and comparison with corresponding gene sequences revealed 80% nucleotide sequence identity between group B rotaviruses and available sequences of porcine isolates. A nucleotide sequence identity of 92% was obtained in group C rotaviruses as compared with the Cowden strain.

Ovine babesiosis is the most important haemoparasitic tick-borne disease of small ruminants in Iran caused by Babesia ovis, B. motasi, and B. crassa. The aim of this study was to characterize the species of ovine Babesia species isolated from different geographical region of Iran. One hundred fifty four blood samples collected from animals, which demonstrated the pale mucous membranes or hyperthermia. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, the morphological and biometrical data of parasite in any infected erythrocyte have been considered. Extracted DNA from each blood samples were used in PCR and semi nested- PCR in order to confirm the presence of the species. Microscopical observation on 154 blood smears determined 38 (24.67%) and 40 (26%) samples were infected by Babesia and Theileria respectively. The mixed infections occurred in four (2.6%) samples. The results of the PCR assays showed nine (5.85%), 81 (53%) and 18 (11.7%) were distinguished as Babesia, Theileria and mixed infection, respectively. Semi nested- PCR did not confirm the presence of B. motasi. The causative organism of many cases of haemoprotozoal diseases, which recorded in previous studies, could be B. ovis or Theileria lestoquardi. The result confirmed that B. ovis was only species which causes babesiosis in the study areas. It seems that the biometrical polymorphisms could exist in B. ovis in Iran. This polymorphism could be a main problem in differentiation between B. ovis and B. motasi and it could be dissolved by specific PCR analysis.

Invasive candidiasis (IC) represents a growing concern worldwide, with a considerable increase in non-albicans  Candida (NAC) species. The study's primary goal was to determine if species identification by semi-nested PCR (sn-PCR) with primers for the five most prevalent Candida species is sufficient to deal with the current trends of Candida infections in cancer patients. Over one year, Candida isolates were collected from samples of patients with hematological and solid organ tumors in a single center. Species of Candida were identified by chromagar and multiplex sn-PCR using specific primers for Candida albicans , Candida tropicalis , Candida glabrata , Candida krusei , and the Candida parapsilosis complex. Most Candida infection episodes are caused by NAC species (70.5% of 105 isolates). Rare species (14 isolates) accounted for 13.3% of isolates and were not identified by sn-PCR using the five most common Candida species primers. More than half of these rare species caused candidemia in cancer patients (57.1%; p  = 0.011). The risk factor for candidiasis was recent surgeries ( p  = 0.020) in adults and chemotherapy in pediatric patients ( p  = 0.006). Prolonged hospitalization and genitourinary tract cancer were significantly associated with invasive infections ( p  = 0.005 and 0.049, respectively). Recent surgery was a significant risk factor associated with C. parapsilosis and C. glabrata infections ( P  = 0.038 and 0.003, respectively), while C. tropicalis was significantly more common in patients with hematological malignancies ( P  = 0.012). Techniques with a broader identification spectrum than the major five Candida species are crucial for the optimal management of cancer patients.

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.