Genotyping of Pneumocystis jirovecii isolates obtained from clinical samples by multilocus sequencing: a molecular epidemiology study conducted in Turkey

PubMed: 322745572-s2.0-85083059533Pneumocystis jirovecii is an opportunistic respiratory pathogen causing Pneumocystis pneumonia (PcP) in immunocompromised patients. The aim of this study was to investigate the genetic diversity of P. jirovecii isolates (n: 84) obtained from PcP patients using multilocus sequencing method based on mt26S, SOD, and CYB loci. Among the 84 clinical samples that were positive for P. jirovecii DNA, 31 (36.90%) of them were genotyped using at least one locus. Of the 31 clinical samples, 26 of them were successfully genotyped using all loci whereas three samples were genotyped using either mt26S/CYB loci or mt26S/SOD loci. Additionally, there were two more clinical samples that were genotyped using CYB or SOD locus. Using mt26S locus, genotypes 2, 3, 7, and 8 were detected. Frequencies of genotype 7 and 8 were higher and both of them were found in 11 (n: 29; 37.93%) clinical samples. Using SOD locus, SOD 1, 2, and 4 genotypes were detected. SOD 1 was the predominant genotype (20/28; 71.42%). During the analyses of CYB locus, CYB 1, 2, 5, 6, and 7 as well as a new CYB genotype were detected. CYB 1 (16/29; 55.17%) and 2 (10/29; 34.48%) were the predominant genotypes. Overall, according to the multilocus sequencing results E, F, M, N, P, and V multilocus genotypes were detected among the PcP patients. In addition, SOD 1 was the predominant genotype and CYB had a more polymorphic locus. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature.2013TIP050, TGA-2019-20253This study was supported by Ege University Scientific Research Projects Coordination Unit (Project Numbers: TGA-2019-20253 and 2013TIP050)

Analysis of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene related to neonatal isoerythrolysis in stray cats of Izmir, Turkey [Türkiye, İzmir sokak kedilerinde neonatal izoeritrolizisle ilişkili sitidin monofosfat-N-asetilnöraminik asit hidroksilaz (CMAH) geninin analizi]

Neonatal isoerythrolysis is a life threatening disease in new born cats. It occurs when type A or type AB kittens are born from a type B queen (female cat). A homozygous 18 bp insertion located in cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene has been detected in type B cats, causing production of inactive CMAH enzyme. Currently, molecular methods are being used to determine type B blood in female cats, which can help prevent neonatal isoerythrolysis in kittens. These molecular assays target the presence of 18 bp insertion in CMAH gene. In this study, we aimed to analyze the potential of neonatal isoerythrolysis among stray cats of İzmir, Turkey using PCR detecting the 18 bp insertion in CMAH gene. During the study, we analyzed 793 cats’ blood sample for the presence of 18 bp insertion in CMAH gene. Three cats known to have blood types A, B, and AB were used as control in PCR. According to the PCR results, blood type A control cat displayed a 175 bp product indicating a homozygous type A cat while blood type control B cat showed a 193 bp product in CMAH gene (with 18 bp insertion) indicating a homozygous type B cat. Interestingly, blood type AB control cat showed a heterozygous pattern for CMAH gene, in which three different bands (175 bp like that of type A, 193 bp product for type B, and the third unique band with approximately 240 bp size) were detected. Among 793 stray cats of İzmir, 791 were homozygous for CMAH gene with 175 bp band size (99.7%). The remaining two stray cats showed heterozygous band pattern like blood type AB cat (0.12%). Overall, 175 bp band displaying type A cats are prevalent contrary to the two cats that have type AB pattern and non-existence of homozygous type B cats. These results show that the potential of neonatal isoerythrolysis in stray cats of İzmir is minimal. Future studies are required to scrutinize the reason(s) for non-existence of type B cats in İzmir and presence of unique band in blood type AB. © 2016, Veteriner Fakultesi Dergisi. All rights reserved

Detection of echinococcus granulosus and echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction [Kist Örneklerinde Yeni Bir Tek Tüp Multipleks Gerçek Zamanli Polimeraz Zincir Reaksiyonu He Echinococcus granulosus ve Echinococcus multilocularis' in Saptanmasi]

PubMed ID: 27175499Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTCTGACCCTGACAT-3') and Echi A (5'-GGTCTTAACTCMCTCATGCAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTMGGTTTTGGTGTAGTMTTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC-000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specificity by distilled water at 106-105-104-103-102-101-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/µl reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specificity of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specificity of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specific, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples