Investigation of resistance against to flumethrin using against Varroa destructor in Türkiye.

The honeybee ectoparasite Varroa destructor is a major threat to apiculture when evaluating bee diseases and pests. While attempting to control this mite, beekeepers often depend on a small selection of authorized synthetic acaricides, such as flumethrin, which is widely used in Türkiye and globally. However, resistance to flumethrin develops due to incorrect and excessive use. In this study conducted at Ordu Beekeeping Research Institute, trial group were established including an untreated control group and group where flumethrin-based pesticides were applied. Dead varroas collected from pollen traps and live varroas collected from bees were obtained from these trial groups for molecular analysis as positive-negative controls. Varroa samples were collected from provinces representing different regions with intensive beekeeping activities such as Adana, Ankara, Bingöl, Mugla, Ordu, Sanliurfa, Tekirdag. Molecular methods were employed to investigate the resistance gene region for pyrethroids (specifically flumethrin) against V. destructor. In our study, individual DNA extractions were performed on dead parasites from colonies subjected to pyrethroid application (resistance negative control) and live parasites (resistance positive control). The DNA samples obtained were used in PCR reactions targeting the region encoding the 925th amino acid of the voltage-gated sodium channel (VGSC) gene, which is responsible for resistance formation. The DNA samples were subjected to gel electrophoresis to observe the amplification products of the expected target region. To examine the nucleotide sequence changes that encode leucine at the 925th amino acid, which is associated with resistance, DNA sequence analysis was applied to the amplification products. Out of 332 V. destructor parasites obtained from different provinces, 279 were analysed using molecular methods. It was observed that 31% of the samples showed sensitivity to flumethrin while 69% exhibited resistance to it. Among the resistant samples: 27% had homozygous isoleucine mutation; 28% had homozygous valine mutation; 2.8% had heterozygous isoleucine mutation; 8.5% had heterozygous valine mutation; and 2.8% had heterozygous methionine mutation, all of which were associated with flumethrin resistance. As a result, the rate of flumethrin resistance in parasites varied between 51% and 94% among different provinces.

Borrelia miyamotoi in wild rodents from four different regions of Turkey.

Borrelia miyamotoi is a tick-borne zoonotic agent that causes hard tick-borne relapsing fever, an emerging disease in humans. Some small mammalian and bird species are reported to be reservoirs of B. miyamotoi. This study aims to examine Borrelia species present in rodents captured from rural areas of Turkey. Blood samples of rodents were initially screened with Borrelia 16S rRNA qPCR. The Borrelia flaB gene was subsequently amplified by conventional PCR, after which all positive samples were sequenced. Borrelia miyamotoi was observed in nine out of 536 blood samples (1.7%) collected from wild rodents. Phylogenetic analysis showed that all positive samples belonged to the European genotype clade of B. miyamotoi. PCR positivity was 5.3%, 3.7%, and 1.8% in Apodemus uralensis, Apodemus flavicollis, and Myodes glareolus, respectively. Borrelia burgdorferi sensu lato that causes Lyme borreliosis in humans could not be detected in the rodents. In this study, presence of B. miyamotoi DNA is reported for the first time in rodents in Turkey.

The first human case of Rickettsia slovaca from Turkey.

A three year-old girl was admitted to our university hospital with fever, muscle and abdominal pain, and painful cervical lymph nodes after a tick bite on scalp. Rickettsia slovaca DNA was detected in eschar tissue taken from the bite site. This is the first clinical case of a R. slovaca infection reported from Turkey.

Genomic Characterization of the Novel Bartonella refiksaydamii sp. Isolated from the Blood of a Crocidura suaveolens (Pallas, 1811).

Bartonella species are reemerging infectious agents that are transmitted by arthropod vectors among animals and/or humans. At least 13 of the 35 currently recognized Bartonella species are pathogenic for humans. Most of the pathogenic species, except Bartonella quintana and Bartonella bacilliformis, are zoonotic agents with animal reservoirs, including cats, dogs, coyotes, foxes, cattle, and rodents. In this study, a novel Bartonella species was isolated from the blood of a Crocidura suaveolens (Pallas, 1811) Lesser shrew that was captured in the Bartin region of Northwestern Turkey. The strain, RSKK 19006, was characterized using whole-genome sequencing and comparison, multilocus sequence typing (gltA, rpoB, ssrA, nuoG, and 16S rRNA) and internal transcribed spacer sequencing, electron microscopy scanning, biochemical tests, and MALDI-TOF MS (matrix assisted laser desorption ionization-time of flight mass spectrometry). This novel Bartonella is a Gram-negative, rod-shaped, microaerophilic bacterium and has neither flagella nor pilus. As a consequence, we propose to name this new species Bartonella refiksaydamii sp. nov. in Bartonella genus. The zoonotic potential of this novel Bartonella species is as yet unknown.

Human anthrax in Turkey: A ten years' experience (2009-2018).

Anthrax is a notifiable disease in Turkey. In order to control the human disease, animal foci are being monitored and prevention and control activities are being implemented by the Ministry of Health in coordination with the Ministry of Agriculture and Forestry. The objective of our study was to evaluate the national surveillance data and control activities in the last decade. A total of 1174 anthrax cases and 9 deaths have been reported. Anthrax was frequent in eastern provinces and in big cities where large animal movements were significant. The incidence rate was 1.5 times higher in males than in females. The disease was more common in the 30-64 age group. The number of cases increased in the summer and autumn seasons. Human anthrax is still being reported though in decreasing numbers in Turkey. A collaborative control programme continues to be needed.

Brucella melitensis and Brucella abortus genotyping via real-time PCR targeting 21 variable genome loci.

Brucella melitensis and Brucella abortus account for almost all cases of brucellosis in Turkish population. We developed a fourplex quantitative real-time PCR (qPCR) assay for the electrophoresis-free, rapid and cost-effective differentiation of B. abortus and B. melitensis from the other Brucella spp. The 4-plex species differentiation assay was combined with a qPCR assay targeting 17 different single nucleotide polymorphism (SNP) loci in Brucella genomes. This combination resulted in a 21 Variable Genome Loci (21-VGL) qPCR assay for high resolution genotyping of B. abortus and B. melitensis. A total of 486 Brucella was analyzed using the qPCR assay to create a 21-VGL profile database. The database contained the profiles of 55 B. abortus, 352 B. melitensis, 3 B. ceti, 6 B. neotomae, 7 B. ovis, 6 B. pinnipedialis, 44 B. suis and 13 B. canis strains. The 21-VGL Brucella genotyping clearly distinguished B. abortus, B. melitensis, B. neotomae and B. ovis. The 21-VGL approach could not distinguish B. pinnipedialis from B. ceti and some B. suis genotypes from B. canis. The results revealed that more than 99% of the Brucella isolates in Turkey were B. melitensis and 21-VGL genotyping can be reduced to 8-VGL B. melitensis genotyping without any loss of genotyping resolution. To our knowledge, we introduced the fastest and the lowest-cost B. abortus and B. melitensis genotyping and species differentiation methodology in the literature.

[Comparison of Multiplex Nested PCR, In-house Real-Time PCR and Commercial Real-Time PCR Methods for the Detection of Plasmodium spp. from Blood Samples]. / Plasmodium spp.'nin Saptanmasinda Multipleks Nested PCR, In-house Gerçek Zamanli PCR ve Ticari Gerçek Zamanli PCR Yöntemlerinin Karsilastirilmasi.

Malaria is a life-threatening parasitic disease caused by the parasites belonging to Plasmodium genus. Microscopic examination of Giemsa stained blood smears is accepted as the gold standard diagnostic method. It is recommended to use more than one method in order to strengthen the laboratory diagnosis of malaria which is an important health problem in our country as in the whole world. In this study, it was aimed to compare the results of three different molecular methods and determine which molecular method could be used in the diagnostic algorithm to be applied. DNA was extracted from 280 whole blood sample stored in EDTA tubes using a commercial kit. Three different polymerase chain reaction (PCR) methods were used for the detection of Plasmodium spp. in DNA samples obtained and the results were compared. First, multiplex nested PCR was applied and then in-house real-time PCR (Rt-PCR) which was validated in our laboratory and a commercial Rt-PCR kit were applied. Multiplex nested PCR was accepted as the gold standard and 182 samples that were evaluated as Plasmodium spp. positive and 98 samples that were evaluated as negative were also studied by in-house and commercial Rt-PCR methods. In multiplex nested PCR's first step reaction 1670 base pairs (bp) band was observed in Plasmodium spp. positive samples and 117 bp band was observed in Plasmodium vivax positive samples in the second step reaction. Tm values of P.vivax positive samples were determined as 78-79 in the melting analysis of the in-house Rt-PCR. CT values of the positive samples in in-house Rt-PCR were between 20.03-31.71 and were between 17.26-34.94 in the commercial Rt-PCR. With the in-house Rt-PCR method 180 cases were determined as positive, while with the commercial Rt-PCR method 178 cases were determined as positive. Two samples with the in-house Rt-PCR and 4 samples with the commercial Rt-PCR were considered as false negative. When the sensitivity and specificity of the both methods were calculated, the sensitivity of the in-house Rt-PCR method was 0.98, the specificity was 0.97, the positive predictive value (PPV) was 98%, the negative predictive value (NPV) was 97%, the sensitivity of the commercial Rt-PCR was 0.97, the specificity was 0.95, the PPV was 97%, the NPV was 95%. A high level of agreement (κ: 0.953) was determined between the in-house and the commercial Rt-PCR methods. In order for a test to be accepted as a confirmatory test, its specificity must be high. It was decided that sensitivity and specificity of the in-house Rt-PCR were suitable for using this method in the laboratory diagnosis of Plasmodium species.

Bacterial and protozoan agents found in Hyalomma aegyptium (L., 1758) (Ixodida: Ixodidae) collected from Testudo graeca L., 1758 (Reptilia: Testudines) in Corum Province of Turkey.

Hyalomma aegyptium (L., 1758) (Ixodida: Ixodidae) is a hard tick and the main host for adults are Palearctic tortoises of the genus Testudo, while larvae and nymphs are less host-specific and nymphs also attach to humans. In the present study, a total of 261 H. aegyptium ticks were removed from 26 Testudo graeca L., 1758 in Corum Province of Turkey. The most prevalent pathogens identified molecularly in the ticks were Hemolivia mauritanica (51.9 %), followed by Rickettsia aeschlimannii (32.6 %), Ehrlichia spp. (30.2 %), and Bartonella bovis (0.8 %). All samples were negative for Coxiella burnetii, Francisella tularensis, Anaplasma phagocytophilum, Babesia spp., Hepatozoon spp. and Theileria spp. Overall, 97.4 % of the examined adult ticks and 26.3 % of nymphs were infected with at least one pathogen, while 40.9 % of all ticks were infected with only one pathogen, 27.4 % with two pathogens, and 9.9 % with three pathogens, concomitantly. Overall, 80.8 % of the examined blood smears of tortoises were H. mauritanica-positive, and the mean intensity of parasitemia was 4.8 % (1-21). As a conclusion, since the examined tortoises were sampled in gardens and vineyards close to human habitation, and as a relatively large percentage of them were infested with ticks carrying pathogenic agents affecting also humans, the importance of tortoises, their ticks and pathogens in terms of the public health should be farther examined.

[Determination of the Subspecies of Francisella tularensis Isolated in Turkey by Molecular Methods]. / Türkiye'de Izole Edilen Francisella tularensis Alt Türlerinin Moleküler Yöntemlerle Belirlenmesi.

Francisella tularensis is a gram-negative, coccobasillus, facultative intracellular bacteria and causes a zoonotic disease, tularemia in humans. F.tularensis has four subspecies, which have different virulences for humans as F.tularensis subsp. tularensis, F.tularensis subsp. holarctica, F.tularensis subsp. mediasiatica and F.tularensis subsp. novicida. F.tularensis subsp. tularensis is the most virulent subspecies and mortality rate is high in human cases. F.tularensis subsp. holarctica, which has been reported in our country to date, has lower virulence than that of subsp. tularensis, and causes rare lethality among untreated patients. According to the erythromycin resistance and the properties of glucose-glycerol fermentation, F.tularensis subsp. holarctica has three biovar as biovar I, biovar II and biovar japonica. F.tularensis subsp. mediasiatica has been reported only in a few central asian countries and its virulence is similar to the F.tularensis subsp. holarctica F.tularensis subsp. novicida is avirulent for immunocompetent individuals but has been observed to cause infection in immunocompromised individuals. The aim of this study was to determine the F.tularensis subspecies in 259 F.tularensis strains isolated from clinical specimens, drinking water and a rodent sample and 517 F.tularensis PCR-positive DNA isolated from clinical specimens between years 2009 and 2014. Conventional PCR was performed using primers specific for the RD1 (Region Difference) region of F.tularensis. Subspecies were differentiated depending on the difference in PCR amplification product size. In our study, F.tularensis subsp. holarctica was detected in 764 samples yielding 922 base pair (bp) amplification product. The DNA samples obtained from one water and 11 lymph aspirates were determined as F.tularensis subsp. holarctica biovar japonica. The DNA sequence analysis of the amplification product of the RD1 region of the isolate from water sample was determined. The 1136 bp nucleotide sequence obtained from the DNA sequence analysis was 100% similar to F.tularensis subsp. holarctica biovar japonica (FCS075 strain-accesion number AF469618) when compared with GenBank data. The whole genome sequence of this isolate was also determined and recorded to GenBank with accesion number CP007148. None of the samples used in our study belonged to other sub-species. F.tularensis subsp. holarctica biovar japonica positive 11 lymph aspirate samples were sent to our center from Ankara (n= 1), Kayseri (n= 1) and Afyon (n= 9) provinces. The results of the current study revealed that F.tularensis subsp. holarctica biovar japonica caused a tularemia outbreak in a village in Afyon province at first time and it was observed sporadically in two other different provinces.

Characterization of Bartonella taylorii Strains in Small Mammals of the Turkish Thrace.

Rodents play role as a reservoir for some Bartonella species which cause different clinical manifestations in humans. Bartonella spp. existence in rodents of Turkish Thrace has been detected for the first time, and the risky habitat types were evaluated for the infection. Ninety individuals belonging to three small rodent species were screened by PCR, and the overall prevalence of Bartonella infection was 22.2%. The strains were characterized molecularly based on the phylogenetic analyses of two housekeeping genes, rpoB and gltA. They clustered with B. taylorii. The significant effects of habitat types and rodent species on Bartonella infections were observed. It was detected that B. taylorii prevalence was the highest in the swamp forest habitat and A. flavicollis species. The present study demonstrates that A. flavicollis is the reservoir of B. taylorii in the European part of Turkey.

[Method Verification of Inhouse Real-time Polymerase Chain Reaction for Detection of Toxoplasma gondii]. / Toxoplasma gondii'nin Inhouse Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Saptanmasinda Yöntemin Dogrulanmasi.

Toxoplasmosis is one of the most common parasitic infection in humans. Serological and molecular methods are used for diagnosis. Molecular methods are becoming increasingly preferred, since they lead to shortening of diagnostic time. In our study, it was aimed to determine Toxoplasma gondii by a cost-effective, quantitative, fast and reliable method without using a commercial kit, and apply method verification. T.gondii strain which was continued by mouse inoculation in our laboratory was used for method verification study. For this purpose DNA extraction was performed using a commercial kit. The limit of detection and, high and low positivity rates were determined by serial dilutions of DNA sample. Accuracy and certainty studies were performed using with TG-F, TG-R primers and TaqMan TG probe for method verification of the test. In the study with serial dilutions of DNA sample, detection limit was determined as 10-3 dilutions (0.028 copies/reaction). Furthermore 10-1 dilution (2.8 copies/reaction) was considered as high positive, 10-2 dilution (0.28 copies/reaction) was considered as low positive and method verification studies were performed. The accuracy of test was determined as 0.62 for high positive samples and 0.14 for low positive samples. CV value of intra-assay certainty was 0.62 for high positive samples and 0.14 for low positive samples, whereas, CV value of inter-assay certainty was calculated as 1.03 for high positive samples and 2.34 for low positive samples. Correlation coefficient was determined as 0.99. The coefficient of variation of inhouse realtime PCR method used in our study was found to be below 15%, and it was decided to be suitable for routine laboratory studies.

[First Isolation of Coxiella burnetii in Turkey from a Patient with Endocarditis; Antigen Production and Phase Change Study]. / Endokarditli Bir Olgudan Coxiella burnetii'nin Türkiye'de Ilk Izolasyonu; Antijen Üretimi ve Faz Degisimi Çalismasi.

Coxiella burnetii is the causative agent of Q fever, a zoonotic infection. The bacteria is a gram-negative, pleomorphic, coccobacilli and capable to survive and proliferate within the host cell's phagolysosome. There are two morphological cell types of C.burnetii including small and large cell variants. C.burnetii is divided into phase I and phase II serologically variants according to LPS structure in the cell wall. Phase I is the natural phase found in infected animals or humans and is highly infectious. Phase II is not very infectious and could be obtained only in laboratories after serial passages in cell cultures or embryonated egg cultures. Q fever can be asymptomatic (in 50% of the cases), acute or chronic. Major presentations of acute Q fever are flu-like illness, pneumonia, and hepatitis, whereas the chronic form presents mainly as infective endocarditis. The aim of this study was to obtain C.burnetii phase II variant from C.burnetii phase I variant by a phase change study. In this study, C.burnetii was isolated by cell culture method from the heart valve tissue of a Q fever endocarditis case. C.burnetii phase I antigen for the indirect fluorescent antibody test (IFAT) was prepared from the isolated strain. For the isolation and identification of C.burnetii, heart valve tissue of the patient was homogenized and DNA was extracted by tissue extraction kit. C.burnetii DNA in the valve tissue was determined by real-time PCR (Rt-PCR). This C.burnetii DNA positive specimen was inoculated into Vero cells by shell vial centrifugation method. The scraped Vero cells were fixed on the slides after one week of incubation and IFAT was performed using C.burnetii phase I IgG positive sera, bacteria that were grown in and surrounding the Vero cells stained apple green were determined microscopically. Infected cells were disrupted by freeze and thaw method to obtain bacterial suspension. The DNA obtained from the bacterial suspension was again found to be positive for C.burnetii by Rt-PCR. Isolation sample was found to be positive in PCR at an earlier cycle compared to heart tissue sample, thus the bacterial growth was also confirmed with PCR. 16S ribosomal RNA gene of our isolate was amplified by PCR using 27F and 1492 primers and then sequenced. The DNA sequences were compared with reference DNA sequences of GeneBank; and the nucleotide sequence of the 16S ribosomal RNA gene of our isolate was found to be 99% similar to C.burnetii strain ATCC VR-615 an accession number NR104916. Serial cell culture passages of the isolated strain were performed to obtain C.burnetii phase II variant from C.burnetii phase I variant. After each passage, presence of phase change was investigated by IFAT using C.burnetii phase I and phase II IgG positive sera. At the end of 17 cell culture passages, phase change could not be observed. C.burnetii phase I IFAT antigen was prepared from the obtained bacterial suspension. In this study, we presented the isolation and identification of C.burnetii by cell culture, molecular and serological methods from the heart valve of a patient with endocarditis for the first time in our country.

Molecular Survey of Babesia microti (Aconoidasida: Piroplasmida) in Wild Rodents in Turkey.

Babesia microti (Aconoidasida: Piroplasmida) (Franca, 1910) is an important tick-borne zoonotic parasite with rodents serving as reservoir hosts. In the present study, 536 rodents were captured from Burdur, Bartin, Giresun, and Yozgat provinces of Turkey between the years 2010 and 2012, and blood samples were examined for the presence of Babesia spp. using conventional PCR which targeted the 18S rRNA gene. The sequence analysis of PCR amplicons was tested for B. microti as well as for Hepatozoon spp., and Sarcocystis spp. Overall, 5.8% of the rodents were positive for B. microti: 41% in Myodes glareolus, 7.7% in Chionomys roberti, and 2% in Apodemus spp., whereas no Babesia DNA was detected in Mus macedonicus and Microtus spp. Six rodents were positive for Hepatozoon spp. and one rodent was positive for Sarcocystis spp. Overall, 14.9 and 4.5% of rodents captured from Bartin and Giresun provinces, respectively, were PCR positive for B. microti, whereas none of rodents captured in Burdur and Yozgat were positive for Babesia spp. The sequence data of B. microti from rodents revealed that all sequences belonged to the zoonotic genotype. Sequences of B. microti obtained from rodents of the Bartin province were genotypically closer to European isolates, whereas those obtained from rodents of the Giresun province were closer to Russian and Mongolian isolates.

[Isolation of Rickettsia slovaca by shell-vial cell culture method from Dermacentor marginatus ticks]. / Shell-vial hücre kültürü yöntemi ile Dermacentor marginatus türü kenelerden Rickettsia slovaca izolasyonu.

Rickettsia species are gram negative, small pleomorphic coccobacilli, obligate intracellular bacteria. The majority of these bacteria are transported by the ticks. Rickettsia slovaca and Rickettsia raoultii are among the Rickettsia species in the spotted fever group and carried by Dermacentor species ticks, that cause tick-borne lymphadenopathy (Tickborne Lymphadenopathy-TIBOLA or Dermacentor-borne necrotic erythema and lymphadenopathy- DEBONEL). Rickettsia species are obligate intracellular bacteria and they can be cultivated in cell cultures. The chance of isolation of Rickettsia species from clinical and tick specimens has been increased by the shell-vial centrifugation cell culture method. The aim of this study was to isolate R.slovaca from two Dermacentor marginatus species ticks which were detected in humans by the use of shell-vial centrifugation cell culture method using VERO cell. Before proceeding to the culture stage, an algorithm including description, disinfection, dissection of ticks and methods of hemolymph, homogenization, DNA extraction and real-time PCR was performed. Iodine-alcohol disinfection was performed following identification of the ticks with the use of a stereo microscope. Ticks hemolymph were obtained with extremity dissection of ticks and indirect fluorescence antibody (IFA) test was performed with Rickettsia positive sera. Ticks identified as containing Rickettsia like bacteria in hemolymph were homogenized and DNA extraction was performed from homogenate of ticks. By real-time PCR, using Rickettsia genus-specific PanR8 primers and probes, PCR positive Rickettsia spp. were determined among the ticks. Vero cells that have formed monolayer in vials were used for Rickettsia culture. Homogenized tick samples which were positive for Rickettsia in hemolymph and real-time PCR methods were inoculated to cell culture vials. Suspended bacteria in the inoculum were allowed to approach the cells by the shell-vial centrifugation method. Cells were scraped after five days of incubation in 5% CO2 at 36°C. An aliquot of the determined cell suspension was fixed to the slides and then IFA was performed using Rickettsia positive sera. In fluorescence microscopy examination, adhered and proliferated bacteria were observed on Vero cells. This cell suspension was again inoculated into the Vero cell cultures to increase bacterial replication and taken for 5-7 days of incubation. DNA was extracted from the suspension of the cultivated bacteria. Conventional PCR of citrate synthase (gltA) and outer membrane protein A (ompA) gene regions were used to identify Rickettsia species. DNA sequence analysis of PCR amplification products were determined. DNA sequence results were compared to Genbank data and found that the gltA sequence was 100%, similar to R.slovaca with accession number AY129301.1 and the ompA sequence was 100%, similar to R.slovaca with accession number KF791242.1.  In addition, the two strains isolated in phylogenetic analysis were found to be R.slovaca. As a result, R.slovaca isolation from of D.marginatus type ticks has been reported for the first time in our country by the cell culture method.

First report of human ehrlichiosis in Turkey.

Emiroglu M, Çelebi B. First report of human ehrlichiosis in Turkey. Turk J Pediatr 2019; 61: 267-270. Ehrlichiosis, a tick-borne infection, can cause severe and fatal disease. A 6-year-old boy was admitted with fever, chills, malaise, headache, anorexia, rhinorrhoea, and cough lasting two days. He had had contact with a dog 10 days prior, and a tick had been removed the day before. Fever, minimal conjunctival injection, oropharyngeal hyperemia and cracked, hyperemic lips were observed. Laboratory tests were normal except for lymphopenia and hyponatremia. Cytoplasmic morulae in the monocytes and granulocytes were seen on peripheral blood smear. Doxycycline was started immediately, and the fever subsided within 48 hours. Given the Ehrlichia was positive but Anaplasma negative by real-time PCR, he was diagnosed as ehrlichiosis, subspecies identification could not be performed. This is the first human ehrlichiosis case in Turkey.

[A Rickettsia case caused by Rickettsia conorii]. / Rickettsia conorii'nin neden oldugu riketsiyal enfeksiyon olgusu.

Rickettsia species are gram-negative intracellular, small pleomorphic coccobacilli in the Rickettsiaceae family. This genus is serologically and genotypically divided into four groups as spotted fever group, typhus group, Rickettsia belli and Rickettsia canadensis. Rickettsia conorii (R.conorii subsp. conorii) in the spotted fever group was reported to cause mediterranean spotted fever in Europe, especially in mediterranean countries including Turkey. The major vectors of Rickettsia species are ticks, and in some species fleas or mites. In this report a case with R.conorii infection was presented. A 46-year-old female patient, who had anorexia, fatigue, muscle aches, chills and high fever was admitted to a health institution. The patient was diagnosed as influenza. There was no regression in the patient's complaints with the recommended treatment. The patient was examined in our infectious diseases clinic and had several symptoms like severe muscle and joint pain with significant headache, and rashes at her body including hands and feet. The patient had a single eschar in the upper midline of the belly that matched tick biting and pink small maculopapular scars on the trunk, arms, legs, feet, and hands. Considering a Rickettsia pre-diagnosis, liquid electrolyte and doxycycline 2 x 100 mg oral treatment was started. On the third day of treatment, high fever, muscle and joint pain were decreased. On the fifth day, active skin lesions were started to fade. R.conorii IgM and IgG were negative in the first serum sample of the patient. In the biopsy sample taken from eschar tissue, Rickettsia spp. was detected as positive with rt-PCR. PCR was used by using the specific regions of the genetically specific gltA and ompA genes in the biopsy specimens and then the PCR products were determined by DNA sequence analysis. The DNA sequence results were compA red with Genbank data and determined that the gltA sequence was 99%, similar to R.conorii with accession number JN182786 and the ompA sequence was 99%, similar to R.conorii with accession number KR401144. When the phylogenetic tree was created, it was observed that the etiological agent was R.conorii. A week after the treatment, in the second serum sample R.conorii IFA IgM 1/192 titer and IgG 1/320 titer were detected as positive. In this case report, we have presented a Rickettsia case, clinically diagnosed as Rickettsia, serologically negative in the acute phase, PCR positive, with post-treatment seroconversion and etiologic agent determined as R.conorii.

Metagenomics Analysis of Bacterial Population of Tympanosclerotic Plaques and Cholesteatomas.

Objective Chronic otitis media can cause cholesteatomas or tympanosclerosis; however, the pathophysiology of such conditions is not completely known. The aim was to identify a bacterial genome that might be present in tympanosclerotic plaques and cholesteatomas using sequence analysis of the gene responsible for the transcription of 16 ribosomal RNA (rRNA). Study Design Metagenomics analysis of the samples. Setting Samples were collected and evaluated at tertiary care centers. Subjects and Methods Sixty-five tympanosclerotic plaques and 37 cholesteatomas were evaluated. The polymerase chain reaction (PCR) was performed using primers designed for the amplification of the gene responsible for the transcription of bacterial 16 rRNA. The PCR-positive samples were sequenced via Sanger method, and 46 selected samples were analyzed with next-generation sequencing (NGS). Results Sanger sequencing revealed the presence of bacterial genomes in a total of 18 of the 102 samples tested. Sequencing of these genomes indicated the presence of Alloiococcus otitis, Staphylococcus aureus, Achromobacter xylosoxidans, Escherichia coli, Staphylococcus sciuri, Staphylococcus caprae, Parvimonas spp., and Bacillus sp. in the tested samples. The NGS showed 1 or more different bacterial genomes in 44 (95.7%) of the 46 samples tested. Predominately, genome of Clostridiales (27 samples), Staphylococcaceae (24 samples), Peptoniphilaceae (12 samples), and Turicella otitidis (9 samples) were identified. Conclusion The middle ear is inhabited by a diverse microbial community than that previously known. With the use of molecular biology, it has become easier to identify the bacterial genomes and improve our understanding of the role of middle ear microbiota in the pathogenesis of chronic inflammatory ear diseases.

Bacterial and protozoal pathogens found in ticks collected from humans in Corum province of Turkey.

BACKGROUND: Tick-borne diseases are increasing all over the word, including Turkey. The aim of this study was to determine the bacterial and protozoan vector-borne pathogens in ticks infesting humans in the Corum province of Turkey. METHODOLOGY/PRINCIPAL FINDINGS: From March to November 2014 a total of 322 ticks were collected from patients who attended the local hospitals with tick bites. Ticks were screened by real time-PCR and PCR, and obtained amplicons were sequenced. The dedected tick was belonging to the genus Hyalomma, Haemaphysalis, Rhipicephalus, Dermacentor and Ixodes. A total of 17 microorganism species were identified in ticks. The most prevalent Rickettsia spp. were: R. aeschlimannii (19.5%), R. slovaca (4.5%), R. raoultii (2.2%), R. hoogstraalii (1.9%), R. sibirica subsp. mongolitimonae (1.2%), R. monacensis (0.31%), and Rickettsia spp. (1.2%). In addition, the following pathogens were identified: Borrelia afzelii (0.31%), Anaplasma spp. (0.31%), Ehrlichia spp. (0.93%), Babesia microti (0.93%), Babesia ovis (0.31%), Babesia occultans (3.4%), Theileria spp. (1.6%), Hepatozoon felis (0.31%), Hepatozoon canis (0.31%), and Hemolivia mauritanica (2.1%). All samples were negative for Francisella tularensis, Coxiella burnetii, Bartonella spp., Toxoplasma gondii and Leishmania spp. CONCLUSIONS/SIGNIFICANCE: Ticks in Corum carry a large variety of human and zoonotic pathogens that were detected not only in known vectors, but showed a wider vector diversity. There is an increase in the prevalence of ticks infected with the spotted fever group and lymphangitis-associated rickettsiosis, while Ehrlichia spp. and Anaplasma spp. were reported for the first time from this region. B. microti was detected for the first time in Hyalomma marginatum infesting humans. The detection of B. occultans, B. ovis, Hepatozoon spp., Theileria spp. and Hemolivia mauritanica indicate the importance of these ticks as vectors of pathogens of veterinary importance, therefore patients with a tick infestation should be followed for a variety of pathogens with medical importance.

[A negleted bacteria with a case: Bartonella henselae]. / Bir olgu ile ihmal edilen bir etken: Bartonella henselae.

Bartonella henselae the causative agent of cat scratch disease (CSD), is a gram-negative, coccobacillus, facultative intracellular bacterium CSD usually presents as a clinical form of benign local lymphadenopathy (LAP) but sometimes it may progress to severe life threatening complications. Despite the fact that CSD is known to be a common disease, which is one of the important causes of local LAPs in the world, there are few publications in our country. For the diagnosis, the clinician should suspect for CSD and has to ask to the patient whether there is a story of cat scratch or not. In our country the diagnosis of CSD is usually done by invasive pathological examination instead of simple serological tests. In this report, a 14 years old case with CSD with antibody titers of 1/384 IgM, 1/2048 IgG B.henselae antibody determined by indirect fluorescent antibody (IFA) method in serum and B.henselae positivity by polymerase chain reaction (PCR) from LAP sample of the patient with axillary LAP was presented. Even though molecular techniques have been used for the diagnosis of the previous reported cases, it is the first B.henselae positive case in our country detected with PCR. In the history of the case it was learned that the patient was scratched by a street cat few months ago and the axillary LAP developed 4-5 weeks later. Axillary ultrasonography shawed abscesses with the largest 22 x 44 mm compatible with LAP. No growth was detected in the LAP biopsy specimen culture. Leucocyte count was normal but sedimentation rate (68 mm/h), and C-reactive protein (41.7 mg/L) were higher.Therapy was started with azitromycin 500 mg/day but two weeks later as there was no regression of LAP, considering the development of resistance, the treatment was changed to doxycycline 2 x 100 mg/day and rifampicin 1 x 300 mg/day. As the LAP was in abscess formation and the titers found in IFA was higher than the predictive value of B.henselae antibody titer for endocarditis, the treatment has been extended to four weeks and the patient has been cured. Especially children and adolescents are at very high risk for zoonotic infections transmitted from pets in our country due to the intense immigration to the city from the rural areas and the unconscious and uncontrolled livelihood of friendship with street animals. We should accept that this is not a rare condition, as the cat scratch disease can change from harmless to very serious forms the diagnosis and treatment should be quickly and carefully performed. Currently, serological examinations for Bartonella are rarely done in some certain reference laboratories in our country. The number of these laboratories should be increased or the usage of the tests in these reference laboratories should be at least expanded.

Evaluation of clinical and serological findings for diagnosis of cutaneous anthrax infection after an outbreak.

PURPOSE: Anthrax, caused by the bacterium Bacillus anthracis, is one of the oldest documented infectious diseases in both livestock and humans. We aimed to evaluate clinical findings and risk factors of patients with cutaneous anthrax infection and report anti-lethal factor (LF) IgG and anti-protective antigen (PA) IgG titers in the serologic diagnosis of disease. METHODS: In this study, serum samples of 18 cutaneous anthrax patients were collected and anti-LF IgG and anti-PA IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Twelve (67%) males and 6 (33%) females, with a mean age of 36.06 ± 16.58 years were included in the study. Risk factors identified in the patient population studied were slaughtering (28%), flaying (56%), chopping meat (67%), burying diseased animal corpses (17%) and milking (6%) livestock. Black eschar formation (94%), pruritus (78%) and painful lymphadenopathy (61%) were first three common clinical signs and symptoms, respectively. Fourteen (78%) patients produced a positive IgG response against PA, 11 (61%) patients produced against LF. Three (17%) patients had no response to either antigen. CONCLUSIONS: A detailed history of contact with sick animals or animal products along with clinical findings should be taken at the first step for the diagnosis of cutaneous anthrax infection. Serologic detection of anti-LF IgG and anti-PA IgG with ELISA may be useful auxillary method for establishing the diagnosis.