[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.

[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.

[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.

[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 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.

[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.

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.

[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.

Oropharyngeal Tularemia Outbreak Associated with Drinking Contaminated Tap Water, Turkey, July-September 2013.

In 2013, an oropharyngeal tularemia outbreak in Turkey affected 55 persons. Drinking tap water during the likely exposure period was significantly associated with illness (attack rate 27% vs. 11% among non-tap water drinkers). Findings showed the tap water source had been contaminated by surface water, and the chlorination device malfunctioned.

Water as Source of Francisella tularensis Infection in Humans, Turkey.

Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey.

Dobrava-Belgrade virus in Apodemus flavicollis and A. uralensis mice, Turkey.

In 2009, human Dobrava-Belgrade virus (DOBV) infections were reported on the Black Sea coast of Turkey. Serologic and molecular studies of potential rodent reservoirs demonstrated DOBV infections in Apodemus flavicollis and A. uralensis mice. Phylogenetic analysis of DOBV strains showed their similarity to A. flavicollis mice-borne DOBV in Greece, Slovenia, and Slovakia.

[Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis]. / Francisella tularensis'in moleküler tanisinda yeni gelistirilen kullanima hazir ticari PCR kitinin etkinliginin degerlendirilmesi.

Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR Toolbox assay were found to be 100%. The lowest detection limit of this method was determined as 100 genomic equivalent per PCR reaction mix. The new PCR kit is a rapid and accurate alternative to the conventional PCR methods since the toolbox includes all of the required chemicals, accessories and equipments. This ready-to-use PCR assay was appraised to be a valuable diagnostic tool for the detection of F.tularensis in the outbreak settings particularly in remote areas with limited resources.

[Investigation of the presence of Mycobacterium tuberculosis in the lymph node aspirates of the suspected tularemia lymphadenitis cases]. / Tularemi lenfadeniti süphesi ile alinan lenf aspirati örneklerinde Mycobacterium tuberculosis s varliginin arastirilmasi

Recently reports of cervical tuberculous lymphadenitis and oropharyngeal tularemia which are the most common infectious causes of granulomatous lymphadenitis, have been significantly increased in Turkey. The differentiation of cervical tuberculous lymphadenitis and oropharyngeal tularemia is usually confusing on the basis of clinical and histopathological findings. Thus, in tularemia endemic areas, the patients are more commonly evaluated in terms of tularemia lymphadenitis leaving tuberculosis out. The aim of this study was to investigate the presence of Mycobacterium tuberculosis in cervical lymph node aspirates, obtained from tularemia suspected cases. A total of 105 oropharyngeal tularemia-suspected cases which were found negative for Francisella tularensis by bacteriological (culture), molecular (PCR) and serological (microagglutination) methods, were included in the study. The samples had been previously studied at National Tularemia Reference Laboratory, Turkish Public Health Institution, between 2009-2011. The study samples were evaluated in terms of M.tuberculosis by culture and real-time PCR (rtPCR) methods in the National Tuberculosis Reference Laboratory. Both Lowenstein-Jensen (LJ) medium and liquid-based MGIT (BD, USA) automated culture system were used for mycobacterial culture. Samples that yielded mycobacterial growth were identified as M.tuberculosis by immunochromotographic test (BD, USA). The lymph node aspirates of 65 patients who were F.tularensis PCR negative but antibody positive, were used as the control group. As a result, M.tuberculosis was found to be positive in 9 (8.6%) of 105 tularemia-negative lymph node aspirates, sent to our laboratory from different geographic regions for the investigation of tularemia. Six of the M.tuberculosis positive cases were male and the age range of the patients was 26-85 years. The presence of M.tuberculosis was detected only by culture in two samples, only by rtPCR in five samples and both by culture and rtPCR in two samples. M.tuberculosis was not identified in the control group specimens. Three of the samples which revealed tuberculosis, were from the tularemia endemic areas. In conclusion, the data of this preliminary study indicated that tuberculous lymphadenitis should be kept in mind in suspected tularemia cases and those patients should also be investigated simultaneously for the presence of tuberculous lymphadenitis.

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.

In vitro susceptibility of isolates of Francisella tularensis from Turkey.

BACKGROUND: Tularemia is an infection caused by Francisella tularensis, which has a wide distribution in the northern hemisphere and diverse clinical manifestations. For decades, the drug of choice for the treatment of tularemia has been streptomycin, with tetracycline and chloramphenicol being used as alternatives. The purpose of the present study was to determine the in vitro antimicrobial susceptibility of a large panel of geographically diverse F. tularensis isolates from Turkey against traditional and newer antimicrobial agents. METHODS: The antibiotic susceptibilities of 250 F. tularensis strains were examined using the Epsilometer test for 9 antimicrobial agents. Each isolate was identified by conventional and molecular techniques. RESULTS: All the strains were confirmed biochemically and using a combination of species- and subspecies-specific polymerase chain reaction (PCR) assays to be F. tularensis subsp. holarctica. One isolate was assigned to F. tularensis subsp. holarctica biovar japonica based on erythromycin susceptibility, an ability to ferment glycerol, and the nucleotide sequence of the region of difference 1 (RD1). All strains were susceptible to aminoglycosides (streptomycin and gentamicin), tetracyclines (tetracycline and doxycycline), chloramphenicol, 2 fluoroquinolones (ciprofloxacin and levofloxacin), and rifampicin. In addition, all isolates except 1 had a minimal inhibitory concentration (MIC) for erythromycin of > 256 µg/ml. CONCLUSIONS: Since the fluoroquinolones showed the lowest MIC values and have advantages such as excellent bioavailability and activity, availability of oral formulations, and lower toxicities, they represent candidate therapeutic options in the first-line treatment of tularemia. To the best of our knowledge, this is the first report of the presence of F. tularensis subsp. holarctica biovar japonica outside Japan.

Francisella tularensis bacteremia: report of two cases and review of the literature.

Isolation of Francisella tularensis in blood culture is exceedingly rare and has been reported infrequently in Europe; a literature review showed 28 documented cases. Herein we report the first cases of bacteremic F.tularensis pneumonia in immunocompetent individuals in Turkey.

[Development of a novel Francisella tularensis antigen stained with tetrazolium-blue for tularemia microagglutination test]. / Tularemi mikroaglütinasyon testi için tetrazolyum mavisi ile boyali Francisella tularensis antijeninin gelistirilmesi.

The isolation of Francisella tularensis in cultures is the reference method for the laboratory diagnosis of tularemia. However, due to the limitations such as the low sensitivity and need for high safety level and equipped laboratories, serologic methods are frequently used as diagnostic tools. F.tularensis-specific antibodies may be demonstrated by several methods, however microagglutination test (MA) remains the most common method with its high sensitivity and specificity. The aim of this study was to develop a novel MA test antigen prepared from whole F.tularensis cells and stained with tetrazolium-blue for more clear and easier evaluation. F.tularensis NCTC 10857 strain was cultured on the cysteine heart agar supplemented with 9% sheep blood and bacterial cells were harvested by scraping, collected in physiological saline (PS) and centrifuged at 1500 rpm for 10 minutes. For preparing stock antigen suspension cell concentration was adjusted to OD600=1.5, spectrophotometrically. Tetrazolium-blue solution (BTC [3,3'-(3,3'-Dimethoxy[1,1'-biphenyl]-4,4'-diyl) bis [2,5-diphenyl-2H-tetrazolium dichloride], T4375 Sigma-Aldrich) at the final concentration of 1% was added to cell suspension and incubated at 37°C for 5 hours for absorption. Then, the living cells were chemically inactivated by formaldehyde. Repeating centrifugations were performed to discard excess dye and formaline, then 0.4% formaline saline was added on the sediment. Optimum concentration of this novel antigen (BTC-Ag) for MA test was determined by plate titration method by using standard serum sample with a known MA titer (1/128). The performance of BTC-Ag in MA test was evaluated by using 100 patient sera positive for F.tularensis antibodies, and 100 tularemia negative patient sera (of them 50 were seropositive for brucellosis). All of the results were compared with standard MA test in which safranin-O stained antigen (SO-Ag) was used. There was 100% agreement between the two tests performed with BTC-Ag and SO-Ag in tularemia seropositive (in ≥ 1/20 titers and when ±1 dilution variation was accepted as normal) and seronegative sera. No significant cross reactivity with Brucella spp. was observed. Accuracy, sensitivity and specificity of BTC-Ag were found to be 100%. In conclusion, newly developed BTC-Ag for MA test provides better agglutination patterns resulting in a clear supernatant in wells, thus provides easy evaluation for the agglutination reaction, and is expected to facilitate tularemia serodiagnosis.

[In Vitro Activity of Tigecycline Against Francisella tularensis Subsp. holarctica in Comparison with Doxycycline, Ciprofloxacin and Aminoglycosides]. / Tigesiklinin Francisella tularensis Alttip holarctica'ya In Vitro Etkinligi; Doksisiklin, Siprofloksasin ve Aminoglikozidlerle Karsilastirilmasi

Francisella tularensis is the etiological agent of tularemia which is a zoonosis of the northern hemisphere. For decades, streptomycin was considered the drug of choice, despite possible side effects, and vestibular toxicity in particular. Alternatives are tetracylines and chloramphenicol which are bacteriostatic agents that are associated with a considerable risk of relapse. The aim of the present study was to assess the in vitro susceptibility of F.tularensis subsp. holarctica biovar II strains to tigecycline, a member of a new class of glycylcyclines. Fourteen F.tularensis strains isolated from patients in Central Anatolia region of Turkey were examined. Minimum inhibitory concentration (MIC) values of tigecycline, doxycycline, streptomycin, gentamicin, and ciprofloxacin were determined using the E-test method on glucose-cysteine blood agar plates. Interpretation of results was made according to CLSI clinical breakpoints. All strains were susceptible to the antibiotics traditionally used to treat tularemia. Tigecycline showed good in vitro activity to all the isolates (MIC range: 0.094-0.38 mg/L). In this study, tigecycline was more active than doxycycline against F.tularensis subsp. holarctica strains, according to MIC50 (0.19 mg/L) and MIC90 (0.25 mg/L) values. Doxycycline (MIC90: 0.38 mg/L) showed good in vitro activity against all the isolates and MIC values interpreted according to the CLSI criteria for potential bioterrorism agents, have shown ranges below the breakpoint for sensitivity determination (S ≤ 4 mg/L). Ciprofloxacin had the lowest MIC50 and MIC90 values. In case the other antibiotics can not be used or intravenous therapy is required, tigecycline may be an important therapeutic alternative agent. However, confinement of tigecycline in the treatment of multi-drug resistant bacterial infections, its parenteral way of administration and overall cost were considered as the major limitations of tigecycline in tularemia treatment.

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.

Synthesis of a monolithic, micro-immobilised enzyme reactor via click-chemistry.

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-L-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography-IMERs.