Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

Evaluation of Intestinal Microbiota in Children With Sickle Cell Disease.

BACKGROUND AND AIMS: Sickle cell disease (SCD) is a chronic hemolytic anemia that may be life-threatening due to multisystemic effects. Identification of the factors which affect the pathophysiology of the disease is important in reducing mortality and morbidity. This study aimed to determine gut microbial diversity in children and adolescents with SCA compared with healthy volunteers and to evaluate the clinical impact of microbiota. MATERIALS AND METHODS: The study included 34 children and young adolescents with SCD and 41 healthy volunteer participants. The microbiome was assessed by 16S rRNA sequencing in stool samples. Laboratory parameters of all participants, such as complete blood count and C-reactive protein values and clinical characteristics of SCD patients, were determined and compared, as well as clinical conditions of the patients, such as vascular occlusive crisis and/or acute chest syndrome, frequency of transfusions, intake of penicillin, hydroxyurea, and chelation therapy were recorded. RESULTS: White blood cell count, hemoglobin, immature granulocyte and C-reactive protein levels were significantly higher in the patient group ( P <0.05). Microbiota analysis revealed 3 different clusters among subjects; controls and 2 clusters in the SCD patients (patient G1 and G2 groups). Bacteroides spp. were more prevalent, while Dialester spp. and Prevotella spp. were less prevalent in SCD compared with controls ( t =2.142, P <0.05). Patient G2 (n=9) had a higher prevalence of Bacteroides and a lower prevalence of Prevotella than patient G1 (n=25). CONCLUSION: In our study, there was a difference between SCD patients and the control group, while 2 different microbiota profiles were encountered in SCD patients. This difference between the microbiota of the patients was not found to affect the clinical picture (such as vascular occlusive crisis, acute chest syndrome).

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents.

AIMS: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. METHODS: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. CONCLUSIONS: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Determination of genetic diversity of multidrug-resistant Mycobacterium tuberculosis strains in Turkey using 15 locus MIRU-VNTR and spoligotyping methods.

Tuberculosis (TB) remains the leading cause of deaths from infectious disease worldwide. Nowadays, the tendency of Mycobacterium tuberculosis complex (MTBC) to spread between continents due to uncontrolled migration movements shows that TB is a global health problem. The number of studies for the detection of MTBC strains' epidemiological features in areas with TB spread risk using molecular-based methods such as spoligotyping and Mycobacterial Interspersed Repetitive Unit (MIRU) Variable Number Tandem Repeats (VNTR) at the clonal level is insufficient. In this study, it was aimed to determine the phylogenetic relationships of MTBC strains at the species level by spoligotyping and 15 locus MIRU-VNTR (MIRU-VNTR15) molecular methods of 96 multidrug-resistant (MDR) MTBC strains isolated from sputum samples of patients with a preliminary diagnosis of pulmonary TB or suspected contact history those sent to National Tuberculosis Reference Laboratory from the centers that are members of the Tuberculosis Laboratory Surveillance Network. The phylogenetic relationship between 96 MDR-TB strains was investigated with the combination of bead-based spoligotyping and MIRU-VNTR15 methods on the MAGPIX® Milliplex Map device. In this study, it was determined that the T1 family is more common in our country and LAM7-TUR family is less common than the Beijing family unlike other studies. It was determined that the strains in the same cluster had different locus profiles, and there was no transmission from the same clone in the clonal typing we performed with spoligotyping and MIRU-VNTR15.

Evaluation of Echinococcosis Pre-Diagnosis Patients Admitted to the National Parasitology Reference Laboratory of Turkey from 2014-2019.

Background: Echinococcosis is a common parasite with zoonotic character created by a small cestode, Echinococcus spp., and is an important public health problem in Turkey as well as all over the world. We aimed to investigate antibodies in serum samples of suspected Echinococcosis patients sent to the National Parasitology Reference Laboratories of the General Directorate of Public Health. Methods: Serum samples of 2390 patients sent to our laboratory between January 1, 2014 and May 01, 2019, evaluated by ELISA, Indirect Hemagglutination Test (IHA) and Western Blot (WB) methods are presented. Our laboratory is the national reference laboratory. All kinds of tests requested from suspected patients can be performed. Results: Overall, 1199 (50.2%) of 2390 serum samples were female and 1191 (49.8%) were male. It was observed that 178 (14.9%) of men and 210 (17.5%) of women were seropositive. There was no statistical difference between the sexes in terms of seropositivity. Of all samples, 1941 (81.2%) were negative, 388 (16.2%) were positive, and 61 (2.6%) were borderline. Results determined as borderline are considered suspicious and a recommendation is made to repeat the test after 15 days. A statistical difference was found in the distribution of seropositivity by years. While seropositivity was lowest in 2014, it was found to be highest in 2018 and 2019. Conclusion: Despite all the precautions taken, it is seen that echinococcosis still continues to exist in Turkey as a zoonotic disease. Hence, CE has been involved in Turkey Zoonotic Diseases Action Plan (2019-2023) and decided to carry out studies for the protection and prevention of the disease.

[A Case of Chronic Q Fever Endocarditis Mimicking Lymphoproliferative Disorders]. / Lenfoproliferatif Hastaliklarla Karisan Bir Kronik Q Atesi Endokarditi Olgusu.

Q fever is a zoonosis caused by Coxiella burnetii. In this report, a case of chronic Q fever endocarditis with pancytopenia and hypergammaglobulinemia mimicking a lymphoproliferative disease was presented. A 39-years-old male living in Çatalca and whose family is engaged in animal husbandry admitted with the complaints of weakness and fatigue. The patient had aortic valve replacement 29 years ago and had aortic valve re-replacement, and ascending aorta grafting because of endocarditis three years ago. It was revealed that the second operation of the patient was due to possible infective endocarditis, but no definitive agent could be identified. He was evaluated for massive hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, presence of M-spike and elevated ß-2 microglobulin levels and was referred to our hematology clinic with a preliminary diagnosis of lymphoproliferative disease. Lymphoplasmacytic lymphoma was excluded with the result of bone marrow biopsy and he was referred to our clinic for the investigation of possible infectious etiologies. We detected hepatosplenomegaly and finger clubbing. His blood analyses were normal except for the following: leukocyte count 3800/µl, platelet count 148000/µl, gamma globulin 5.9 gr/dl, rheumatoid factor (RF) and antinuclear antibody (ANA) positivity. Chronic Q fever endocarditis was suspected and C.burnetii Phase I IgG test was found positive in 1/132071 titers. Although transesophageal echocardiography showed no lesion of endocarditis, positron emission tomography/computed tomography revealed increased fluorodeoxyglucose uptake around the prosthetic heart valve and graft. The patient was diagnosed as having Q fever endocarditis and graft infection. He refused hospitalization and was started on hydroxychloroquine and doxycycline treatment. The patient stopped taking these antibiotics by himself seven days after the diagnosis. He was admitted with a headache to another hospital and operated for an intracranial hemorrhage and died shortly after. Apart from unfamiliarity, wide range of clinical presentations of disease could also lead to delayed diagnosis. Among patients with chronic Q fever, continuous bacteremia and antigenic stimulus causes inflammatory syndrome with hepatosplenomegaly, hypergammaglobulinemia and, presence of autoantibodies which leads to misdiagnoses of rheumatologic, autoimmune or hematologic diseases Chronic Q fever should be investigated in patients with known valvulopathy and chronic hepatomegaly or splenomegaly, pancytopenia, hypergammaglobulinemia, and unexplained autoantibody positivity.

Pertussis antibody levels in infants and their mothers receiving combined tetanus-diphtheria toxoid and acellular pertussis vaccine during pregnancy in Turkey.

OBJECTIVE: Pertussis is an important cause of morbidity and mortality in infants under two months of age and these high risk babies are dependent on maternally derived antibodies until completion of their first immunization series. This study aimed to evaluate the vaccine response of late preterm and term newborns as well as their mothers who underwent combined tetanus-diphtheria toxoid and acellular pertussis (Tdap) vaccination during pregnancy. STUDY DESIGN: A total of 70 pregnant women were administered Tdap vaccine (Boostrix®, GSK) between 27 and 33 gestational weeks of pregnancy. The IgG antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) in maternal blood before vaccination and in both maternal and umbilical cord blood after vaccination were evaluated using the in-house ELISA method. The geometric mean concentrations (GMC) and placental transfer ratios of antibodies were measured. RESULTS: Participants' with a mean age of 29.59 ± 4.70 years received Tdap vaccine at an average 28.6 ± 1.31 gestational weeks. Average pre and post vaccination levels of anti-PT IgG GMCs and anti-FHA IgG GMCs were 8.01 IU/ml vs 39.48 IU/ml (p = 0.001) and 122.24 IU/ml vs 183.97 IU/ml (p < 0.001), respectively. The anti-PT and anti-FHA IgG GMCs of cord blood after vaccination was 25.15 IU/ml and 118.77 IU/ml, respectively (p < 0.001 and p = 0.064). Placental transfer ratios of anti-PT ve anti-FHA IgG antibodies were detected as 0.65 and 0.62, respectively. CONCLUSION: Immunization of pregnant women with Tdap at the third trimester results in high maternal and infant antibody levels. Maternal immunization during each pregnancy seems to be the best strategy in revealing the highest maternal and infant antibodies and in narrowing the gap between birth and immune system maturation in infants. Pregnant women in our country should also get the Tdap vaccine during pregnancy especially in the early third trimester.

Evaluation of Seropositivity of Toxoplasmosis Suspected Patients Admitted to the National Parasitology Reference Laboratory Between 2009 and 2019

Objective: Toxoplasmosis caused by Toxoplasma gondii (T. gondii), which is an obligatory intracellular parasite, is a worldwide zoonotic parasitic disease. In this study, results of T. gondii test conducted between January 2009 and May 2019 were analysed. This study aimed to evaluate the results of T. gondii test of patients who were admitted to the General Directorate of Public Health, National Parasitology Reference Laboratory between 2009 and 2019. Methods: The results of anti-T. gondii IgG, IgM, IgG avidity and Sabin-Feldman dye tests (SFDT), which are used to detect the presence of T. gondii, were examined. ELISA was used for anti-T. gondii IgG, IgM and IgG avidity tests. SFDT, which is the reference test in the diagnosis of T. gondii, is still the gold standard. In addition to laboratory analyses, information on gender, age, city of origin, year distribution of all cases and type of sample sent was also collected. Results: Of the 2.778 patients evaluated, 25.4% were males and 74.6% were females. Moreover, 47.1% and 10.2% of the patients were positive for anti-T. gondii IgG and anti-T. gondii IgM antibodies, respectively. In SFDT, 1.228 (52%) patients were found to be positive, including 319 (59.4%) of 537 men and 909 (49.8%) of 1.824 women. In this 10-year study, the most common seropositivity titre of SFDT was at the level of 1/64. In our study, IgG levels were found to be positive in all cases in which IgG avidity was studied when all the cases in which all three of the anti-T. gondii IgG, IgM and IgG avidity tests were studied together in one patient were evaluated. In addition, of the 293 patients with positive anti-T. gondii IgG, 62.8% had high avidity, 24.2% had a limit value and 13% had a low avidity. In cases involving both mother and baby, anti-T. gondii IgG and IgM seropositivity rates were 80% and 5% for both, respectively. These high rates support the transfer of antibodies from the mother to the baby. Regarding the distribution of provinces from which the samples originated, the highest number of cases came from Ankara (80.7%). Blood is the most predominant sample, followed by cerebrospinal fluid. Conclusion: T. gondii maintains its importance in public health, owing to its high positivity rates. This study, in which 10-year data were collected, showed that despite an increase in awareness, high seropositivity still continues. Therefore, systematic collection and evaluation of laboratory analysis results for toxoplasmosis diagnosis will contribute in taking control measures.

Gingival Myiasis on Oral Squamous Cell Carcinoma in Turkey: A Case Report

Myiasis is a disease caused by tissue invasion of diptera larvae and eggs. Oral myiasis is mostly related to old age, poor oral hygiene, suppurative lesions, anatomical disorders and cancer cases. Oral squamous cell carcinoma (OSCC) is an important risk factor for myiasis. This report presents the case of an 82-year-old woman who presented with gingival myiasis developing on the background of OSSC. The patient was diagnosed with OSSC in the hospital. Myiasis larvae were identified and sent to the National Parasitology Reference Laboratory for identification. Thus, development of myiasis on OSCC background was shown in Turkey for the first time. Myiasis larvae have been identified as the 3rd phase of the larvae Sarcophaga sp. development (Diptera: Sarcophagidae). As a result, myiasis cases are sporadic in Turkey, and it can be avoided by controlling fly population and by paying attention to hygiene. Controlling myiasis is an important public health problem and should be considered in a single health concept, as it causes health problems in both humans and animals. The findings of this case will draw attention to the importance of dealing with myiasis factors, which is a public health problem.

Investigation of antibiotic susceptibilities of Brucella Strains isolated from various clinical samples in eastern Turkey.

BACKGROUND: Brucellosis is a worldwide zoonotic disease that causes serious public health problems. This study aimed to identify Brucella strains isolated from various clinical samples by conventional and molecular methods and to determine antimicrobial susceptibilities against doxycycline (DOX), streptomycin (STR), ciprofloxacin (CIP) and rifampicin (RIF) by the gradient strip (E test) test method. METHODS: A total of 87 Brucella strains isolated from various clinical specimens between 2004 and 2018 were included in this study. While four of the 87 strains included in the study were identified only at the genus level, the remaining 83 strains were identified at the species level by the Real-Time Multiplex PCR (M-RT-PCR) method and conventional methods were used for biotyping. RESULTS: According to molecular identification results, 83 strains were identified as B. melitensis by the M-RT-PCR method, with 82 strains identified as Brucella melitensis biovar (bv) 3 and one as B. melitensis bv 1 according to the conventional biotyping method. Among the antibiotics studied, CIP was found to be the most active agent according to the minimum inhibitory concentrations (MIC)90 values. This was followed by DOX and STR, respectively. While all of the isolates were sensitive to CIP, DOX and STR, 18 (20.7%) strains were found to be moderately susceptible to RIF, with the highest values of MIC50 and MIC90. CONCLUSIONS: In our study, all strains were identified as B. melitensis. DOX, STR, CIP and RIF used in the treatment of brucellosis were found to be effective.

Whole-genome sequencing of a Toxoplasma gondii strain from a Turkish isolate using next-generation sequencing technology.

PURPOSE: Toxoplasma gondii is an intracellular parasite that can affect all vertebrae and is the causative agent of toxoplasmosis. At present, the United States CDC (Centers for Disease Control and Prevention) recognizes this infection as a neglected disease. Toxoplasma gondii infection profiles exhibit differences because of the different regional and climatic responses to these parasites in Turkey, and these protozoan infections are notably common in this country. In this study, we attempted to obtain the whole-genome sequence of T. gondii using next-generation sequencing technology. METHODS: Toxoplasma gondii isolates were isolated from an infant with congenital toxoplasmosis by Ekmen et al. (1974) in Ankara, Turkey. Whole-genome sequencing (WGS) was performed using the Illumina HiSeq 2500 and HiSeq SBS Kit v2. A T. gondii library was created on this device in the initial stage. After the completion of the library phase, sequence analysis was begun with a next-generation sequencing device. The resulting fragments were combined using paired-end (PE) reading and converted into a single DNA fragment. Bioinformatic analysis was performed using the Geneious 2.1. RESULTS: In our study, WGS was successfully performed on T. gondii. The T. gondii whole-genome sequence has a coverage value of 50x, a size of 61,5763 Mb and a GC ratio of 52.6%. Data from this sequence were submitted to the National Center for Biotechnology Information (NCBI) GenBank (www.ncbi.nlm.nih.gov) database under the name Toxoplasma gondii TR01 (TG_TR01). The accession number of the genome obtained in this study is WOEV00000000.1. The biological sample access number is SAMN13338796. The genome of the T. gondii strain obtained in this study was compared with the reference genome, and 8312 CDSs (coding sequences), 183 tRNAs, 294 rRNAs and 8789 genes were identified. Among the 8312 CDSs, 4284 encoded hypothetical proteins (hypothetical protein CDSs/proteins of unknown function). The entire genome sequence of T gondii TR01 was compared with that of Toxoplasma gondii ME49. The results of this comparison demonstrate that the analyzed genome was 99,98% similar to the reference genome. The accession numbers of 14 chromosomes belonging to the genome sequences of T. gondii TR01 (TG_TR01) are CM019722.1, CM019723.1, CM019724.1, CM019725.1, CM019726.1, CM019727.1, CM019728.1, CM019729.1, CM019730.1, CM019731.1, CM019732.1, CM019733.1, CM019734.1, and CM019735.1. CONCLUSION: In this study, a whole-genome sequences of T. gondii was conducted for the first time in Turkey. The analyzed strain was named T. gondii TR01. The data obtained from this study may contribute to a better understanding of T. gondii. T. gondii is an important pathogen with an unusual population structure. Although T. gondii is highly zoonotic and has a complicated life cycle, some strains of this parasite have exhibited high genetic sequence similarity, and our study supports this knowlegde. The characterization of this strain may be very useful for the scientific community of our country and may help to establish a foundation for further research investigating the genome of T. gondii.

[The Epidemiology of Carbapenemases in Escherichia coli and Klebsiella pneumoniae Isolated in 2019 in Turkey]. / Türkiye'de 2019 Yili Içinde Izole Edilen Escherichia coli ve Klebsiella pneumoniae Izolatlarinda Karbapenemaz Epidemiyolojisi.

Antibiotic resistance is one of the most important public health problem and one of the most critical steps in preventing resistance is the monitorization of the resistance. Local, regional and global monitoring enables the spread of antibiotic resistance to be understood more clearly. In this study, it was aimed to evaluate the results of the pilot study for the establishment of molecular-based carbapenem surveillance system in Escherichia coli and Klebsiella pneumoniae isolates and to investigate the carbapenemase epidemiology in Turkey. Hospitals (n= 28) from 26 different statistical level II regions from Turkey were included in the study. The hospitals participated in the study submitted ten carbapenem susceptible and ten carbapenem resistant E.coli and K.pneumoniae isolates to our laboratory that were isolated in two different periiods of six-month either between 1 March-31 August or 1 April-30 September 2019. A total of 509 isolates were collected from 26 of the 28 participating hospitals in the study. Isolates were identified by matrix assisted laser desorptionization-time of flight mass spectrophotometry (MALDI TOF MS) (Bruker Daltonics, Germany) method and antibiotic susceptibility tests for imipenem, meropenem and colistin were studied by broth microdilution. Moreover, susceptibilities to amikacin, amoxicillin-clavulanic acid, ampicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, ertapenem, gentamicin, piperacillin-tazobactam, tobramycin and trimethoprim-sulfamethoxazole were determined by disc diffusion method. The resistance genes were investigated in isolates which were found to be phenotypically resistant to carbapenem and colistin, in house method was used to investigate carbapenemase genes and a commercial colistin resistant real-time PCR kit (Biospeedy, Turkey) was used for colistin resistance genes. In total, 493 of the 509 isolates collected from hospitals were identified as E.coli (25.7%, n= 127) and K.pneumoniae (74.3%, n= 366) and included in the study. It was determined that 31% of the isolates evaluated were from community-acquired infections and 69% were either from healthcare-associated infections or from colonization sites. Among the tested isolates, 248 (50.3%) were susceptible to carbapenems and 245 (49.7%) were resistant. The types of carbapenemases in carbapenemase-producing were OXA-48 (52.2%), KPC (16.1%), NDM-1 (15%), OXA-48 + NDM-1 (12.6%), KPC + NDM-1 (2.8%) and VIM (0.5%) and OXA-48+VIM (0.5%). Resistance to colistin was detected in 23.3% of the isolates but mcr1-8 genes were not detected. It was found that all colistin resistant isolates are resistant to at least one of the carbapenems. The importance of a molecular-based antimicrobial resistance surveillance system in our country was demonstrated with this pilot study. It is thought that continuous monitoring of these epidemiological features will contribute to the management of infections due to carbapenemase-producing organisms.

[Experience of the National HIV-AIDS Reference Center of Turkey, in the Transition to the New HIV Diagnostic Algorithm; Comparative Analysis of Line-Immunoassay Test and Bio-Rad Geenius™ HIV-1/2 Antibody Confirmatory Assay]. / Yeni HIV Tani Algoritmasina Geçis Sürecinde Ulusal HIV-AIDS Referans Merkezi'nin Deneyimi: Line-Immunoassay Test ve Bio-Rad Geenius™ HIV-1/2 Antikor Ayirt Edici Hizli Dogrulama Testleri Karsilastirmali Analizi.

Shortly after the first detection of human immundeficiency virus (HIV) infection in USA in 1981, the number of cases have increased gradually from all around the world. Turkey's high capacity for tourism and the unique geographic location extending between Europe and Asia, provides convenience for the passage of individuals across the countries and sexually transmitted infections including HIV, as well. According to the official data of the Ministry of Health; there are 25809 HIV positive and 1958 AIDS cases as of November 30, 2020, after the epidemic started in 1985 in Turkey. Despite the decrease in the number of newly detected HIV cases as a result of serious measures taken for the transmission of infection worldwide, the increase in the number of cases still continues in our country. Shortening the reporting period and starting treatment as soon as possible in the diagnosis of infection is critical for the control of the epidemic. For this purpose, Centers for Disease Control and Prevention (CDC) published a new test algorithm in 2010, which suggested the use of the Geenius™ HIV ½ supplemental assay test instead of western blot tests, which have been used for many years to verify HIV screening test positivity. In this study, we aimed to report the experience of the National HIV-Acquiner Immundeficiency Syndrome(AIDS) and Viral Hepatitis Reference Laboratories of Turkey in the first year of transition to the new HIV algorithm and to evaluate the diagnostic performance of Geenius™ HIV ó and line immunassay (LIA) s. A total of 2090 anti-HIV positive patient sera sent to National HIV-AIDS and Viral Hepatitis Reference Laboratories of Turkey, Ankara for HIV confirmation were included in the study. All samples were retested with a fourth-generation enzyme linked immunosorbent assay (ELISA) test (VIDAS® HIV-1/2 Duo Ultra assay, BioMerieux, France) followed by the confirmatory tests; Geenius™ HIV 1/2 confirmatory assay (BioRad, Redmond, WA) and Line-immunoassay (INNO-LIA HIV ½ Score, Fujirebio, reverse transcriptase polymerase chain reaction (RT-PCR) (artus HI Virus-1 RT-PCR, Qiagen, Germany) test and in-house HIV-2 RNA and proviral DNA PCR. The sensitivity, specificity, and the agreement of the each assay were compared. Cohen's Kappa analysis was used for the evaluation of the agreement between the tests. According to the new algorithm which recommended Geenius™ test besides HIV-1 RNA test, 1707 (81.7%) HIV-1 positive samples were identified. Of these samples; 95.9% and 95.02% were identified as HIV-1 positive by GeeniusTM and INNO-LIA, respectively. However, 2.5% of the positive samples were negative with Geenius™ and 3.5% with INNO-LIA. One and a half percentage (1.5%) of these samples were detected with Geenius™ and 1.4% with INNO-LIA as indeterminant. When all the positive samples determined with ELISA were evaluated; it was detected that,1.3% were indeterminate by Geenius™ test and 2.4% by the INNO-LIA test. When the INNO-LIA test was regarded as the gold standard method; sensitivity, specificity, positive predictive and negative predictive values of the Geenius™ test were as follows; 99.7%, 96.1%, 98.9%, and 99.1%. The agreement between INNO-LIA and Geenius™ tests was found to be 98.95% (κ= 0.969; very good). When the Geenius™ and HIV-1 PCR tests were evaluated together for the confirmation; the sensitivities of Geenius™ and INNO-LIA tests were 99.8% and 98.3%, specificities were 89.8% and 85.3%, respectively. Slight positive bands were detected in the gp36 or gp140 bands, the HIV-2 specific envelope proteins, were detected in seven samples, However, the positivity disappeared after the dilution of the samples and it was accepted as false positivite reaction due to the absence of HIV-2 RNA and proviral DNA in these samples. In conclusion; we concluded that Geenius ™ and INNO-LIA tests have a perfect agreement in HIV diagnosis and due to the rapid and reliable results provided for the HIV test protocol, Geenius™ test can be used safely as an alternative to the immunoblot tests. HIV-1 RNA testing must be performed in all HIV confirmation centers in order to detect acute HIV cases in the fast and early period which are the main reason for the updates in HIV diagnosis.

Investigation of immunity against Bordetella pertussis in pregnant women and an overview of the vaccination schedule in Turkey.

BACKGROUND: Pertussis caused by Bordetella pertussis, is a disease leading to significant morbidity and mortality in neonates and infants. Direct protection of the infant may be achieved by maternal and neonatal vaccination. Despite primary vaccination, infants under six months pose the greatest risk of infection with pertussis. Maternal immunization provides a high level of infant protection from birth until immunity is achieved by active vaccination. There is no routine Tdap vaccination recommendation for pregnant women in Turkey. This study was carried out to determine pertussis antibody levels in pregnant women and provide data for improving vaccine planning. METHODS: The study was carried out with 133 pregnant women in Turkey. Antibody titers to pertussis toxin (anti-PT) and filamentous hemagglutinin (anti-FHA) were measured by the commercially available ELISA. RESULTS: Among 133 participants, 93 (69.9%) were found to be immune according to anti-PT IgG antibody levels. According to anti-FHA IgG antibody levels, 123 (92.5%) participants were considered to be immune. A positive correlation was observed between PT and FHA and the findings were statistically significant (P < 0.001, r = 0.343). In the study group, the ages of the participants varied between 17 and 44 years. The mean age of those who were immune was 27.3±5.6, the mean age of non-immune patients was 29.1±6.2 and the difference was not statistically significant (P= 0.14). CONCLUSIONS: Our results reveal that approximately one-third of pregnant women were not immune to pertussis, reflecting many young infants to be vulnerable to pertussis infection until the onset of primary vaccinations, although childhood pertussis vaccination coverage has been high for a long time. We conclude that Tdap vaccine recommendation for pregnant women regardless of previous immunization history may be beneficial for the protection of infants in their first six months.

The Effect of Acromioplasty or Bursectomy on the Results of Arthroscopic Repair of Full Thickness Rotator Cuff Tears: Does the Acromion Type Affect These Results?

Objectives: The aim of this study was to investigate the effect of subacromial decompression on the results of full thickness rotator cuff repair applied arthroscopically. Examination was also made of the effect of acromion type on the subacromial decompression procedure in patients applied with arthroscopic rotator cuff repair. Methods: The study included a total of 150 patients, comprising 102 (68%) females and 48 (32%) males with a full thickness rotator cuff tear repaired arthroscopically. The patients were separated into three groups of 50. Group A comprised those with acromioplasty and bursectomy applied additional to the repair. In Group B, only bursectomy was performed additional to the repair and in Group C, only rotator cuff repair was applied. Evaluation was made of the post-operative long-term pain and functional results. Results: The mean age of the cases was 65.63±9.22 years (range, 46-86 years). The affected side was right side in 95 (63.3%) cases and left side in 55 (36.7%). No statistically significant difference was determined between the groups according to the post-operative Constant Murley and ASES scores (p>0.05). In the paired comparisons, the post-operative VAS scores of Group C were higher than those of Groups A and B (p=0.018, p=0.029, p<0.05). No statistically significant difference was determined between Group A and Group B in respect of the post-operative VAS scores (p>0.05). Conclusion: In the arthroscopic repair of full thickness rotator cuff tears, neither acromioplasty, coracoacromial ligament loosening nor bursectomy were determined to have any positive effect on the results. Whatever the acromion type, there is no need for an additional subacromial decompression procedure after rotator cuff repair, in respect of pain and functional outcomes. Only acromial spurs should be gently removed paying attention to the coraco-acromial ligament.

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.

[Direct Identification of Microorganisms from Positive Blood Cultures by MALDI-TOF MS Using an in-House Tween® 80 Method: Experimental and Clinical Study]. / "In-House" Tween® 80 Yöntemi ile Pozitif Kan Kültürlerinden Mikroorganizmalarin MALDI-TOF MS Yöntemi ile Dogrudan Tanimlanmasi: Deneysel ve Klinik Çalisma.

It has been reported that direct identification from blood culture bottles with positive signals and reporting the results to the clinics earlier has positive effects on mortality and morbidity. Extraction methods especially using detergents are used for the direct identification from the bottles which give positive signal. For this purpose, in-house methods developed based on the usage of saponin are widely available in the literature. In this study, it was aimed to develop a simple, easy-to-apply and reliable protocol for identifying the agent directly from the blood culture bottle that gives positive signal with the use of detergent Tween® 80, and to study the obtained protocol in clinical samples in a routine microbiology laboratory and to evaluate the results. The study was carried out in two stages, the experimental stage where the method was developed and the clinical stage where the method was applied. In the experimental stage, blood culture bottles were created with standard strains and isolates previously diagnosed with the 16S rRNA method. 10% solution of Tween® 80 was prepared with distilled water. 1 ml sample was transferred from the bottle that gave positive signal to the microcentrifuge tube, 100 µl of 10% solution of Tween® 80 was added, vortexed for 10 seconds and then incubated for 5 minutes at room temperature. The tubes were centrifuged for 5 min at 14.000 rpm, the supernatant was discarded and the pellet was washed with 1 ml of distilled water and centrifuged at 14.000 rpm for 5 minutes in three times. Samples taken from the pellets were rubbed on the slide and dried on air. Firstly, 1 µl of 70% formic acid, then 1 µl, of matrix solution was added and it was used after drying. In the second stage of the study, the method was applied to the 502 vials giving positive signal in the Microbiology Laboratory of Ankara University Faculty of Medicine Ibni Sina Hospital between 17 April 2018-31 August 2018 and the results were compared with the subculture results. The results obtained at the end of extraction in the experimental stage were compared with the subculture results and no statistical difference was found. In 383 (82.9%) bottles among 462 (92.1%) bottles with monomicrobial positive cultures, compatible results with the subculture results were obtained. Of the microorganisms correctly identified, 350 (91.3%) were bacteria and 33 (8.7%) were fungi. On the other hand, 216 (56.4%) of the bacteria were gram positive and 134 (34.9%) of them were gram negative bacteria. At least one microorganism was correctly identified in 19 (47.5%) of 40 (7.9%) bottles with polymicrobial blood cultures. Their distribution was gram negative (n= 10) and gram positive (n= 8) and yeast (n= 1). No microorganisms were identified in six bottles with polymicrobial cultures. According to the results, we believe that this in-house method developed using Tween® 80 will be a routinely applicable method for blood culture bottles that give positive signal in microbiology laboratories and it will contribute to the early diagnosis.

Coronaviruses and SARS-COV-2

Coronaviruses (CoVs) cause a broad spectrum of diseases in domestic and wild animals, poultry, and rodents, ranging from mild to severe enteric, respiratory, and systemic disease, and also cause the common cold or pneumonia in humans. Seven coronavirus species are known to cause human infection, 4 of which, HCoV 229E, HCoV NL63, HCoV HKU1 and HCoV OC43, typically cause cold symptoms in immunocompetent individuals. The others namely SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) were zoonotic in origin and cause severe respiratory illness and fatalities. On 31 December 2019, the existence of patients with pneumonia of an unknown aetiology was reported to WHO by the national authorities in China. This virus was officially identified by the coronavirus study group as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the present outbreak of a coronavirus-associated acute respiratory disease was labelled coronavirus disease 19 (COVID-19). COVID-19's first cases were seen in Turkey on March 10, 2020 and was number 47,029 cases and 1006 deaths after 1 month. Infections with SARS-CoV-2 are now widespread, and as of 10 April 2020, 1,727,602 cases have been confirmed in more than 210 countries, with 105,728 deaths.

Evaluation of the diagnostic performance and optimal cutoff value of a fourth-generation ELISA, VIDAS HIV-1/2 Duo Ultra assay, in a low-prevalence country.

In this study, we described the largest analysis to date conducted with VIDAS® HIV Duo Ultra assay. Additionally, we analyzed the diagnostic performance and cutoff values (TV) of HIV Duo Ultra assay and total cost analysis for HIV testing. Of 11,642 enzyme-linked immunosorbent assay (ELISA)-positive samples referred to our center for confirmation, 2000 were positive with HIV Duo Ultra, and of these, 87% were HIV-1 positive and 0.6% were HIV-1 indeterminate with the confirmatory test. Overall, the false-positivity rate was 1.75% for HIV Duo Ultra assay. The sensitivity and specificity were 100% and 99.1%, respectively, when the TV was set at the recommended cutoff value. Even increasing the cutoff value four times, sensitivity and specificity remained high, pointing out that a TV of 0.99 is highly indicative of HIV positivity. Retesting samples with HIV Duo Ultra assay decreased 80% of the confirmatory tests, revealing a significant decrease of 78% in the total costs and reporting time.

[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.