[Investigation of Efflux Pump Genes in Resistant Mycobacterium tuberculosis Complex Clinical Isolates Exposed to First Line Antituberculosis Drugs and Verapamil Combination]. / Birinci Seçenek Antitüberküloz Ilaçlara ve Verapamil Kombinasyonuna Maruz Birakilan Dirençli Mycobacterium tuberculosis Kompleks Klinik Izolatlarindaki Efluks Pompa Genlerinin Arastirilmasi.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt­PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 µg/mL, 1-128 µg/mL, 2-32 µg/mL, 4-16 µg/mL and 15.62-250 µg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.

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.

[Investigation of pncA, rpsA and panD Gene Mutations Associated with Resistance in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates and Spoligotyping]. / Pirazinamid Dirençli Mycobacterium tuberculosis Izolatlarinda Direnç ile Iliskili pncA, rpsA ve panD Gen Mutasyonlari ve Spoligotiplerin Arastirilmasi.

Pyrazinamide (PZA) is one of the first-line anti-tuberculous drugs used in the treatment of tuberculosis (TB). Considering the ability of PZA to shorten the treatment period from 9-12 months to six months by eliminating persistent bacilli, it appears to be an important cornerstone of TB therapy. While the main mechanism causing the PZA resistance is pncA mutations at a rate of 70-97%, it has been determined that rpsA and panD mutations can also cause resistance. In this study, we aimed to investigate the pncA, rpsA and panD gene mutations, the efficiency of the pyrazinamidase (PZAse) enzyme test in determining PZA resistance, the drug susceptibility and their families in PZA-resistant Mycobacterium tuberculosis isolates. Totally 46 PZA resistant M.tuberculosis isolates were included in the study. The pncA, rpsA and panD mutations caused by PZA resistance were investigated by in-house PCR followed by DNA sequencing method. Drug susceptibility was determined with Bactec MGIT 960 (Becton Dickinson, USA) system, the presence of PZAse was evaluated by colorimetric PZAse enzyme assay and the families were determined by the spoligotyping method. Of the 46 PZA-resistant isolates, 24 (52.2%) were identified as PZA monoresistant, 11 (23.9%) multidrug resistant (MDR)-TB and 11 (23.9%) poly drug resistant (PDR)-TB. Gene mutations associated with resistance were detected in 73.9% (34) of PZA-resistant M.tuberculosis isolates. The pncA, rpsA and panD mutations were found in 71.7% (33), 28.2% (12) and 4.3% (2) of the isolates, respectively. The coexistence of pncA/rpsA and pncA/panD gene mutations were determined in 12 and two isolates, respectively. The pncA gene mutations were observed in 3 (33.3%) of 9 (19.6%) isolates whose enzyme presence was detected by the colorimetric PZAse test. In the pncA gene, eight different point mutations in the form of missense mutation;A226C (27.3%), A152C (24.2%), C169G (21.2%) A422C (9.1%), G145A (6.1%), A29G (6.1%), A424G (3%) and T464G (3%) were detected. In the rpsA gene, A636C (42.9%) silent and G1318A (42.9%) missense mutations and in the panD gene, C66G (50%) nonsense and A145G (50%) missense mutations were the most common mutations detected. As a result of genotyping of PZA resistant isolates, the most common genotypes were found in T1 cluster with 17 (36.9%) isolates; followed by the families of Beijing with 7 (15.2%) isolates, H3 with 6 (13%) isolates, TUR with 5 (10.9%) isolates, and LAM 9 with 4 (8.7%) isolates, respectively. In addition, 2 (4.3%) isolates belonging to the ORPHAN family and one isolate belonging to each of LAM TUR, LAM 2, LAM 7, T2, T5-RUS1 families were identified. Our study is the first to investigate all pncA, rpsA and panD gene mutations that have been found to cause PZA resistance in Turkey. Epidemiological studies on PZA resistance will make important contributions to the determination of resistance mechanism and the development of methods that will provide rapid diagnosis for the detection of resistance.

[Antibody Response After Two Doses of Inactivated SARS-CoV-2 Vaccine in Healthcare Workers with and without Previous COVID-19 Infection: A Prospective Observational Study]. / COVID-19 Enfeksiyonu Geçiren ve Geçirmeyen Saglik Çalisanlarinda Iki Doz Inaktive SARS-CoV-2 Asisi Sonrasi Antikor Yaniti: Prospektif Gözlemsel Çalisma.

In this study, we aimed to investigate the anti-S1 IgG response that occurs after inactivated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine and the factors affecting this response in healthcare workers (HCWs) who are in the risk group for coronavirus disease 2019 (COVID-19). A total of 276 HCWs, of whom 82 previously exposed to COVID-19 infection and 194 naive who are working in Kafkas University Faculty of Medicine, Health Research and Application Center were included in this study. Anti-SARS-CoV-2 QuantiVac ELISA IgG (Euroimmun, Lubeck, Germany) kit, coated with recombinant S1 antigen including the receptor binding domain of the SARS-CoV-2 S protein was used for quantitative determination of the humoral immune response in serum samples 28 days after the first and second dose of vaccination. The antibody responses and the mean antibody levels of 194 naive HCWs 28 days after the first and second doses of vaccine were determined to be 27.2%, 98.5% and 19.72 ± 38.73 BAU, 222.09 ± 119.18 binding antibody unit (BAU), respectively. The mean antibody levels of 82 HCWs previously exposed to COVID-19 infection 28 days after the first and second doses of vaccine were 268.27 ± 112.91 BAU and 309.45 ± 112.75 BAU, respectively. The antibody response reached 100% after the first dose of vaccination. A statistically significant difference was observed between the antibody levels after the first and second doses of vaccine in both groups (p<0.001). It was determined that the mean antibody level formed after the first dose of vaccine administration of HCWs who had COVID-19 infection was statistically higher than the mean antibody level formed after the second dose of vaccine in naive individuals (p= 0.003). There was no significant difference in mean antibody levels after the first (269.10 ± 117.24 BAU and 266.59 ± 105.65 BAU, respectively) and second doses (309.98 ± 113.17 and 308.36 ± 114.04 BAU, respectively) between individuals previously exposed to COVID-19 infection in the last three months and more than three months (p= 0.925 and p= 0.951, respectively). The highest antibody levels were detected in the 19-30 age group (251.50 ± 119.81 BAU), women (249.03 ± 118.57 BAU), body mass index (BMI)<20 kg/m2 group (267.08 ± 137.51) and non-smokers (237.85 ± 124.83 BAU) in naive HCWs after the second dose of vaccination. A statistically significant difference was found between age, gender, BMI, smoking and vaccine response (p= 0.003, p= 0.005, p= 0.008 and p= 0.036, respectively). The post-vaccination adverse effects after the first and second doses of vaccination were not observed in 76.1% and 73.9% of HCWs, respectively. There was no increase in the frequency of post-vaccination adverse events after the first and second doses in individuals previously exposed to COVID-19 infection and naive persons (p= 0.46 and p= 0.43, respectively). It was determined that the mean antibody level after the first dose of vaccination in HCWs previously exposed to COVID-19 infection was higher than naive individuals after the second dose vaccination. Prospective clinical studies on antibody levels and their duration of protection are needed for the prevention of COVID-19 infection.

Molecular characterization, phylogenetic and variation analyses of SARS-CoV-2 strains in Turkey.

Aims: We present the sequence and single-nucleotide polymorphism (SNP) analysis for 47 complete genomes for SARS-CoV-2 isolates on Turkish patients. Methods: The Illumina MiSeq platform was used for sequencing the libraries. The SNPs were detected by using Genome Analysis Toolkit - HaplotypeCaller v.3.8.0 and were inspected on GenomeBrowse v2.1.2. Results: All viral genome sequences of our isolates were located in lineage B under the different clusters, such as B.1 (n = 3), B.1.1 (n = 28) and B.1.9 (n = 16). According to the Global Initiative on Sharing All Influenza Data nomenclature, all of our complete genomes were placed in G, GR and GH clades. In our study, 549 total and 53 unique SNPs were detected. Conclusion: The results indicate that the SARS-CoV-2 sequences of our isolates have great similarity with all Turkish and European sequences.

[Mortality Risk Factors for Invasive Candidiasis in Pediatric Intensive Care Unit]. / Çocuk Yogun Bakim Ünitesinde Invaziv Kandidiyaz Için Mortalite Risk Faktörleri.

Invasive Candida infections are one of the most important risk factors for the increasing mortality of immunocompromised patients with comorbidities in intensive care units. In this study, it was aimed to evaluate the mortality rate and risk factors affecting mortality in patients followed up with the diagnosis of invasive candidiasis in our pediatric intensive care unit. Patients who were between the ages of 1 month and 18 years followed up in the paediatric intensive care unit with invasive candidiasis between 2014 and 2018, were included in the study. The demographic characteristics of the patients, fever and hypotension, the Candida species, use of broad-spectrum antibiotics, blood transfusion, parenteral nutrition, invasive interventions, use of mechanical ventilation and laboratory test results were retrospectively analyzed and the relationship with mortality was statistically determined. A total of 85 patients, 45 girls, and 40 boys were included in the study. The death rate was 38.8% (n= 33). Candida albicans (48%) was the most common species for all isolates followed by Candida parapsilosis (21%), Candida tropicalis (15%), and others (16%). No statistically significant relationship was detected between the central venous catheter, broad-spectrum antibiotic and corticosteroid treatment, parenteral nutrition, gender difference, surgical operation, patient culture samples, isolated Candida species, and mortality (p> 0.05). A statistically significant relationship was found between blood transfusion, thrombocytopenia, and leukopenia, the first positive culture time since hospitalization, and the duration of antibiotic treatment and mortality (p<0.05). A statistically significant correlation was found with the presence of hypotension, one of the clinical markers associated with mortality (p<0.05) but the same relationship was not found with the presence of fever (p> 0.05). The mortality rate is high in candidiasis patients in pediatric intensive care units. Blood transfusions, long-term use of broad-spectrum antibiotics, and hypotension increase mortality.

Prevalence of COVID-19 in 10,000 samples from 7853 patients in Eastern Turkey by positive real-time PCR.

Aim: COVID-19, caused by SARS-CoV-2, started in December 2019 and has spread across the world. Materials & methods: We analyzed real-time PCR results of 10,000 samples from 2 April to 30 May 2020 in three neighbor cities located in the East of Turkey. The final study population was 7853 cases, after excluding screening tests. Results: Real-time PCR was performed to detect the SARS-CoV-2-specific RNA-dependent-RNA-polymerase gene fragment. The number of total positive samples out of 7853 were 487; however, the number of nonrepeating positive patient was 373 (4.8%). Cough and fever were the most common symptoms in positive cases. Conclusion: Epidemiologic studies should be performed about the prevalence of SARS-CoV-2 infection to better understand the effect of the virus across the world.

Saliva samples for detection of SARS-CoV-2 in mildly symptomatic and asymptomatic patients.

BACKGROUND: The ongoing coronavirus disease 2019 (Covid-19) pandemic has been rapidly spreading throughout the world with confirmed case numbers already exceeding 75 million. Although nasopharyngeal swabs are the most commonly utilized samples for based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection, collecting these specimens requires healthcare workers and necessitates the use of personal protective equipment as it presents a nosocomial transmission risk. We aimed to assess the diagnostic value of saliva samples in mildly symptomatic and asymptomatic patients with confirmed Covid-19. METHODS: We performed a cohort study to validate the use of saliva for SARS-CoV-2 detection in mildly symptomatic and asymptomatic patients with a confirmed diagnosis of Covid-19. Saliva samples of the patients were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: In May 2020, 28 asymptomatic and 25 mildly symptomatic patients were enrolled in the study. The median age was 37 years (range 4-70). None of the patients had a fever on presentation. Among 53 patients with SARS-CoV-2 detected in the nasopharyngeal sample, the real-time RT-PCR was positive in the saliva specimens in 48 (90.56%) patients. The mean cycle threshold (CT) values for nasopharyngeal and saliva specimens (27.80 ± 3.44 and 30.64 ± 2.83, respectively) were significantly correlated between the two sample types (p = .016). The mean CT values of nasopharyngeal and saliva samples in mildly symptomatic and asymptomatic patients (27.18 ± 3.53 and 30.24 ± 3.29 vs. 28.36 ± 3.31 and 30.98 ± 2.39, respectively) were not significantly different (p = .236 and p = .733, respectively). CONCLUSIONS: Saliva specimens can be considered as a reliable and less resource-intensive alternative to nasopharyngeal specimens for screening asymptomatic SARS-CoV-2 infections.

[Detection of virulence factors and antifungal susceptibilities of Fusarium strains isolated from keratitis cases]. / Keratit olgularindan elde edilen Fusarium izolatlarinin virülans faktörlerinin ve antifungal duyarliliklarinin belirlenmesi.

Fusarium species have gained importance as a cause of keratitis. The pathogenicity and virulence factors of genus Fusarium remain largely unknown. Several putative virulence factors have been reported for fungal pathogens, including biofilm formation, production of proteinases and other hydrolytic enzymes. It has been emphasized that Fusarium species are generally resistant to antifungals but the resistance may vary depending on the species and even according to the isolate. For this reason, pathogenic features and antifungal susceptibility of the clinical isolates gained importance for the management of keratitis cases. The aim of this study was to identify clinical Fusarium isolates, to evaluate their virulence factors and to show antifungal susceptibility patterns. The identification of Fusarium was made on genus level isolated from 25 keratitis cases. Among them, 13 of the isolates were identified by ITS sequencing on species complex level. The production of hemolytic activity, caseinase, esterase, proteinase and phospholipase activity were investigated in 13 of the isolates. Biofilm production was searched among all 25 isolates. Galleria mellonella larvae was used as in vivo infection model. Antifungal susceptibility for amphotericin B, itraconazole, voriconazole and posaconazole was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 microdilution assay guidelines. As the subcommittee on antifungal susceptibility tests did not determine the clinical resistance breakpoints (CBP) specific to Fusarium species complex, the epidemiological cut off values (ECV) were used for the interpretation of the minimum inhibitory concentration (MIC) values of the antifungal drugs. Isolates were identified as six F.oxysporum, six F.solani species complex and one F.brachygibbosum. One F.solani, one F.oxysporum were positive for hemolytic activity; all isolates were caseinase positive; three F.oxysporum and two F.solani isolate were esterase positive; one F.solani isolate was proteinase positive; five F.oxysporum and two F.solani isolates were phospholipase positive; biofilm activity was positive in 52% of the 25 isolates. The larvae survived for seven days after Fusarium inoculation in the G.mellonella larvae model. MIC range was 0.5-8 µg/ml for amphotericin B, 2-32 µg/ml for itraconazole, 0.5-8 µg/ml for voriconazole, 0.5-16 µg/ml for posaconazole and according to the ECV values F.solani and F.oxysporum isolates were determined as wild type for four antifungal agents. As a result, it was shown that Fusarium isolates have some virulence factors, there was a concordance between in vitro virulence properties and in vivo virulence characteristics and some of the isolates were classified as antifungal susceptible wild type isolates.

[Serological investigation of phlebovirus exposure in blood donors from the Mediterranean Province of Mersin, Turkey]. / Mersin bölgesi kan donörlerinde fl ebovirus maruziyetinin serolojik olarak arastirilmasi.

Phleboviruses are enveloped segmented RNA viruses, capable of inducing febrile disease and/or meningoencephalitis in exposed individuals, according to the infecting strain, following transmission via arthropods. Prototype medically-important phlebovirus strains responsible for sandfly fever are sandfly fever Sicilian virus (SFSV) and sandfly fever Naples virus (SFNV), where the SFSV variant sandfly fever Cyprus virus (SFCV) is also detected in individuals with febrile disease. Toscana virus (TOSV) is unique among phleboviruses as the cause of infections involving central nervous system. In this seroepidemiological study, human exposure to selected medically-important phleboviruses was investigated in healthy adult residents of the Mersin province, Mediterranean Anatolia, Turkey, where the current data on phlebovirus epidemiology is scarce. A total of 1784 healthy individuals (mean age: 34.7±9.6 years; 97.3% were male), accepted as blood donors at the Mersin University Center for Health Research and Application Blood Bank were included in the study after informed consent during a seventeen month period between July 2011 to November 2012. All participants were requested to fill out a questionnaire to reveal risk factors for vector exposure. SFSV, SFNV, SFCV and TOSV IgG antibodies in serum were investigated via a commercial indirect immunofluorescence test (IIFT) (Sandfly Fever Virus IgG Mosaic I; Euroimmun, Germany). Sera interpreted as positive or strong positive for TOSV or SFNV+TOSV in IIFT were evaluated via TOSV virus neutralization test (VNT) for specificity confirmation. IIFT seroreactivity for at least one of the tested phleboviruses was present in 66.8% (1192/1784) of the samples. The most frequently-detected phlebovirus strain was SFSV (51.6%; 920/1784), followed by SFNV (46.4%; 827/1784), TOSV (43.7%; 779/1784) and SFCV (47.3%; 843/1784). Among the reactive sera, 6.6% (79/1192) were positive for a single virus serotype, whereas in 39.8% (475/1192) antibodies reacting with all tested virus serotypes were revealed. A total of 187 sera was included in the TOSV VNT and neutralizing antibodies were detected in 13.9%. According to the IIFT reactivity, residing in rural areas was observed as a statistically significant risk factor for exposure in all phleboviruses tested (p values for SFSV, SFNV, TOSV and SFCV were 0.002, 0.001, <0.001 and 0.003, respectively). TOSV exposure is more frequently detected via IIFT in individuals having pets or domestic farm animals around the living quarters (p=0.005). As a result, frequent exposure to SFSV/SFCV or antigenically similar phlebovirus strains and viruses of the SFNV species were determined in healthy blood donors in Mersin province, located in the Mediterranean region of Turkey. Furthermore, TOSV neutralizing antibodies were detected in selected samples with IIFT reactivity, confirming previous reports suggesting TOSV activity in the region. TOSV and other phleboviruses must be included in the diagnostic work-up in cases with febrile diseases and viral central nervous system infections during the sandfly-active months.

Human papillomavirus genotype distribution and E6/E7 oncogene expression in Turkish women with cervical cytological findings.

BACKGROUND: Infection with certain human papillomavirus (HPV) genotypes is the most important risk factor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPV infection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women with different cervical cytological findings in Mersin province, Southern Turkey. MATERIALS AND METHODS: A total of 476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in the study. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNA expression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ(®)HPV v1.1). RESULTS: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR. The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL (6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were, in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and 83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%). CONCLUSIONS: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes 66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Because of the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the role of mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive and cytology negative. These epidemiological data will be important to determine the future impact of vaccination on HPV infected women in our region.