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Staphylococcus aureus is responsible for life-threatening conditions, while in the meantime it has rapidly acquired resistance to several antibiotic classes. In the context of an effective empirical antibiotic therapy, an accurate evaluation of the resistance rates of S. aureus may be critical. The aim of this study was to determine the resistance rates of S. aureus in the years 2015-2018 and to assess the impact of specimen stratification on the resistance rates. We have retrospectively analysed S. aureus strains isolated from blood, bronchial aspirate, pus, sputum and urine collected from hospitalized and ambulatory care patients. The comparison between resistance rates from 2015 to 2018 and among different specimens was assessed by Fisher's exact test followed by Benjamini and Hochberg's correction of the p-values. Higher resistance rates were detected for penicillin followed by oxacillin, levofloxacin, erythromycin and clindamycin. Differences in the annual resistance rates were not statistically significant after the BH's correction. The comparison between cumulative S. aureus resistance rates stratified by specimens showed some statistically relevant differences among the five specimen types. In particular, p-values were statistically significant for clindamycin, erythromycin, gentamicin, levofloxacin, oxacillin, penicillin and vancomycin. Annual resistance rates of S. aureus clinical isolates remained constant over the course of time. Moreover, the stratification of the data by specimen may significantly impact on the evaluation of the resistance rates, at least for some antibiotics. Therefore, if the number of data is high, stratification by specimens may be recommendable to better approach an empirical antibiotic therapy.

Klebsiella pneumoniae is a common nosocomial pathogen involved in many infectious diseases such as bacteraemia, urinary and respiratory tract infections. It is responsible for the rise in morbidity and mortality rates since most clinical isolates exhibit resistance to several antibiotics. Moreover, the epidemiology of these nosocomial infections is variable across countries and regions. From January 2015 to December 2017 we retrospectively analysed the bloodstream infections caused by K. pneumoniae strains in hospitalised patients with the aim of studying the temporal trends of wild type (WT), multi-drug resistant (MDR), extended drug resistant (XDR), pan-drug resistant (PDR) and carbapenem-resistant (CR) strains. In all, 439 K. pneumoniae isolates from 356 patients were collected from all units of the Policlinico of Bari. The majority of clinical isolates were collected from the intensive care unit (125, 28.47%), haematology (34, 7.74%), rehabilitation (27, 6.15%) and cardiac surgery wards (25, 5.69%). Moreover, the majority of the isolates were classified as CR (325, 74.03%, 95%CI: 69.61-78.19) and XDR (255, 58.09%, 95%CI: 53.31-62.72). Annual prevalence rates and monthly counts were analysed using the Chi Squared test for trends and the Poisson regression with multiple p-value correction according to Benjamini and Hochberg's procedure. The annual relative frequencies of the XDR and CR K. pneumoniae isolates decreased significantly from 63.37% to 48.44% and from 78.48% to 63.28% respectively, while WT K. pneumoniae significantly increased from 13.95% to 23.44%. Poisson regression analysis confirmed the presence of a decreasing monthly trend for the XDR and CR K. pneumoniae count series. In order to control the spread of antibiotic resistance, more inclusive surveillance data will be needed to either confirm these results or improve antibiotic stewardship measures.

Clostridium difficile is an emerging cause of healthcare-associated infections. The increasing frequency and severity is attributed to highly virulent ribotypes such as 027. The aim of this study was to retrospectively analyze the prevalence of CDI and ribotype 027 in 481 clinical samples collected from hospitalized patients and sent to the laboratory of molecular biology, UOC Microbiology and Virology, Azienda Ospedaliera-Universitaria, Policlinico of Bari, Italy. Toxins A+B and DNA C. difficile detections were performed using immunochromatographic test and a multiplex real-time PCR assay, respectively. Overall, 37/366 (10.11%) patients were positive at the immunochromatographic assay. This result was confirmed in 31 (8.47%) samples from 31 different patients by molecular assay. Logist regression confirmed age >50 years (adjusted odds ratio [aOR]: 4.29, 95%CI:1.44-18.50) and hospitalization in the Infectious Diseases (aOR: 3.77, 95%CI: 1.34-9.85) ward were risk factors for CDI. The associated 027 ribotype deletion D117tcd was detected in seven (22.58%) of 31 positive patients. Exploratory analysis of monthly prevalence of 027 ribotype suggested a slight increase after August 2015. Our results show that a monitoring program is needed to either better assess the diffusion of CDI and ribotype 027 or also to establish the risk factors associated with the transmission in our healthcare facilities.

Human papillomavirus (HPV) is considered the most important risk factor for the development of ano-genital region cancer in both women and men. Whereas low-risk genotypes are responsible for cutaneous and genital lesions, high-risk genotypes are associated with ano-genital cancer. The aim of this study was to retrospectively analyse the prevalence, genotype distribution and temporal dynamics of HPV infection in 2312 specimens from 2312 subjects (2149 women and 163 men) who attended the laboratory of molecular biology, UOC Microbiology and Virology, Azienda Ospedaliera-Universitaria, Policlinico of Bari, Italy. HPV DNA detection and genotyping was performed using a multiplex real-time PCR assay. In all, 1123/2312 subjects (48.57%) resulted positive for HPV DNA. In particular, HPV DNA was detected in (1056) 49.14% of females and (67) 41.10% of males. HPV co-infections were detected in 565 (24.44%) patients. High-risk and low-risk HPV genotypes were detected in 887 (38.37%) and 600 (25.95%) patients, respectively. The most prevalent HPV genotypes were HPV-42 (10.29%), HPV-16 (8.56%), HPV-31 (7.40%) and HPV-53 (7.14%). Statistically significant differences between female and male patients were not detected. Moreover, HPV prevalence remained constant in time while HPV-16, but not HPV-6, 11 and 18, showed a decreasing trend from 2013 (11.24%) to 2016 (6.67%). Other HPV genotypes showed some complex and different patterns. Our data showed an unusually high frequency of HPV-42 and a high prevalence of HPV infection in the patients analysed. Although evidence of a decreasing trend of HPV-16 could be a consequence of anti-HPV vaccination, corroboration from further studies will be needed. Moreover, the small number of studied males and the similarity to females in terms of HPV prevalence suggest that more active HPV screening and anti-HPV vaccination in the male population should be considered important tools to eliminate HPV sexual transmission.

This study evaluated the diagnostic performances of an ELISA method and a molecular method for the detection of verotoxin in faecal samples during an outbreak of haemolytic-uraemic syndrome (HUS) occurring in Apulia, Southern Italy. Two of the 16 faecal samples were positive for verotoxin when analysed by ELISA and resulted PCR positive for stx1, stx2, eaeA and serogroup O26. The other 14 faecal samples resulted negative with both tests. The detection of verotoxin in faecal samples by ELISA is a simple, sensitive, specific and rapid method (2 hours) of considerable utility for routine clinical testing laboratories without access to more specialized diagnostic procedures.

The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.

BDProbeTec ET (Becton Dickinson, Sparks, Md, USA) is a fully automated walkaway system based on strand displacement amplification (SDA) technology that provides a method for the direct detection of Mycobacterium tuberculosis complex (MTBC) target sequence. The purpose of this study was to evaluate the ability of BDProbeTec ET system to detect MTBC directly from clinical specimens and compare the results with staining and culture. From February 2002 through December 2003 a total of 1521 [pulmonary (n=1329) and extrapulmonary (n=192)] specimens from 1518 patients were examined by BDProbeTec ET system for the detection of MTBC and the results were compared to those obtained by microscopy and liquid culture (BACTEC 9000 MB, Becton Dickinson). MTBC was cultivated from 65 specimens (60 pulmonary and 5 extrapulmonary) of which 43 (66.2%) (42 pulmonary and 1 extrapulmonary) were smear positive and 22 (33.8%) (18 pulmonary and 4 extrapulmonary) were smear negative. BDProbeTec ET detected MTBC in 58 (55 pulmonary and 3 extrapulmonary) of the 65 culture-positive specimens. Although the BDProbeTec ET system gave five false-negative results among the 18 smear-negative culture-positive pulmonary specimens, our results demonstrate that the BDProbeTec ET system is a reliable tool in smear-positive samples and given its technical characteristics it can be used for the rapid detection of MTBC in either pulmonary or extrapulmonary samples.

This study aimed to evaluate the incidence of Chlamydophila pneumoniae antibodies in patients with community acquired pneumonia (CAP) by a new ELISA test (EIA CP-IgG, IgA, IgM--Eurospital, Trieste, Italy). From January 1999 to July 2001 141 patients with clinical signs of CAP were enrolled in sixteen Italian Hospitals. Specific IgM and IgG antibodies anti-C. pneumoniae in serum and IgA in both serum and sputum were detected. At a primary inspection (time T-0) serum and sputum samples were taken from 115/141 patients, whereas serum was collected from only 100/141 patients after 30 days (time T-30). At T-0 24/115 (20.8%) patients showed serological markers thus suggesting an acute C. pneumoniae infection. In 23/24 patients the overall serological pattern found at T-0 was confirmed at T-30. In 32/115 patients (27.8%) serological markers of C. pneumoniae past infection were found positive and were confirmed 30 days later. These data support the role of C. pneumoniae as an important aetiological agent of CAP throughout different geographic areas of Italy. The test was suitable for the laboratory diagnosis of C. pneumoniae infection. In particular, the presence of specific IgA anti- C. pneumoniae in both serum and sputum proved useful to define different stages and evolution of infection.

The nasopharyngeal colonization rate of Streptococcus pneumoniae and its antibiotic susceptibility was determined in a given population of 317 young children (ages 1-7 years) in the area of Bari, Italy. 18.29% of the cultures were positive for S. pneumoniae. 8.62% of the strains were intermediately resistant to penicillin. Erythromycin-(65.51%) and cotrimoxazole-(17.24%) resistance was also observed whereas all the strains resulted uniformely susceptible to cefotaxime and ceftriaxone. The high rate of nasopharyngeal carriage of Streptococcus pneumoniae along with the resistance to antibiotics widely used in the community suggests the importance of epidemiological surveillance as well as the application of new vaccine strategies.