Epidemiological evaluation of human papillomavirus genotypes and their associations in multiple infections.

The aim of this study was to determine the frequency of multiple type human papillomavirus (HPV) infections, and whether any types are involved in multiple HPV-type infections (mHPV) more or less frequently than expected. From January 2012 to February 2018, 2848 cervico-vaginal swabs were analysed in the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested-polymerase chain reaction (PCR) and subsequently multiplex real-time PCR assay. 1357/2848 samples (47.65%) were HPV DNA positive and 694/1357 (51.14%) showed mHPVs. The median number of mHPVs was 2 (interquartile range: 2-3). HPV-types more frequently detected were 42 (9.97%), 16 (8.92%), 53 (7.23%) and 31 (7.16%). Each detected HPV-type was involved in mHPVs in more than 50% of cases. Statistical analysis showed significant associations for all HPV-types except for 33, 43, 51, 58 and 82 HPV-types. The major number of significant pairwise associations were detected for the types 42 and 70. Only positive associations were detected. Further data are necessary to evaluate the clinical impact of the single combinations.

Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and weakness.

The occurrence of Malassezia spp. bloodstream infections (BSIs) in neonatal intensive care unit was evaluated by using pediatric Isolator, BacT/Alert systems and central venous catheter (CVC) culture. The efficacy of BacT/Alert system in detecting Malassezia was assessed by conventional procedures, culturing 1 ml of bottle content before incubation and by studying the survival of Malassezia spp. strains in BacT/Alert bottles. Of the 492 neonates enrolled, blood was collected by pediatric Isolator (290 patients; group I) or by BacT/Alert bottles (202 patients; group II). The survival of Malassezia furfur and Malassezia pachydermatis in BacT/Alert bottles was evaluated by culturing the inoculum suspension (from 106 to 10 colony-forming units, cfu/ml) and assessing the cfu/ml for 15 days. In total, 15 Malassezia BSIs were detected, of which six (2.1%) from both blood and CVC culture in Dixon agar (DixA) in patients belong to group I (blood collected by paediatric Isolator tube) and nine (4.4%) only from CVC culture in DixA in patients of group II (blood collected by BacT/Alert bottle). Only one patient (0.5%) from group II scored positive for M. furfur also by culturing in DixA 1 ml blood content of BacT/Alert bottle before incubation in BacT/Alert system.M. furfur population size in BacT/Alert bottles decreased during the incubation time, whereas that of M. pachydermatis increased. The BacT/Alert system detected M. pachydermatis even at very low concentration (i.e., 10 cfu/ml) but not any positive blood culture for M. furfur. For a correct diagnosis of Malassezia furfur BSI, the blood should be culture in lipid-enriched fungal medium, and the BacT/Alert system implemented by adding lipid substrates to increase the method sensibility. Finally, CVC cultures on lipid-supplemented media may be proposed as a routine procedure to diagnose the Malassezia fungemia.

Paecilomyces lilacinus Keratitis in a Soft Contact Lens Wearer.

We describe a case of keratitis caused by Paecilomyces lilacinus in a contact lens wearer with a history of diabetes.

Simultaneous detection and identification of STI pathogens by multiplex Real-Time PCR in genital tract specimens in a selected area of Apulia, a region of Southern Italy.

PURPOSE: Genital tract infections are globally a major cause of morbidity in sexually active individuals. The aim of this study was to investigate the prevalence and associations of co-infections of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis (MH), Mycoplasma genitalium, Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP) in specimens collected from female (SF) and male (SM) patients. METHODS: 1575 samples from 1575 individuals from the geographical area around Bari, Apulia region in Southern Italy, were collected and analyzed by a multiplex Real-Time PCR (mRT-PCR) (AnyplexTM II STI-7, Seegene, Inc., Seoul, Korea) assay. RESULTS: 455/1575 (28.89%) samples resulted positive for at least one of the targets named above. Statistically significant differences in prevalence of the pathogens between SF and SM were not detected except for UP (24.92% in SF vs 8.91% in SM). Prevalence of co-infections was 6.84 and 3.96% in SF and SM, respectively. Moreover, MH presence in SF, but not in SM, was associated with UU and UP. CONCLUSIONS: Our data suggest different patterns of infections between females and male and the importance of an increased vigilance of sexually transmitted pathogens to reduce the burden on general population and the sequelae or the complications on reproductive organs.

Prevalence of Clostridium difficile and ribotype 027 infection in patients with nosocomial diarrhoea in Southern Italy.

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.

Species Distribution and In Vitro Azole Susceptibility of Aspergillus Section Nigri Isolates from Clinical and Environmental Settings.

Aspergillus section Nigri includes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two black Aspergillus strains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and their in vitro triazole susceptibilities investigated. Aspergillus tubingensis was isolated from humans, animals, and environmental settings, whereas Aspergillus awamori and Aspergillus niger were isolated exclusively from humans. Phylogenetic analyses of ß-tubulin and calmodulin gene sequences were concordant in differentiating A. tubingensis from A. awamori and A. niger Voriconazole and posaconazole (PSZ) were the most active triazoles. One A. tubingensis strain was resistant to itraconazole and PSZ and one A. niger strain to PSZ. Sequence analysis of the cyp51A gene revealed different sequence types within a species, and A. tubingensis strains were also phylogenetically distinct from A. awamori/A. niger strains according to the strain origin and susceptibility profile. Genetic analysis of the cyp51A sequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in black Aspergillus isolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. The cyp51A sequence is useful for the molecular identification of black Aspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.

Comparison of two methods for the detection of verotoxin producing E.coli in human faecal samples during an outbreak of HUS in Apulia, Southern Italy.

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.

High-dose, extended-interval colistin administration in critically ill patients: is this the right dosing strategy? A preliminary study.

BACKGROUND: Gram-negative bacteria susceptible only to colistin (COS) are emerging causes of severe nosocomial infections, reviving interest in the use of colistin. However, consensus on the most effective way to administer colistin has not yet been reached. METHODS: All patients who had sepsis due to COS gram-negative bacteria or minimally susceptible gram-negative bacteria and received intravenous colistimethate sodium (CMS) were prospectively enrolled. The CMS dosing schedule was based on a loading dose of 9 MU and a 9-MU twice-daily fractioned maintenance dose, titrated on renal function. For each CMS course, clinical cure, bacteriological clearance, daily serum creatinine clearance, and estimated creatinine clearance were recorded. RESULTS: Twenty-eight infectious episodes due to Acinetobacter baumannii (46.4%), Klebsiella pneumoniae (46.4%), and Pseudomonas aeruginosa (7.2%) were analyzed. The main types of infection were bloodstream infection (64.3%) and ventilator-associated pneumonia (35.7%). Clinical cure was observed in 23 cases (82.1%). Acute kidney injury developed during 5 treatment courses (17.8%), did not require renal replacement therapy, and subsided within 10 days from CMS discontinuation. No correlation was found between variation in serum creatinine level (from baseline to peak) and daily and cumulative doses of CMS, and between variation in serum creatinine level (from baseline to peak) and duration of CMS treatment. CONCLUSIONS: Our study shows that in severe infections due to COS gram-negative bacteria, the high-dose, extended-interval CMS regimen has a high efficacy, without significant renal toxicity.

Trends in the Incidence and Antibiotic Resistance of Enterococcal Bloodstream Isolates: A 7-Year Retrospective Multicenter Epidemiological Study in Italy.

The spread of resistance to vancomycin and other last-resort drugs in Enterococcus spp. remains of concern. In Italy, surveillance data for enterococcal bloodstream isolates in humans are scant. The aim of our study was to assess the incidence trends of bacteremias due to Enterococcus species and their prevalence trends of antimicrobial resistance. We retrospectively included all consecutive not-duplicate Enterococcus species isolated from blood cultures, in patients from 11 Italian hospitals (2011-2017). Incidence was defined as the number of isolates per 10,000 patient-days, while resistance prevalence was defined as the number of resistant strains divided by the number of tested strains. We included 4,858 isolates (59%, 36%, and 5% due to Enterococcus faecalis, E. faecium, and other Enterococcus spp., respectively). Over the study period, the incidence of bacteremias due to E. faecalis (incidence rate ratio [IRR]: 1.02, 95% confidence interval [CI]: 1.00-1.04, p = 0.008) and E. faecium increased (IRR: 1.03, 95% CI: 1.01-1.05, p < 0.001) alongside with the whole enterococcal bacteremias trend (IRR: 1.02, 95% CIs: 1.01-1.04, p = 0.002). A progressive increase in vancomycin-resistant E. faecium (VREfm) bacteremias was observed. Resistance to tigecycline and linezolid was rarely reported. The incidence of enterococcal bloodstream isolates is increasing in Italy, together with the prevalence of VREfm. Resistance to linezolid, a cornerstone drug used in the treatment of VRE bloodstream infection, remains negligible.

Streptococcus pneumoniae meningitis in a child vaccinated with pneumococcal heptavalent conjugate vaccine.

Pneumococcal meningitis is still today a life threatening disease among children under-5 worldwide. Although the heptavalent vaccine has demonstrated its ability to reduce the incidence of pneumococcal disease its efficacy is limited due to the restricted number of serotypes included. We report a case of a child with a Streptococcus pneumoniae meningitis despite the use of heptavalent conjugate vaccine.

Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay.

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.

Trends in the antibiotic resistance of S. aureus clinical isolates: a 4 years retrospective study in a teaching hospital in South Italy.

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.

Trends in Klebsiella pneumoniae strains isolated from the bloodstream in a teaching hospital in southern Italy.

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.

Leukocytoclastic Vasculitis Associated with HHV6-A/ciHHV6-A and HHV6-B Coinfection in an Immunocompetent Woman.

BACKGROUND AND OBJECTIVE: Leukocytoclastic vasculitis (LCV) is a small vessel vasculitis that can be limited to the skin but may also affect other organs. Often, its cause is unknown. LCV has previously been reported to occur with the reactivation of human herpesvirus 6 (HHV-6). Here, we report a second instance of HHV-6 reactivation in a 43-year-old woman with idiopathic cutaneous LCV. CASE DESCRIPTION: In this case, the patient was immunocompetent, and testing revealed that she had inherited chromosomally integrated human herpesvirus 6 variant A (iciHHV6-A) with a parallel skin infection of HHV-6B. The integrated ciHHV-6A strain was found to be transcriptionally active in the blood, while HHV-6B late antigen was detected in a skin biopsy. The patient's rash was not accompanied by fever nor systemic symptoms and resolved over four weeks without any therapeutic intervention. CONCLUSION: In light of the transcriptional activity documented in our case, further examination of a possible role for HHV-6 in the etiology of LCV is warranted.

Prevalence and genotypes identification of human papillomavirus infection in a population of South Italy.

BACKGROUND: A limited number of human papillomavirus (HPV) types account for the majority of invasive cervical cancer cases. OBJECTIVES: To assess, in a southern Italian region, where HPV infection had not yet been investigated, the prevalence of type-specific HPV infection. STUDY DESIGN: Multiplex PCR was used to test cervical specimens from 871 asymptomatic women. RESULTS: The HPV infection rate was 23.1%, with the highest prevalence being observed in women aged 20-30 years (32.6%). Type 16 was the most frequent HPV type detected either in mono-infected (39.8%) or in multi-infected (46.3%) women. CONCLUSIONS: The HPV infection rate was higher than reported from other Italian areas. Our results further emphasise the importance of vaccinations to immunize females before they acquire HPV infection.

Chronic endometritis in patients with unexplained infertility: Prevalence and effects of antibiotic treatment on spontaneous conception.

PROBLEM: The correlations between chronic endometritis and unexplained infertility are unexplored. METHOD OF STUDY: We performed a retrospective study on consecutive patients referred to our hysteroscopy service due to unexplained infertility. All women underwent endometrial sampling with histological and cultural examinations. If chronic endometritis was diagnosed, patients received antibiotic therapy, and chronic endometritis resolution was subsequently ascertained by histological examination. We aimed to estimate chronic endometritis prevalence and the effects of antibiotic therapy on spontaneous conception during the year following hysteroscopy. RESULTS: A total number of 95 women were included. Pooled prevalence of chronic endometritis was 56.8%. Antibiotic therapy resulted in chronic endometritis resolution in 82.3% of patients, while in 17.6% disease was persistent. Women with cured chronic endometritis showed higher pregnancy rate and live birth rate in comparison with both women with persistent disease and women without chronic endometritis diagnosis (pregnancy rate = 76.3% vs 20% vs 9.5%, P < .0001; live birth rate = 65.8% vs 6.6% vs 4.8%, P < .0001). CONCLUSION: Chronic endometritis is highly prevalent in patients with unexplained infertility. Diagnosis and treatment of chronic endometritis improve spontaneous pregnancy rate and live birth rate in such patients.

Antimicrobial profiles of Prevotella spp. and Fusobacterium nucleatum isolated from periodontal infections in a selected area of southern Italy.

In view of the increasing antibiotic resistance of anaerobic Gram-negative bacteria, we determined the antimicrobial profile of 55 periodontal anaerobic Gram-negative bacteria correlated with human infections, comprising 16 strains of Fusobacterium nucleatum and 39 strains of Prevotella spp. isolated from periodontal pockets of 26 adults suffering from chronic periodontitis. All the strains of F. nucleatum were susceptible to amoxicillin/clavulanic acid, doxycycline, metronidazole, moxifloxacin and levofloxacin, whilst 2/16 strains were both resistant to amoxicillin and beta-lactamase-positive and 11/16 were resistant to clarithromycin. All of the Prevotella strains were susceptible to amoxicillin/clavulanic acid, doxycycline and metronidazole, whereas 7/39 strains were beta-lactamase-positive and resistant to amoxicillin, 5/39 were resistant to clarithromycin and 3/39 were resistant to both moxifloxacin and levofloxacin. Our findings confirm that there is an increasing need to encourage practitioners to use laboratory investigations to limit the risk of an incorrect therapeutic approach and to avoid the overuse of antimicrobial agents.

Utility of gas chromatography for rapid identification of mycobacterial species frequently encountered in clinical laboratory.

Over the last years, the clinical importance of mycobacteria has been raised. In this regard, it is important their identification in order to establish either the clinical significance or the appropriate therapy of the disease. Biochemical tests are usually time consuming until the report of results, that is why more rapid techniques are needed. As an alternative identification method, we have used a commercially available system for microbial identification based on whole cellular fatty acids analysis using gas-chromatography (GC). Sixty-eight strains of Mycobacterium tuberculosis, Mycobacterium gordonae, Mycobacterium xenopi, Mycobacterium kansasii, Mycobacterium fortuitum, and Mycobacterium avium-intracellulare were clearly identified by their unique fatty acid profile using the Sherlock Microbial Identification System (MIS). The results were in agreement with those obtained with traditional methods. This method is highly automated, rapid, easy to perform with a sample preparation for lipid analysis which is neither time consuming nor requiring a particular expertise. On this basis the MIS-GC method for the identification of some clinically important mycobacteria appears to be suitable for routine clinical use.

Atypical Herpetic Whitlow: A Diagnosis to Consider.

CONTEXT: Herpetic whitlow is caused by herpes virus (type1 or 2) during primary infection or as result of autoinoculation. Commonly, it is caused by HSV-2 in adults with positive history for genital infection. CASE DESCRIPTION: We report the case of a 44-year-old woman that came to our attention with a 3- year history of recurrent cutaneous eruption on the ring finger of her left hand associated to lymphangitis of the homolateral arm. Laboratory exams including PCR on blood and cutaneous swab allowed to diagnosis it as a rare case of herpetic whitlow. CONCLUSION: The case here reported demonstrates that herpetic whitlow should be kept in mind by physicians in recurrent cases of fingers infection. Advanced diagnostic techniques as PCR are required to help clinicians to achieve a definite diagnosis and to choose the right treatment.

Prevalence, genotype distribution and temporal dynamics of human papillomavirus infection in a population in southern Italy.

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.