Successful Control of an Outbreak by Phenotypically Identified Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae in a Neonatal Intensive Care Unit.

BACKGROUND: Premature newborns represent a vulnerable population, at high risk of acquiring nosocomial infections during neonatal intensive care unit (NICU) admission. Multidrug-resistant organisms represent the greatest concern due to their intrinsic virulence and the limited therapeutic options. Resistant Enterobacterales are a growing threat for critically ill neonates, with increasing numbers of NICU outbreaks caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales being described. This study reports the early detection and successful control of an outbreak caused by ESBL-producing Klebsiella pneumoniae (ESBL-KP) in an Italian NICU in February 2021. RESULTS: A total of 13 newborns tested positive for ESBL-KP between 2-9 February 2021, of whom four (31%) had a bloodstream infection. Two were critically ill, extremely premature newborns who died because of multiple comorbidities, and two were cured after treatment with meropenem. All other patients survived and were either discharged home or moved to other hospitals/wards in good clinical condition. ESBL-KP ST45 was found in all isolates by multilocus sequence typing (MLST) analysis. An outbreak control plan was set, including surveillance cultures for all neonates, NICU environments, and medical devices, along with the extended use of contact precautions and cohorting. In addition, the infection control plan was carried out through reinforcement and enhancement measures to guarantee maximal compliance. The outbreak was successfully controlled in seven days, given that no further cases were identified after 9 February. The source of the ESBL-KP outbreak was not identified through environmental sampling. CONCLUSIONS: Thanks to multidisciplinary management, a threatening outbreak of ESBL-KP in a NICU was controlled in few days. The prompt recognition of the event onset and the adoption of infection control interventions helped contain the bacteria spread on the ward.

Anal and oral human papillomavirus (HPV) infection in HIV-infected subjects in northern Italy: a longitudinal cohort study among men who have sex with men.

BACKGROUND: A study including 166 subjects was performed to investigate the frequency and persistence over a 6-month interval of concurrent oral and anal Human Papillomavirus (HPV) infections in Human Immunodeficiency Virus (HIV)-infected men who have sex with men (MSM). METHODS: Patients with no previously documented HPV-related anogenital lesion/disease were recruited to participate in a longitudinal study. Polymerase chain reaction (PCR) was performed to detect HPV from oral and anal swabs and to detect Human Herpes Virus 8 (HHV-8) DNA in saliva on 2 separate specimen series, one collected at baseline and the other collected 6 months later. A multivariate logistic analysis was performed using anal HPV infection as the dependent variable versus a set of covariates: age, HIV plasma viral load, CD4+ count, hepatitis B virus (HBV) serology, hepatitis C virus (HCV) serology, syphilis serology and HHV-8 viral shedding. A stepwise elimination of covariates with a p-value > 0.1 was performed. RESULTS: The overall prevalence of HPV did not vary significantly between the baseline and the follow-up, either in the oral (20.1 and 21.3%, respectively) or the anal specimens (88.6 and 86.3%). The prevalence of high-risk (HR) genotypes among the HPV-positive specimens was similar in the oral and anal infections (mean values 24.3% and 20.9%). Among 68 patients with either a HR, low-risk (LR) or undetermined genotype at baseline, 75% had persistent HPV and the persistence rates were 71.4% in HR infections and 76.7% in LR infections. There was a lack of genotype concordance between oral and anal HPV samples. The prevalence of HR HPV in anus appeared to be higher in the younger patients, peaking (> 25%) in the 43-50 years age group. A decrease of the high level of anal prevalence of all genotypes of HPV in the patients > 50 years was evident. HHV-8 oral shedding was positively related to HPV anal infection (p = 0.0046). A significant correlation was found between the persistence of HHV-8 shedding and HIV viral load by logistic bivariate analysis (Odds Ratio of HHV-8 persistence for 1-log increase of HIV viral load = 1.725 ± 0.397, p = 0.018). CONCLUSIONS: A high prevalence of HPV infection was found in our cohort of HIV-infected MSM, with a negative correlation between anal HPV infection and CD4 cell count.

Distribution of human papillomavirus types in the anogenital tract of females and males.

Human papillomavirus (HPV) infection is the most common sexually transmitted infection in both men and women, but there are limited data comparing the prevalence of HPV infection between genders and in different anogenital sites. This cross-sectional analysis describes the distribution of HPV types in the genital tract of 3,410 consecutive females and 1,033 males undergoing voluntary screening for HPV and referred to a single institution. The relationship between specific HPV types and the presence of anogenital lesions was examined. In both females and males, the overall prevalence of HPV infection was about 40%. A wide variety of HPV types was identified, but the prevalence of different types was remarkably similar in the two genders, even when considering different anatomical sites. HPV-6 was the most frequent (prevalence 13%) type in all anogenital sites in men followed by HPV-16 (7%), while HPV-16 was the most common type in women (about 6%), either in the cervix, vagina, or vulva, followed by HPV-6. In addition to HPV-16, HPV-58, HPV-33, HPV-31, and HPV-56 were the carcinogenic types detected most commonly and were significantly associated with high-grade squamous intraepithelial cervical lesions, while HPV-53 and HPV-66 were the most common among possibly carcinogenic types. In both genders, anogenital warts were associated with HPV-6 and HPV-11 infection, and, less frequently, with other types, like HPV-54, HPV-62, and HPV-66. These results show that genital HPV infection involves numerous HPV types, which have similar distribution patterns in females and males and in different anogenital anatomical sites.

Measles Virus Infection and Immunity in a Suboptimal Vaccination Coverage Setting.

Despite efforts to improve surveillance and vaccination coverage, measles virus (MeV) continues to cause outbreaks also in high-income countries. As the reference laboratory of the Veneto Region, Italy, we analyzed changes in population immunity, described measles outbreaks, investigated MeV genetic diversity, and evaluated cross-protection of measles vaccination against MeV epidemic strains. Like most European areas, the Veneto Region has suboptimal measles vaccination coverage and is facing a growing public mistrust of vaccination. A progressive decline of measles vaccine uptake was observed during the last decade in the Veneto Region, leading to immunity gaps in children and young adults. Measles outbreaks were caused by the same MeV genotype B3, D4, and D8 strains that were circulating in other European countries. Eleven cases of measles were observed in immunized subjects. These cases were not associated with particular MeV genotypes nor with mutations in epitopes recognized by neutralizing antibodies. Accordingly, sera from fully vaccinated subjects cross-neutralized epidemic MeV strains, including the genotypes B3, D4, and D8, with the same high efficiency demonstrated against the vaccine strain. In fully vaccinated subjects, high MeV IgG antibody titers persisted up to 30 years following vaccination. These results support the use of the current measles-containing vaccines and strategies to strengthen vaccination.

Drug resistance in B and non-B subtypes amongst subjects recently diagnosed as primary/recent or chronic HIV-infected over the period 2013-2016: Impact on susceptibility to first-line strategies including integrase strand-transfer inhibitors.

OBJECTIVES: To characterize the prevalence of transmitted drug resistance mutations (TDRMs) by plasma analysis of 750 patients at the time of HIV diagnosis from January 1, 2013 to November 16, 2016 in the Veneto region (Italy), where all drugs included in the recommended first line therapies were prescribed, included integrase strand transfer inhibitors (InNSTI). METHODS: TDRMs were defined according to the Stanford HIV database algorithm. RESULTS: Subtype B was the most prevalent HIV clade (67.3%). A total of 92 patients (12.3%) were expected to be resistant to one drug at least, most with a single class mutation (60/68-88.2% in subtype B infected subjectsand 23/24-95.8% in non-B subjects) and affecting mainly NNRTIs. No significant differences were observed between the prevalence rates of TDRMs involving one or more drugs, except for the presence of E138A quite only in patients with B subtype and other NNRTI in subjects with non-B infection. The diagnosis of primary/recent infection was made in 73 patients (9.7%): they had almost only TDRMs involving a single class. Resistance to InSTI was studied in 484 subjects (53 with primary-recent infection), one patient had 143C in 2016, a total of thirteen 157Q mutations were detected (only one in primary/recent infection). CONCLUSIONS: Only one major InSTI-TDRM was identified but monitoring of TDRMs should continue in the light of continuing presence of NNRTI-related mutation amongst newly diagnosed subjects, sometime impacting also to modern NNRTI drugs recommended in first-line therapy.

Difficulties in the mutation analysis of plasminogen gene: a study in two patients with ligneous conjunctivitis.

The absence or very low levels of plasminogen cause a rare disabling disease called ligneous conjunctivitis, characterized by the growth of fibrin-rich pseudomembranes in the conjunctiva and on other mucosal surfaces. Several mutations have been detected in the plasminogen gene of patients affected with ligneous conjunctivitis. The human plasminogen gene, located on chromosome 6, has a marked homology with the genes belonging to the plasminogen-apo(a) family, and with a number of pseudogenes and plasminogen-like genes located on chromosome 2. This work describes a series of nucleotide variations related to genes other than the plasminogen one, found during the genetic characterization of plasminogen defect in two unrelated patients with ligneous conjunctivitis. The results of automated sequences of each exon and intron-exon boundaries were compared with those of the human plasminogen gene from the NCBI gene bank. In particular, a co-amplified gene on chromosome 2 mimicking a 14 bp deletion in exon 5 of the plasminogen gene was identified by sequencing two different bands obtained from a long run of the PCR exon 5 product in NuSieve agarose gel, and by PstI restriction enzyme analysis of the same amplicons. Moreover, 21 single nucleotide exchanges due to plasminogen-like genes co-amplification were observed, namely one in exon 1, two in exon 4, three in exons 3, 5 and 16, four in exon 13, and five in exon 17. In conclusion, these data confirm the difficulty of plasminogen genetic analysis and may help researchers to better identify the true plasminogen gene mutations causing molecular defects.

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin.

OBJECTIVES: Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV-HCV co-infected patients. METHODS: This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV-HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing. RESULTS: DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16. CONCLUSIONS: Sub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients.

Viral infections of the central nervous system in elderly patients: a retrospective study.

OBJECTIVES: Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old). METHODS: This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein-Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection. RESULTS: A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥ 80 years and in 35 (9.6%) patients aged 65-79 years (p=0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p=0.03). CONCLUSIONS: HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥ 80 years.

Conformation sensitive gel electrophoresis for detection of factor X gene mutations.

Fifteen patients from five families with laboratory data suggesting factor X (FX) deficiency were screened for causative mutations by conformation sensitive gel electrophoresis (CSGE) followed by sequencing. All exonic and flanking intronic regions of factor X gene were amplified using PCR. After heteroduplex formation, samples were analyzed onto a polyacrylamide gel for possible mismatch. An abnormal CSGE profile indicating an heteroduplex was identified in 10/15 cases. All the 10 patients with a patter of migration suggesting a mismatch had a laboratoristic pattern of FX deficiency whereas the five cases with a normal CSGE aspect referred to the normal components of the families who did not carry any FX defect. Sequencing demonstrated that the 10 exons, which showed a suspect CSGE pattern, harbored a mutation responsible for the factor X defect. Of the five mutation identified, two were recognized to be novel mutations (a 871C>T substitution and a 1169G>T transversion in exon 8), both located in the catalytic portion of FX. CSGE may be an effective and simple procedure for screening factor X gene mutations.

Decreasing trends of drug resistance and increase of non-B subtypes amongst subjects recently diagnosed as HIV-infected over the period 2004-2012 in the Veneto Region, Italy.

The present study was designed to prospectively monitor transmitted drug resistance mutations (TDRMs) in the Veneto Region, Italy. Genotypic resistance testing was conducted on the plasma of 1882 patients consecutively enrolled at the time of diagnosis of human immunodeficiency virus (HIV) infection from 2004 to 2012. TDRMs were defined according to the Stanford HIV database algorithm. In total, 214 (16.1%) B subtype-infected and 58 (10.5%) non-B subtype-infected individuals were identified as having a primary or recent HIV-1 infection. In subtype B-infected subjects in 2004-2006, the prevalence of TDRMs was 20.0% in chronic infections and 25.5% in recent infections; in 2007-2009 the rates were 11.5% and 5.3%, respectively; and in 2010-2012 they were 11.3% and 15.2%, respectively. In non-B subtype-infected subjects in 2004-2006, the prevalence of TDRMs was 18.0% in chronic infections and 16.5% in recent infections; in 2007-2009 the rates were 5.7% and 0%, respectively; and in 2010-2012 they were 6.2% and 8.7%, respectively. Protease inhibitor resistance and combined resistance to two or three classes of drugs declined during the three study periods. The observed decrease in TDRMs and a simplification of the resistance patterns may reflect a change over time in the characteristics of the infecting subjects who are often unaware of their infection and transmit a wild-type strain.

Human papillomavirus genotyping by 454 next generation sequencing technology.

BACKGROUND: An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies. OBJECTIVES: Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology. STUDY DESIGN: Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA). RESULTS: In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/µL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen. CONCLUSIONS: NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.

Both human immunodeficiency virus cellular DNA sequencing and plasma RNA sequencing are useful for detection of drug resistance mutations in blood samples from antiretroviral-drug-naive patients.

Genotypic antiretroviral testing is recommended for newly infected drug-naive subjects, and the material of choice is plasma RNA. Since drug resistance mutations (DRMs) may persist longer in cellular DNA than in plasma RNA, we investigated whether the use of peripheral blood mononuclear cell (PBMC) human immunodeficiency virus (HIV) DNA increases the sensitivity of genotypic testing in antiretroviral-drug-naive subjects. We compared the rate of primary drug resistance in plasma RNA and PBMC DNA in 288 HIV type 1-infected drug-naive persons tested at a single clinical virology center from June 2004 to October 2006. Resistance in the plasma compartment to at least one drug was detected for 64 out of 288 (22.2%) naive patients and in the PBMC compartment for 56 (19.4%) patients. Overall, DRMs were found in 80 out of 288 (27.8%) patients. PBMC DNA [corrected] DRMs were present in [corrected] 16 subjects with wild-type virus in their plasma RNA [corrected] Another nine patients had additional DRMs in their PBMC DNA [corrected] with respect to those detected in their [corrected] plasma RNA. On the other hand, extra plasma RNA [corrected] DRMs were detected in [corrected] 24 and 8 subjects with wild-type and drug-resistant virus in their PBMC DNA [corrected] respectively. Resistance to more than one class of antiretroviral drug was detected by plasma and PBMC analysis for 25.0% and 36.2% of the subjects, respectively. Our data support the potential utility of genotypic resistance testing of PBMC DNA in conjunction with the currently recommended plasma RNA analysis.

Comparative evaluation of three computerized algorithms for prediction of antiretroviral susceptibility from HIV type 1 genotype.

OBJECTIVES: To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility. METHODS: We applied three genotypic interpretation algorithms to 478 reverse transcriptase (RT) and 410 protease sequences for which phenotypic data were available. Sequences were obtained from clinical practice and from published sequences in the Stanford HIV-1 RT and Protease Sequence Database. The genotypic interpretation algorithms included: Stanford HIVdb program (HIVdb), the Visible Genetics/Bayer Diagnostics Guidelines 6.0 (VGI) and a genotypic interpretation program (AntiRetroScan, ARS) developed at the University of Siena, Italy. Genotypic interpretations were normalized to a three-level output: susceptible, intermediate and resistant. Discordances were defined as differences between genotype and phenotype for the same virus isolate. Discordances for which an isolate was considered susceptible by one test but resistant by another test were considered major discordances. RESULTS: The frequency of major discordances between genotype and phenotype was 10.6, 13.7 and 15.7% for ARS, VGI and HIVdb, respectively (P < 0.0001 for ARS versus HIVdb and for ARS versus VGI; P = 0.002 for VGI versus HIVdb). The correlation between genotype and phenotype was highest for non-nucleoside RT inhibitors and lowest for nucleoside RT inhibitors. Half of the major discordances involved stavudine, didanosine and zalcitabine. The concordance among the three genotypic algorithms was high, with weighted Kappa values ranging between 0.76 and 0.84 for the pairwise comparisons between each of the algorithms. CONCLUSIONS: Genotype interpretation algorithms correctly predict phenotype in 85-90% of cases, but the rate of concordance is not uniformly distributed among different drugs. These data provide insight into the potential additional benefit derived from phenotyping.