Risk factors for candidaemia in hospitalized patients with liver cirrhosis: a multicentre case-control-control study.

OBJECTIVES: The aim of this study was to evaluate the risk factors for candidaemia in patients with liver cirrhosis. METHODS: This was a case-control-control (1:2:2) study performed in four Italian tertiary centres from 2006 to 2015. Cases were patients with liver cirrhosis developing candidaemia. For every case of candidaemia we enrolled two additional patients undergoing blood cultures for suspected infection yielding isolation of a bacterial pathogen (control A) and two additional patients undergoing blood cultures for suspected infection yielding negative results (control B). Patients were matched according to age, sex and model for end stage liver disease at hospital admission. RESULTS: During the study period 90 cases, 180 controls A and 180 controls B were included. At multivariate analysis assessed by means of multinomial conditional regression models, factors independently associated with candidaemia were previous (<30 days) acute-on-chronic liver failure (relative risk ratio (RRR) 2.22 (95% confidence interval (CI) 1.09-4.54), p = 0.046), previous(<30 days) gastrointestinal endoscopy (RRR 2.38 (95% CI 1.19-4.78) p = 0.014), previous(<30 days) antibiotic treatment for at least 7 days (RRR 2.74 (95% CI 1.00-7.48), p = 0.049), presence of central venous catheter (RRR 2.77 (95% CI 1.26-6.09, p = 0.011), total parenteral nutrition (RRR 3.90 (95% CI 1.62-9.40), p = 0.002) at infection onset and length of in-hospital stay >15 days (RRR 4.63 (95% CI 2.11-10.18), p <0.001] Conversely, rifaximin treatment was associated with lower rate of candidaemia (RRR 0.38 (95% CI 0.19-0.77), p = 0.007). Multivariable analysis for 30-day mortality showed that patients with isolation of Candida spp. from blood cultures had worse outcome when compared with controls even though the difference did not reach a statistical significance (hazard ratio 1.64 (95% 0.97-2.75) p = 0.06). CONCLUSIONS: We identified previous antibiotic use, gastrointestinal endoscopy or acute-on-chronic liver failure and presence of central venous catheter especially for parenteral nutrition as independent factors associated with candidaemia. Surprisingly, chronic rifaximin use was a protective factor.

Serum bactericidal titres for monitoring antimicrobial therapy: current status and potential role in the management of multidrug-resistant Gram-negative infections.

BACKGROUND: Serum bactericidal titres (SBTs) were widely used in the 1970s and 1980s to monitor antimicrobial therapy but are now seldom recommended. It is the only laboratory test that integrates drug pharmacodynamics, host pharmacokinetics and synergistic or antagonistic interactions of antimicrobial combinations into a single index of antimicrobial activity. We hypothesized that SBTs could play a renewed role in monitoring antibiotic treatment of multidrug-resistant Gram-negative infections. However, the last critical appraisal of the test was published over 30 years ago. OBJECTIVES: This narrative review provides an updated assessment of the SBT test and its methodological limitations. We performed a diagnostic meta-analysis to estimate the value of SBTs for predicting clinical failure or death during antibiotic treatment. SOURCES: A comprehensive literature search of PubMed including all English publications was performed in December 2019 using the Medical Subject Headings (MeSH search terms "serum", "bactericidal", "inhibitory", "titre", "monitoring", "anti-infective agents" "antimicrobial therapy" and "therapeutic drug monitoring"). CONTENT: Although standardized methods for performing SBTs were approved in 1999, the test remains labour intensive, and results may not be available until 72 hr. However, the use of non-culture-based endpoints (i.e. spectrophotometric or fluorescent) may shorten test time to 24 hr. Despite considerable heterogeneity in published studies, a meta-analysis of 11 evaluable studies published from 1974 to 2007 indicated a critical SBT result (peak SBT ≤1:8 or trough ≤1:2) is associated with a diagnostic odds ratio for clinical failure during antibiotic treatment of 12.27 (95% confidence interval 5.28-28.54) and a 5.32 (95% 1.32-21.42) odds of death. IMPLICATIONS: SBTs have prognostic value for identifying patients at high risk for antibiotic treatment failure, but the slow turnaround time of the current test limits its clinical utility. Standardization of a more rapid SBT testing method is needed.

Follow-up blood cultures are associated with improved outcome of patients with gram-negative bloodstream infections: retrospective observational cohort study.

OBJECTIVES: We examined factors associated with follow-up blood cultures (FUBCs) in patients with monomicrobial Gram-negative (GN) bloodstream infection (BSI) and investigated the impact of FUBCs on therapeutic management and patient outcome. METHODS: A retrospective cohort analysis was conducted of adult patients diagnosed with GN-BSI at a tertiary-care university hospital during 2013-2016. FUBCs performed between 24 hours and 7 days after index BCs was the exposure variable. Risk factors for 30-day mortality were analysed by multivariate Cox analysis on the overall cohort, including FUBCs as a time-varying covariate and on 1:1 matched patients according to Sequential Organ Failure Assessment (SOFA) score and time to FUBC. RESULTS: In 278 (17.6%) of 1576 patients, FUBCs were performed within a median of 3 and 2 days after index BCs and active antibiotic therapy initiation. Persistent BSI was found in 107 (38.5%) of 278 patients. FUBCs were performed in more severely ill patients, with nonurinary sources, difficult-to-treat pathogens and receipt of initial inappropriate therapy. Source control and infectious disease consultation rates were higher among patients with preceding FUBCs and was associated with longer treatment duration. Thirty-day mortality was 10.4%. Independent risk factors for mortality were Charlson comorbidity index (hazard ratio (HR) 1.12) SOFA (HR 1.11), septic shock (HR 2.64), urinary source (HR 0.60), central venous catheter source (HR 2.30), complicated BSI (HR 2.10), carbapenem resistance (HR 2.34), active empiric therapy (HR 0.68), source control (HR 0.34) and FUBCs (HR 0.48). Association between FUBCs and lower mortality was confirmed in the 274 matched pairs. CONCLUSIONS: FUBCs were performed in more severe GN-BSIs, yielding a high rate of persistent BSI. In this context, FUBCs were associated with lower mortality.

Bloodstream infection caused by KPC-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam: epidemiology and genomic characterization.

OBJECTIVES: The aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. METHODS: We collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. RESULTS: During the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime-avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime-avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. CONCLUSIONS: Our results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

First report of Methylobacterium radiotolerans bacteraemia identified by MALDI-TOF mass spectrometry.

Methylobacterium radiotolerans is a fastidious, pink-pigmented, obligate aerobic Gram-negative bacillus commonly isolated from various environmental sources, and only occasionally from clinical samples, mostly in immunocompromised patients or associated with intravascular devices and haemodialysis. It grows poorly on commonly used culture media and its identification is time-consuming by conventional means. In this study, we present a case of M. radiotolerans bacteraemia in an individual affected by end-stage renal failure, identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The species identification was further confirmed by biochemical and molecular methods. The susceptibility to various antimicrobial agents is also presented and discussed.

The impact of carbapenemase-producing Enterobacteriaceae colonization on infection risk after liver transplantation: a prospective observational cohort study.

OBJECTIVE: To investigate the impact of colonization with carbapenemase-producing Enterobacteriaceae (CPE) on the CPE infection risk after liver transplantation (LT). METHODS: Prospective cohort study of all adult patients undergoing LT at our centre over an 8-year period (2010-2017). Individuals were screened for CPE colonization by rectal swabs at inclusion onto the waiting list, immediately before LT and weekly after LT until hospital discharge. Asymptomatic carriers did not receive decolonization, anti-CPE prophylaxis or pre-emptive antibiotic therapy. Participants were followed up for 1 year after LT. RESULTS: We analysed 553 individuals who underwent a first LT, 38 were colonized with CPE at LT and 104 acquired colonization after LT. CPE colonization rates at LT and acquired after LT increased significantly over the study period: incidence rate ratios (IRR) 1.21 (95% CI 1.05-1.39) and 1.17 (95% CI 1.07-1.27), respectively. Overall, 57 patients developed CPE infection within a median of 31 (interquartile range 11-115) days after LT, with an incidence of 3.05 cases per 10 000 LT-recipient-days and a non-significant increase over the study period (IRR 1.11, 95% CI 0.98-1.26). In multivariable analysis, CPE colonization at LT (hazard ratio (HR) 18.50, 95% CI 6.76-50.54) and CPE colonization acquired after LT (HR 16.89, 95% CI 6.95-41.00) were the strongest risk factors for CPE infection, along with combined transplant (HR 2.60, 95% CI 1.20-5.59), higher Model for End-Stage Liver Disease at the time of LT (HR 1.03, 95% CI 1.00-1.07), prolonged mechanical ventilation (HR 2.63, 95% CI 1.48-4.67), re-intervention (HR 2.16, 95% CI 1.21-3.84) and rejection (HR 2.81, 95% CI 1.52-5.21). CONCLUSIONS: CPE colonization at LT or acquired after LT were the strongest predictors of CPE infection. Prevention strategies focused on LT candidates and recipients colonized with CPE should be investigated.

Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

OBJECTIVES: A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. METHODS: Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. RESULTS: Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor-recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacter aerogenes. CONCLUSIONS: This study showed a low risk of donor-recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories.

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level. The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well. The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Treatment duration for Escherichia coli bloodstream infection and outcomes: retrospective single-centre study.

OBJECTIVES: To investigate the impact of treatment duration on mortality and on relapse in patients with Escherichia coli bloodstream infection (BSI). METHODS: Retrospective single-centre study of patients diagnosed with E. coli BSI at our centre over a 4-year period. EXCLUSION CRITERIA: age <18 years, clinical data not available, polymicrobial BSI, failure to receive in vitro active therapy, and death while receiving antibiotic therapy. Exposure variable was treatment duration dichotomized into short (≤10 days) and long (>10 days) therapy. Primary end point was all-cause mortality within 90 days after index BSI. Secondary end point was relapse, defined as repeat isolation of E. coli from blood cultures within 90 days after index BSI, in patients with documented clinical cure and completion of therapy for the initial episode. RESULTS: Of the 856 analysed patients: 426 received short and 430 received long therapy. All-cause mortality at day 90 occurred in 47 patients; on multivariate analysis, short therapy was not associated with a higher risk of mortality, also after adjusting the model for the propensity score of receiving short therapy. Relapse occurred in 42 patients. Independent risk factors for relapse using death as competing risk were immunosuppression (subhazard ratio 4.67, p < 0.001), and end-stage liver disease (subhazard ratio 2.58, p 0.013). The propensity-weighted estimation of the average treatment effect for relapse reduction with long therapy (>10 days) was -1.6% (p 0.26) in the total population, and -7.1% (p 0.18) in immunocompromised patients. CONCLUSIONS: We could not identify shorter treatment duration as a risk factor for mortality and for relapse in patients with E. coli BSI.

Previous bloodstream infections due to other pathogens as predictors of carbapenem-resistant Klebsiella pneumoniae bacteraemia in colonized patients: results from a retrospective multicentre study.

INTRODUCTION: the purpose of this retrospective multicenter study was to assess whether the risk of developing bloodstream infections (BSI) due to carbapenem-resistant Klebsiella pneumoniae (CRKP) in colonized patients is influenced by the occurrence of BSI due to other pathogens. METHODS: from January 2012 to March 2014, all patients with at least one rectal swab positive for CRKP and at least 30 days of previous hospital stay were included in the study. The primary outcome measure was CRKP BSI, defined as a time-to-event endpoint. The role of potential predictors was evaluated through univariable and multivariable Cox regression analyses, considering previous BSI as a time-dependent variable. RESULTS: during the study period, 353 patients met the inclusion criteria. Thirty-seven developed a CRKP BSI (11%). A higher incidence of CRKP BSI was observed in presence rather than in absence of previous BSI. In the final multivariable model of risk factors for CRKP BSI, multisite colonization (hazard ratio [HR] 13.73, 95% confidence intervals [CI] 3.29-57.32, p < 0.001), ICU stay (HR 3.14, 95% CI 1.19-8.31, p = 0.021), and previous BSI (p = 0.026, with the overall effect being mainly due to Enterococcus spp. BSI vs absence of BSI, HR 6.62, 95% CI 2.11-20.79) were associated with the development of CRKP BSI, while an inverse association was observed for age (HR 0.98, 95% CI 0.95-1.00, p = 0.027). CONCLUSIONS: previous BSI due to other pathogens were associated with an increased risk of CRKP BSI that was independent of other factors in colonized patients with prolonged hospital exposure.

Ease-of-use protocol for the rapid detection of third-generation cephalosporin resistance in Enterobacteriaceae isolated from blood cultures using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

An ease-of-use protocol for the identification of resistance against third-generation cephalosporins in Enterobacteriaceae isolated from blood culture bottles was evaluated using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. A cefotaxime hydrolysis assay from chocolate agar subcultures using antibiotic discs and without inoculum standardization was developed for routine work flow, with minimal hands-on time. This assay showed good performance in distinguishing between cefotaxime-susceptible and cefotaxime-resistant strains, with excellent results for Escherichia coli (sensitivity 94.7%, specificity 100%). However, cefotaxime resistance was not detected reliably in Enterobacteriaceae expressing AmpC genes or carbapenemase-producing Klebsiella pneumoniae.

Risk factors for bloodstream infections due to colistin-resistant KPC-producing Klebsiella pneumoniae: results from a multicenter case-control-control study.

The increasing prevalence of colistin resistance (ColR) Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (Kp) is a matter of concern because of its unfavourable impact on mortality of KPC-Kp bloodstream infections (BSI) and the shortage of alternative therapeutic options. A matched case-control-control analysis was conducted. The primary study end point was to assess risk factors for ColR KPC-Kp BSI. The secondary end point was to describe mortality and clinical characteristics of these infections. To assess risk factors for ColR, 142 patients with ColR KPC-Kp BSI were compared to two controls groups: 284 controls without infections caused by KPC-Kp (control group A) and 284 controls with colistin-susceptible (ColS) KPC-Kp BSI (control group B). In the first multivariate analysis (cases vs. group A), previous colistin therapy, previous KPC-Kp colonization, ≥3 previous hospitalizations, Charlson score ≥3 and neutropenia were found to be associated with the development of ColR KPC-Kp BSI. In the second multivariate analysis (cases vs. group B), only previous colistin therapy, previous KPC-Kp colonization and Charlson score ≥3 were associated with ColR. Overall, ColR among KPC-Kp blood isolates increased more than threefold during the 4.5-year study period, and 30-day mortality of ColR KPC-Kp BSI was as high as 51%. Strict rules for the use of colistin are mandatory to staunch the dissemination of ColR in KPC-Kp-endemic hospitals.

Impact of a hospital-wide multifaceted programme for reducing carbapenem-resistant Enterobacteriaceae infections in a large teaching hospital in northern Italy.

We performed a quasi-experimental study of a multifaceted infection control programme for reducing carbapenem-resistant Enterobacteriaceae (CRE) transmission and bloodstream infections (BSIs) in a 1420-bed university-affiliated teaching hospital during 2010-2014, with 30 months of follow-up. The programme consisted of the following: (a) rectal swab cultures were performed in all patients admitted to high-risk units (intensive-care units, transplantation, and haematology) to screen for CRE carriage, or for any room-mates of CRE-positive patients in other units; (b) cohorting of carriers, managed with strict contact precautions; (c) intensification of education, cleaning and hand-washing programmes; and (d) promotion of an antibiotic stewardship programme carbapenem-sparing regimen. The 30-month incidence rates of CRE-positive rectal cultures and BSIs were analysed with Poisson regression. Following the intervention, the incidence rate of CRE BSI (risk reduction 0.96, 95% CI 0.92-0.99, p 0.03) and CRE colonization (risk reduction 0.96, 95% CI 0.95-0.97, p <0.0001) significantly decreased over a period of 30 months. After accounting for changes in monthly census and percentage of externally acquired cases (positive at ≤72 h), the average institutional monthly rate of compliance with CRE screening procedures was the only independent variable associated with a declining monthly incidence of CRE colonization (p 0.002). The monthly incidence of CRE carriage was predictive of BSI (p 0.01). Targeted screening and cohorting of CRE carriers and infections, combined with cleaning, education, and antimicrobial stewardship measures, significantly decreased the institutional incidence of CRE BSI and colonization, despite endemically high CRE carriage rates in the region.

Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture.

In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures.

Outbreak of NDM-1-producing Enterobacteriaceae in northern Italy, July to August 2011.

Between July 2011 and August 2011, the New Delhi metallo-beta-lactamase 1 (NDM-1) gene was detected in Klebsiella pneumoniae and Escherichia coli isolates obtained from six patients hospitalised in four healthcare facilities in northern Italy. The patient who had been hospitalised in New Delhi, India, from February to May 2011 and subsequently in the Bologna area, Italy, from May to July 2011, may have been the source of the outbreak. Our findings suggest ongoing spread of this carbapenem-resistance gene in Italy and highlight the need for intensive surveillance.

Rapid increase of carbapenemase-producing Klebsiella pneumoniae strains in a large Italian hospital: surveillance period 1 March - 30 September 2010.

The first case of carbapenemase-producing Enterobacteriaceae in Italy was reported in 2009. We performed a study over a period of seven months in 2010 to survey the circulation of Klebsiella pneumoniae carbapenemases (KPC) ina 1,500-bed university hospital in northern Italy and report the presence and rapid increase of these multidrug-resistant bacteria. The results raise a major concern about these pathogens and demonstrate the urgent need for infection control and antibiotic stewardship programmes.

Killing of Treponema denticola by mouse peritoneal macrophages.

Treponema denticola has been identified as an important cause of periodontal disease and hypothesized to be involved in extra-oral infections. The objective of this study was to investigate the role of T. denticola cell length and motility during mouse peritoneal macrophages in vitro uptake. Macrophages, incubated under aerobic and anaerobic conditions, produced a similar amount of TNF-alpha when stimulated with Escherichia coli LPS. The uptake of FlgE- and CfpA-deficient mutants of T. denticola was significantly increased compared with the wild-type strain, due to cell size or lack of motility. Opsonization with specific antibodies considerably improved the treponemes' uptake. These results suggest that macrophages, in addition to other phagocytes, could play an important role in the control of T. denticola infection, and that the raising of specific antibodies could improve the efficacy of the immune response toward T. denticola, either at an oral site or during dissemination.

Multiplex chemiluminescence microscope imaging of P16(INK4A) and HPV DNA as biomarker of cervical neoplasia.

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16(INK4A) (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.

Assessment of the presence of mucosal human papillomaviruses in malignant melanomas using combined fluorescent in situ hybridization and chemiluminescent immunohistochemistry.

BACKGROUND: The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR. OBJECTIVES: To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. METHODS: We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. RESULTS: The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60-80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. CONCLUSIONS: The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.