Non-cholera Vibrio spp. invasive infections in the summer following May 2023 flood disaster in Romagna, Italy: a case series.

Non-cholera Vibrio spp. includes ubiquitous organisms living in aquatic environments. Their occurrence is associated with global warming and meteorological disasters. In May 2023 the Romagna region, Italy, was affected by severe floods. In the following 15 weeks we observed 5 patients with invasive infections caused by V. vulnificus (3/5) and V. harveyi (2/5). All patients (median age 77 years) had medical comorbidities and shared exposure to seawater. Two patients needed surgery; 2 died. In conclusion, we observed an increased burden of Vibrio spp. invasive infections after May 2023 floods, affecting old patients with predisposing medical conditions.

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.

Population-based frequency assessment of HPV-induced lesions in patients with borderline Pap tests in the Emilia-Romagna Region: the PATER study.

BACKGROUND: The PATER study assessed the frequency of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) in HPV-induced lesions in patients with borderline cytology. METHODS: This retrospective observational cohort study was designed to evaluate ASCUS patients detected through a local cervical cancer screening programme and referred to the Department of Gynaecology and Obstetrics at the S. Orsola-Malpighi University Hospital in Bologna, in the period between January 2000 and December 2007. RESULTS: In 1047 patients aged 38.4 ± 9.6 years (range 23-65 years), 34.8% (n = 364) was positive for HR- or LR-HPV DNA. The mean age of women with HPV infection was significantly lower compared with the negative group (36.8 ± 9.4 versus 39.3 ± 9.6 years; p < 0.001). Overall, 357 (34.1%) women had cervical lesions: 279 (26.6%) had CIN1, 18 (1.7%) CIN2, and 60 (5.7%) CIN3+. HR-HPV genotype was detected in 83.3%, and 91.5% of patients with CIN2 and CIN3+ respectively. Among the 124 CIN1 HPV-positive women, 8.9% harboured LR-HPV genotypes, 80.6% HR-HPV and 10.5% a combination of HR- and LR-HPV. HPV-6 and 11 accounted for 19.4% of all HPV-positive CIN1 lesions. CONCLUSION: Our study suggest that: in ASCUS patients over 40 years there is a low risk of positivity for HPV infection; the HPV DNA testing in patients with CIN3+ and a mean age close to 40 years is highly sensitive (98.3%) and acceptably specific (75.5%); the frequency of LR-HPV (alone or in combination with HR) in ASCUS cytology is not negligible. A tetravalent-based HPV vaccination alongside the screening programme would provide considerable clinical, organizational, and economic benefits.

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.

Molecular testing for detection of in vitro infectivity of plasma pools contaminated with B19 virus.

B19 virus can be transmitted by contaminated blood or blood products. Recent observations, in healthy volunteers, suggest that active B19 infection can follow the administration of plasma pools with a concentration > or =10(7) genome equivalents/ml (geq/ml) of B19 DNA. However, patients receiving batches with levels of virus DNA lower than 10(4) geq/ml do not show any evidence of transmission of the virus. The aim of the study was to show, by in vitro assays, a threshold of viral load in B19 contaminated plasma pools over which the infection can be transmitted. Twenty plasma pools, each containing 960 single donations, were tested to correlate the viral load and the level of antibodies anti-B19 with the in vitro infectivity and expression of B19 virus. All the plasma pools, titrated for B19 viral load by competitive PCR, were inoculated into KU812Ep6 erythroid human cell line. Five of the nine contaminated plasma pools, with a B19 DNA concentration > or =3.60 x 10(6) geq/ml, were able to infect KU812Ep6 cells. In vitro infectivity was shown in KU812Ep6 cells at 24 h post-infection by in situ hybridisation and amplification assays for viral DNA and RNAs. Plasma pools with a viral load in the range of 6.00 x 10(3)-8.96 x 10(4) geq/ml did not show infectivity when inoculated into KU812Ep6 cells. Medium-high titres of IgG antibodies anti-B19 were detectable in all the plasma pools and the neutralising activity associated with specific IgG anti-B19 may explain the lack of infectivity of plasma pools contaminated with a low viral load. In conclusion, in situ hybridisation and amplification assays for viral DNA and RNAs in KU812Ep6 cells inoculated with plasma pools can be valid assays to test for the presence of infectious virus in the production of B19-safe material.

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

BACKGROUND AND OBJECTIVES: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. STUDY DESIGN: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. RESULTS: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. CONCLUSIONS: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Relevance of B19 markers in serum samples for a diagnosis of parvovirus B19-correlated diseases.

In order to evaluate the optimal and essential diagnostic test(s) for a correct diagnosis of B19 diseases, 344 consecutive serum samples were tested from 344 patients with clinical suspicion of B19 infection during an epidemic period (early Spring-Autumn 2000). Sera were tested for B19 DNA by a standardized competitive polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and dot-blot hybridization and for specific IgM and IgG by ELISA. Of 344 patients examined, 125 were positive for markers of B19-associated disease: 49 had both B19 DNA and IgM, 50 had B19 DNA without IgM, and 26 had IgM without B19 DNA. After examination of the different patterns of B19 markers as diagnostic tools for B19 infection, IgM determination detected only 60% of B19-documented infections. IgM tests were nevertheless fundamental, as they were the unique diagnostic marker in 20.8% of documented infections (26 of 125 patients), in the diagnosis of recent, but still symptomatic infections when B19 DNA was no longer detectable. The determination of B19 DNA with PCR permitted detection of 79.2% of infections and therefore represented an essential test. PCR was fundamental for the diagnosis of B19 disease, as the unique diagnostic marker in 32% of documented infections (50 of 125 patients), both in acute infections at the onset of symptoms before the appearance of immunological response, and during the course of persistent B19 infections in which IgM had cleared. The contemporaneous determination of B19 DNA by PCR and specific IgM appears to be the most appropriate diagnostic protocol for the correct laboratory diagnosis of B19 infection.

Evaluation of commercial kits for the detection and typing of human papillomavirus in cervical swabs.

Human papillomaviruses (HPV) detection by MY consensus primers amplification within the L1 region and typing of prevalent genital HPVs by reference and commercial sets of probes was compared by PCR-ELISA systems. The specificity of commercial probes used in the L1 HPV Geno-Kit with respect to the reference probes proved to be 100%, with an overall agreement of 97.6%. The discordant results concerned only the detection of HPV 16, both as single genotype present in the sample and as multiple infections. The analytical sensitivity of the commercial probe for HPV 16 proved to be slightly less sensitive than the reference probe in the hybridisation conditions of the PCR-ELISA system. The L1 PCR-ELISA reference system was evaluated further in comparison with commercial E6/E7 consensus PCR and microplate hybridisation by typing kit. Amplified products of both the L1 and E6/E7 consensus regions were also analysed by agarose gel electrophoresis. An overall concordance of 95.2% was found. On account of its specificity and sensitivity the E6/E7 commercial system proved to be particularly useful for diagnostic laboratory, as it detects only the prevalent high risk genotypes. The agarose gel detection can therefore be used as screening test, thus reducing the costs, while the E6 E7 HPV Geno-Kit High Risk can be used when specific typing is required.

Diagnostic procedures in B19 infection.

In immunologic normal hosts, both children and adults, B19 can cause acute, generally self-limiting diseases. The infection leads to a viremia that can be present, at high titre, for about one week, then the onset of a specific immune response controls the infection. B19 infection in pregnancy can be associated with non-immunologic foetal hydrops or foetal death. In immunocompromised hosts, B19 can persist over several months and sometimes years. Persistent or recurrent B19 infections can be associated with chronic clinical manifestations or with transient clinical syndromes, generally related to the recrudescence of viral replication. Since the infection has been associated with a wide variety of clinical manifestations and some clinical features of B19 infection, such as anemia, artropathy and rash, can be common to other pathogens, a laboratory diagnosis of B19 infection is required. A diagnostic protocol must consider both the type of pathology and the type of patient. In immunocompetent individuals serological and virological testing is complementary, while in immunocompromised patients viral detection is the diagnosis of choice. Viral detection methods are generally based, nowadays, on the direct detection of B19 genome in clinical specimens. B19 DNA is mainly detected by hybridizations assays and by the most sensitive PCR assays. Serological diagnosis of B19 infection is generally achieved by detection of IgM and IgG antibodies to the B19 structural proteins VP1 and VP2. IgM detection is most often performed by capture assays, both in EIA and RIA formats, IgG are mainly detected by indirect EIA and immunofluorescence tests.

Detection of adeno-associated virus DNA in female genital samples by PCR-ELISA.

Adeno-associated viruses (AAV) are human parvoviruses that require helper function for their replication. Several studies have demonstrated that AAV DNA sequences can be found in the female genital tract but the incidence of infection seems very variable. A PCR-ELISA method detecting AAV DNA was developed for combining the specificity and the sensitivity of conventional PCR with an objective interpretation of the results. In the PCR-ELISA, a defined number of cells from cervical specimens were digested and amplified with concomitant digoxigenin labeling. Digoxigenin-labeled amplified products hybridized to a specific biotinylated probe were captured in streptavidin-coated microtiter wells by a biotin-streptavidin binding and were visualized by colorimetric immunoenzymatic reaction. PCR-ELISA was carried out in 110 cervical cytological specimens of women with or without the concomitant detectable presence of papillomavirus (HPV) DNA and the results were compared with those obtained by conventional PCR followed by dot blot hybridization. When compared to conventional PCR considered as reference standard, PCR-ELISA was found to be 98% sensitive and 96% specific. Out of the total 110 samples examined, 52.7% were positive for AAV DNA by both techniques, demonstrating a high prevalence of AAV infection in the uterine cervix. When analyzing samples with or without the presence of HPV DNA, 63.2 % of the samples were positive for HPV DNA and 41.5% of the samples were negative for HPV proved positive for AAV DNA by both PCR-ELISA and conventional PCR. Hence, PCR-ELISA, which can be completed in 1 day, proved to be a reliable method for an objective detection of AAV DNA in clinical samples. The present study showed a frequent infection of the cervical epithelium with AAV both in the presence and absence of HPV infection.

Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA.

AIMS: To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions. METHODS: One hundred and seventy six cytological specimens from women with different cervical lesions were investigated. For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Semiquantitative determinations were achieved in relation to an external reference titration curve. RESULTS: HPV DNA was detected in 60.2% of the samples. HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45. HPV-11 was not detected. HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease. Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease. CONCLUSIONS: Only HPV-16 load appears to be associated with the severity of cervical disease.

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples.

Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.