Polyomaviruses shedding in stool of patients with hematological disorders: detection analysis and study of the non-coding control region's genetic variability.

Fragmented data are available on the human polyomaviruses (HPyVs) prevalence in the gastrointestinal tract. Rearrangements in the non-coding control region (NCCR) of JCPyV and BKPyV have been extensively studied and correlated to clinical outcome; instead, little information is available for KIPyV, WUPyV and MCPyV NCCRs. To get insights into the role of HPyVs in the gastrointestinal tract, we investigated JCPyV, BKPyV, KIPyV, WUPyV and MCPyV distribution among hematological patients in concomitance with gastrointestinal symptoms. In addition, NCCRs and VP1 sequences were examined to characterize the strains circulating among the enrolled patients. DNA was extracted from 62 stool samples and qPCR was carried out to detect and quantify JCPyV, BKPyV, KIPyV, WUPyV and MCPyV genomes. Positive samples were subsequently amplified and sequenced for NCCR and VP1 regions. A phylogenetic tree was constructed aligning the obtained VP1 sequences to a set of reference sequences. qPCR revealed low viral loads for all HPyVs searched. Mono and co-infections were detected. A significant correlation was found between gastrointestinal complications and KIPyV infection. Archetype-like NCCRs were found for JCPyV and BKPyV, and a high degree of NCCRs stability was observed for KIPyV, WUPyV and MCPyV. Analysis of the VP1 sequences revealed a 99% identity with the VP1 reference sequences. The study adds important information on HPyVs prevalence and persistence in the gastrointestinal tract. Gastrointestinal signs were correlated with the presence of KIPyV, although definitive conclusions cannot be drawn. HPyVs NCCRs showed a high degree of sequence stability, suggesting that sequence rearrangements are rare in this anatomical site.

Rapid detection of carbapenemase-producing Klebsiella pneumoniae strains derived from blood cultures by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE), particularly carbapenemase-producing Klebsiella pneumoniae isolates, are important causative agents of nosocomial infections associated with significant mortality rates mostly in critical wards. The rapid detection and typing of these strains is critical either for surveillance purposes and to prevent outbreaks and optimize antibiotic therapy. In this study, the MALDI-TOF MS method was used to detect rapidly these isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains. RESULTS: Fifty-five K. pneumoniae strains were tested. Identification and carbapenemase-production detection assay using Ertapenem were performed both from bacterial pellets extracted directly from BCs flasks and from subcultures of these strains. For all isolates, a complete antimicrobial susceptibility testing and a genotypic characterization were performed. We found 100% agreement between the carbapenemase-producing profile generated by MALDI TOF MS and that obtained using conventional methods. The assay detected and discriminated different carbapenemase-producing K. pneumoniae isolates within 30 min to 3 h after incubation with Ertapenem. CONCLUSIONS: MALDI-TOF MS is a promising, rapid and economical method for the detection of carbapenemase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic workflow of clinical microbiology laboratories.

Microbiological Infections in Women With Cervical Cytological Reports of Atypical Squamous Cells of Undetermined Significance.

OBJECTIVE: To study the role of cervicovaginal infections in women with cytological reports of atypical squamous cells of undetermined significance (ASC-US). MATERIALS AND METHODS: The study included 220 women admitted to the Clinic of Microscopy, Cervicovaginal and Vulvar Pathology of the Department of Gynecology and Obstetrics of the Tor Vergata University Hospital, Rome, Italy, enrolled between October 2012 and July 2013. RESULTS: Among the enrolled women, 105 women (47.7%) had ASC-US cytology, whereas 115 women (52.3%) had negative cytology. Microscopy showed infections more frequently in women with ASC-US than in those with negative cytology: 70.5% (74/105) vs 36% (41/115); p < .001. Cocci were present in 73.3% (77/105) of the women with ASC-US and in 43.5% (50/115) of those with negative cytology; p < .001. According to Ison score, 84% (88/105) of ASC-US was grade 0 vs 22% (25/115) of negative cytology, p < .001. Human papillomavirus was detected in 35% of the women with ASC-US. A statistically significant correlation between high pH and vaginal infections was found in women aged 20 to 29 (p = .003) and those 50 years or older in both cytological report groups; p < .001. CONCLUSIONS: Cervicovaginal infections are associated with a cytological report of ASC-US. Direct microscopy of vaginal specimens allowing immediate evaluation of the vaginal microflora and infectious agents may be a useful tool in managing women with cytological reports of ASC-US.

Evaluation of BiesseBioscreen as a new methodology for bacteriuria screening.

Urinary tract infection is a common disease diagnosed from symptoms and clinical signs, and bacterial count per volume of urine. This study have evaluated the BiesseBioscreen analyzer as a new way to analyze urine samples en- abling fast screening of urine, prior to reference standard methods currently utilized in microbiology analysis labo- ratory. We analyzed 962 urine samples from outpatients and inpatients of the Tor Vergata (TV) University Hospital of the University of Rome "Tor Vergata". All samples were processed both with the BiesseBioscreen and with the standard methodology adopted by the clinical microbiology laboratory of TV Hospital and the results were com- pared. Of the samples analyzed 54.9% were concordant negative with the reference method and 21.6% concordant positive, 23.3% resulted false positive and 0.2% false negative. The results obtained from BiesseBioscreen showed a sensitivity of 99.0%, indicating it as a system suitable to rule out urinary tract infection. BiesseBioscreen could represent a valid method for screening negative samples to exclude from culture test with a potential reduction in time, workload and costs of the diagnosis.

A novel culturing system for fluid samples.

BACKGROUND: Large-volume culture methods for sterile body fluids employing automated blood-culture systems increase the recovery of microorganisms compared with traditional plate medium methods. However, in many instances a laboratory receives only small-volume samples. MATERIAL/METHODS: The URO-QUICK system (now HBandL), originally used to process urine samples, was evaluated for organism enrichment and determination of microbial count of fluid samples. Fluid specimens were also evaluated for their residual antimicrobial activity (RAA). The procedures were compared with results from a conventional culture procedure. The 546 samples included 106 endotracheal aspirate, 63 bronchoalveolar lavage, 139 sputum, 47 blood, 105 pleural fluid, 26 cerebrospinal fluids, and 41 peritoneal fluid samples as well as 19 other fluids including synovial fluid (n=5), ascitic fluid (n=9), fluids from the drainage of an infected central venous catheter (n=3), abdominal drainage fluid (n=1), and cholecystic fluid (n=1). RESULTS: The URO-QUICK system allowed the culture of an additional 44 samples (8%, p=0.007) compared with the traditional culturing method. The RAA test demonstrated good concordance with the reference method, showing specificity and positive predictive value of 100% for each, while the sensitivity and the negative predictive value were 67% and 76%, respectively. The microbial counts evaluated using the URO-QUICK system showed excellent agreement with traditional enumeration methods. CONCLUSIONS: The URO-QUICK system may well represent an excellent alternative to solid medium-based recovery and enumeration methods.

Proteomic investigation in A549 lung cell line stably infected by HPV16E6/E7 oncogenes.

BACKGROUND: Data have accumulated implicating the involvement of oncogenic human papillomaviruses (HPVs) in bronchial carcinogenesis. We recently described the presence of oncogenic HPV transcripts in non-small cell lung cancers. OBJECTIVE: To investigate the role of oncogenic HPVs in lung carcinogenesis. MATERIAL AND METHODS: The lung cell line A549 stably infected with HPV16E6, HPV16E7 and HPVE6/E7 constructs was used to investigate the protein profile changes associated with the expression of these oncogenes. Replicated two-dimensional gel electrophoresis gels from uninfected and stably HPV16E6-, E7-, and E6/E7-infected A549 cells were compared for changes in protein profile. Protein identification was achieved by peptide mass fingerprinting by MALDI-TOF-MS and nLC-ESI-Q-TOF-MS/MS peptide ladder sequencing. RESULTS: We identified 17 different polypeptides whose average normalized spot intensity was statistically significant (p < 0.05) and differed by 2-fold. Relationships between differentially expressed proteins and the HPV-induced infection mechanism have been clustered by knowledge-base database functional association network analysis. CONCLUSION: The impact of Hsp27, annexin III, annexin IV, Gp96 and TPT1 on the cellular response mechanism to HPV infection is presented and discussed.

Performance Evaluation of a New Culture Colorimetric Detection Assay.

OBJECTIVE: To evaluate the performance of the culture colorimetric detection assay MYCO WELL D-ONE® (MWD-ONE), designed to detect sexually transmitted infections using real-time polymerase chain reaction (PCR) as a reference method. MATERIALS AND METHODS: One hundred and ten urogenital samples were screened for Gardnerella vaginalis (GV), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma spp., and Ureaplasma urealyticum (UU)/Ureaplasma parvum (UP) using the MWD-ONE and real-time PCR assays Gardnerella vaginalis/Lactobacillus species Real-TM Quant and Anyplex II STI-7 Detection, respectively. RESULTS: GV was detected in 33 samples by both the MWD-ONE and real-time PCR, while 6 samples gave discordant results. TV was detected by both MWD-ONE and Anyplex II STI-7 Detection kits in 3 samples, while 107 were negative. MH was detected by both methods in 5 cases, 4 samples gave discordant results, and 101 were negative. Mycoplasma genitalium (MG) was detected by Anyplex II STI-7 in 2 cases, 1 of which was detected as Mycoplasma spp. by MWD-ONE. Ten samples were positive by MWD-ONE, and 98 were negative with both assays. With regard to UU/UP, 24 cases were detected by MWD-ONE and Anyplex PCR, 25 by PCR only, 4 by MWD-ONE, and 57 tested negative with both methods.The positive predictive values (PPV) and negative predictive values (NPV) of the MWD-ONE assay for the pathogens tested were as following: GV, PPV 94.3%, NPV 94.7%; TV, PPV and NPV 100%; MH, PPV 71.4%, NPV 98.1%; Mycoplasma spp., PPV 9.1%, NPV 98.9%; and Ureaplasma spp., PPV 85.7 %, NPV 69.5 %. The agreement between the MWD-ONE and PCR was strong for GV and MH (k=0.8 and 0.7, respectively); perfect for TV (k=1); and moderate for UU/UP (k=0.4). CONCLUSION: MWD-ONE assay appears to be suitable for routine testing of sexually transmitted infections.

Co-expression of HSV2 and Chlamydia trachomatis in HPV-positive cervical cancer and cervical intraepithelial neoplasia lesions is associated with aberrations in key intracellular pathways.

OBJECTIVE: Oncogenic human papillomaviruses (HPVs) are the etiological agents of cervical cancer. Different cofactors might be needed for malignant transformation, but they still remain elusive. METHODS: To delineate the role of Chlamydia trachomatis (CT) and herpes simplex virus type 2 (HSV2) in HPV-positive cervical intraepithelial neoplasia (CIN) lesions and cervical carcinoma a series of 149 cervical cancer and CIN biopsies were analyzed for CT and HSV2 DNA by PCR, and HPV genotyped by InnoLipa. Monitoring of aberrations in key intracellular pathways due to CT/HSV2 and HPV co-expression were analyzed with 13 biomarkers. RESULTS: Of the 149 samples tested, 136 were HPV DNA positive; 32/136 contained also CT DNA and 29 HSV2 DNA. Detection of CT was significantly (p = 0.0001) related to multiple-type HPV infections, while HSV2 was of borderline significance (p = 0.053). Of the 13 biomarkers tested, cytoplasmic and nuclear NF-kappaB and VEGF-C were significantly increased in CT+/HPV+ lesions; p = 0.023, p = 0.045, and p = 0.020 as well as survivin, p = 0.026. Survivin was the only marker that was overexpressed also in HSV2+/HPV+ lesions, p = 0.027. CONCLUSIONS: CT infection favors the entry and persistence of multiple HR-HPV types, which leads to viral integration, inhibition of apoptosis, overexpression of E6/E7 oncogenes and cell transformation.

Predicting high-risk human papillomavirus infection, progression of cervical intraepithelial neoplasia, and prognosis of cervical cancer with a panel of 13 biomarkers tested in multivariate modeling.

Comprehensive multivariate models were used to disclose whether any of our previously analyzed 13 markers would be independent predictors of intermediate end point markers in cervical carcinogenesis. The expression of the following biomarkers, E-cadherin, extracellular signal-regulated kinase 1, 67-kd laminin receptor (LR67), matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 2, nuclear factor-kappaB, nm23-H1, p16, proliferating cell nuclear antigen, survivin, human telomerase reverse transcriptase, topoisomerase 2alpha, and vascular endothelial growth factor (VEGF) C in 150 cervical cancer (CC) and 152 cervical intraepithelial neoplasia (CIN) lesions were determined immunohistochemically. Multivariate models were constructed to test predictive power of the markers for 3 outcomes: (1) high-grade CIN, (2) high-risk human papillomavirus (HR-HPV), and (3) CC survival. Performance indicators were calculated and compared by the areas under receiver operating characteristic (ROC) curve. Three marker panels were identified consisting of 5 independent predictors of CIN2 (E-cadherin, extracellular signal-regulated kinase 1, LR67, topoisomerase 2alpha, and VEGF-C), 3 predictors of HR-HPV (survivin, p16, and human telomerase reverse transcriptase), and 2 predictors of CC survival (nm23-H1 and tissue inhibitor of metalloproteinase 2). In predicting CIN2, the best balance between sensitivity (SE) and specificity (SP) was obtained by combining the 2 most powerful predictors in panel 1 (VEGF-C and LR67), giving the area under ROC curve, 0.897 (95% confidence interval [CI], 0.847-0.947); odds ratio, 86.27 (95% CI, 19.71-377.47); SE, 86.0%; SP, 93.3%; positive predictive value (PPV), 99.1%; and negative predictive value (NPV), 43.1%. In a hypothetical screening setting (10,000 women; CIN2 prevalence, 1%), this marker combination should theoretically detect CIN2 with 86.0% SE, 100% SP, 99.1% PPV, and 99.6% NPV, area under ROC curve of 0.930 (95% CI, 0.909-0.951), and odds ratio, 29998.0 (95% CI, 7,879.0-37,338.0). Combining 2 markers (LR67 and VEGF-C) enables accurate detection of high-grade CIN in a clinical setting. However, testing the performance of this marker combination in a screening setting necessitates their analysis in cytological samples.

Detection of oncogenic DNA viruses in colorectal cancer.

BACKGROUND: As a part of our search for oncogenic viruses as potential etiological agents in human malignancies, our studies on human papillomaviruses (HPV) were extended to analysis of the 3 polyomaviruses (SV40, BKV and JCV) in colorectal carcinomas. PATIENTS AND METHODS: Archival tumour samples from 71 patients with colorectal cancer were analyzed for the sequences of SV40, BKV, JCV and HPV using PCR-based techniques. HPV genotypes were determined using sequencing and reverse blot hybridization (InnoLipa). RESULTS: Amplification of BKV and JCV with the primer pair PEP-1 and PEP-2 and subsequent restriction digestion of the amplified products with BamH I disclosed BKV in 6/66 (9%) of the samples, whereas none contained JCV. SV40 was amplified in 10/66 (15.1%) samples and confirmed by sequencing analysis. In pair-wise analysis for co-infections, the samples were significantly different in their BKV-JCV and JCV-SV40 status, in contrast to their BKV-SV40 co-infection status. HPV DNA was detected in 22/66 (33.3%) of the samples analysed with either the MY09/11 or SPF primer mix. Of these 22 HPV infections, 7 were single-type infections and 15 contained multiple HPV types. HPV detection or type distribution showed no relationship to the gender of the patients or histological grade of the tumour. HPV status was not significantly related to detection of BKV, JCV or SV40. Similarly, in pair-wise analysis for co-infections, the samples were significantly different in their status of HPV-BKV (p=0.0006), HPV-JCV (p=0.0001), and HPV-SV40 (p=0.019), implicating that HPV and the 3 polyomaviruses are rarely detected concomitantly in the same samples. CONCLUSION: Taking the known molecular mechanisms of action of these individual viruses, there is a chance that these viruses could alter the mechanisms of cell cycle control and inhibit apoptosis, thus potentially causing chromosomal instability and promoting colorectal oncogenesis.

Acinetobacter baumannii in intensive care unit: a novel system to study clonal relationship among the isolates.

BACKGROUND: The nosocomial infections surveillance system must be strongly effective especially in highly critic areas, such as Intensive Care Units (ICU). These areas are frequently an epidemiological epicentre for transmission of multi-resistant pathogens, like Acinetobacter baumannii. As an epidemic outbreak occurs it is very important to confirm or exclude the genetic relationship among the isolates in a short time. There are several molecular typing systems used with this aim. The Repetitive sequence-based PCR (REP-PCR) has been recognized as an effective method and it was recently adapted to an automated format known as the DiversiLab system. METHODS: In the present study we have evaluated the combination of a newly introduced software package for the control of hospital infection (VIGI@ct) with the DiversiLab system. In order to evaluate the reliability of the DiversiLab its results were also compared with those obtained using f-AFLP. RESULTS: The combination of VIGI@ct and DiversiLab enabled an earlier identification of an A. baumannii epidemic cluster, through the confirmation of the genetic relationship among the isolates. This cluster regards 56 multi-drug-resistant A. baumannii isolates from several specimens collected from 13 different patients admitted to the ICU in a ten month period. The A. baumannii isolates were clonally related being their similarity included between 97 and 100%. The results of the DiversiLab were confirmed by f-AFLP analysis. CONCLUSION: The early identification of the outbreak has led to the prompt application of operative procedures and precautions to avoid the spread of pathogen. To date, 6 months after the last A. baumannii isolate, no other related case has been identified.

Rapid and cost-effective identification and antimicrobial susceptibility testing in patients with Gram-negative bacteremia directly from blood-culture fluid.

Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) in bacteremia cases or sepsis could improve patient prognosis. Thus, it is important to provide timely reports, which make it possible for clinicians to set up appropriate antibiotic therapy during the early stages of bloodstream infection (BSI). This study evaluates an in-house microbiological protocol for early ID as well as AST on Gram negative bacteria directly from positive monomicrobial and polymicrobial blood cultures (BCs). A total of 102 non-duplicated positive BCs from patients with Gram-negative bacteremia were tested. Both IDs and ASTs were performed from bacterial pellets extracted directly from BCs using our protocol, which was applied through the combined use of a MALDI-TOF MS and Vitek2 automated system. The results of our study showed a 100% agreement in bacterial ID and 98.25% categorical agreement in AST when compared to those obtained by routine conventional methods. We recorded only a 0.76% minor error (mE), 0.76% major error (ME) and a 0.20% very major error (VME). Moreover, the turnaround time (TAT) regarding the final AST report was significantly shortened (ΔTAT = 8-20 h, p < 0.00001). This in-house protocol is rapid, easy to perform and cost effective and could be successfully introduced into any clinical microbiology laboratory. A final same-day report of ID and AST improves patient management, by early and appropriate antimicrobial treatment and could potentially optimize antimicrobial stewardship programs.

Detection of oncogenic viruses SV40, BKV, JCV, HCMV, HPV and p53 codon 72 polymorphism in lung carcinoma.

As a part of our continuous search for oncogenic viruses in bronchial cancer, we extended our HPV studies to analyse also SV40, BKV, JCV and HCMV sequences in bronchial cancer and related these data with p53 codon 72 polymorphism. Fresh tumor samples from 78 patients with lung cancer were analysed for SV40, BKV, JCV, HCMV and HPV sequences by PCR. HPV genotypes were determined using reverse blot hybridization and sequencing, and all HPV-positive tumors were tested for the presence of E6/E7 transcripts by RT-PCR. All samples were analysed for p53 codon 72 polymorphism, using PCR-based RFLP method. Of the 78 cases studied, 11 (14.1%) were positive for T-Ag gene of SV40, while BKV and JCV sequences were both amplified in 1 tumor only. Altogether, 10/78 lesions were HPV-positive; six HPV16, one HPV31, two HPV6/53 and one HPV16/18. All HPV DNA-positive samples except one also expressed E6 and E7 transcripts. HCMV was amplified in 18 (23%) cases. RFLP analysis of p53 codon 72 revealed 32 homozygotes for arg/arg allele (50.8%), 26 heterozygotes for arg/pro allele (41.3%), and 5 homozygotes for pro/pro allele (7.9%). P53 codon 72 polymorphism was not significantly different between cases (n=63) and controls (n=50) (p=0.455), among virus positive and negative patients, nor was it related to HPV genotypes (p=0.384), expression of E6 (p=0.384) and E7 oncogenes (p=0.293). Of all possible combinations of virus co-detection, only SV40-HCMV association was statistically significant (OR=5.500, 95%CI 1.43-21.02; p=0.015). Taken the known mechanisms of these individual viruses, there is a chance that these viruses could affect cell cycle control and inhibit apoptosis, thus potentially causing genetic instability and promote oncogenesis.

Thymosin alpha 1: from bench to bedside.

After the initial dramatic effects, observed in a Lewis lung carcinoma animal model, using a combination of thymosin alpha 1 (Talpha1) and interferon (IFN) after cyclophosphamide, a number of other preclinical models in mice (Friend erythroleukemia and B16 melanoma) and in rats (DHD/K12 colorectal cancer liver metastasis) have confirmed the efficacy of the combination therapy with Talpha1 and either IFN or IL-2 plus chemotherapy. These results provided the scientific foundation for the first clinical trials using Talpha1 in combination with BRMs and/or chemotherapy. Pivotal trials in advanced non-small cell lung cancer (NSCLC) and melanoma with Talpha1 and IFN-alpha low doses after cis-platinum or dacarbazine produced the first evidence of the high potentiality of this approach in the treatment of human cancer. The combination of Talpha1 and IFN-alpha was also used in patients affected by chronic B and C hepatitis including IFN-nonresponders and infected by precore mutants or genotype 1b. Further studies demonstrated additional biological activities clarifying the mechanism of action of Talpha1, partially explaining the synergism with IFN. It has been shown the capacity of activating infected dendritic cells through Toll-like receptor signaling, thus influencing the inflammation balance, and of increasing the expression of tumor, viral, and major histocompatibility complex (MHC) I antigens. Dose-response studies suggested the possibility of improving the efficacy of this molecule reducing the overall toxic. Based on these information two clinical trials are ongoing: a large phase II on advanced melanoma patients treated with Talpha1 at different doses after dacarbazine and a phase III one, on IFN-resistant hepatitis C virus (HCV) patients treated with a triple combination (IFN, ribavirin, and Talpha1).

In vivo and in vitro studies support that a new splicing isoform of OLR1 gene is protective against acute myocardial infarction.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the "non-risk" disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

Human papillomavirus infections in lung cancer. Detection of E6 and E7 transcripts and review of the literature.

Lung cancer is the leading cause of cancer related death in Western countries. Several factors have been implicated in its aetiology: cigarette smoking, environmental pollution, asbestos and genetic factors. The possible involvement of human papillomavirus (HPV) in bronchial squamous cell lesions was first suggested in 1979 by Syrjänen. Since then, several studies have confirmed the presence of HPV DNA in about 20% of lung cancer cases examined, with HPV16 and 18 as the two most frequently found oncogenic viral types. More recently, these data have been supported by the detection of E6 and E7 transcripts in HPV-positive lung cancer cases, reinforcing the hypothesis that oncogenic HPVs could act as cofactors in bronchial carcinogenesis. This published literature is briefly reviewed and new data of the authors on detection of E6 and E7 transcripts in lung cancer samples are presented.

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens.

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.

Apoptotic cell signaling in lymphocytes from HIV+ patients during successful therapy.

The impact of antiretroviral therapy (ART) on immune-reconstitution and its relationship with the complex scenario of multiple cell signaling associated with apoptosis in HIV infection has not yet been fully elucidated. Here we report the results of the analysis of the expression of 13 genes involved in the apoptotic pathway, simultaneously detected by RNA-protection assay in peripheral blood mononuclear cells (PBMCs) of 12 HIV-1-infected responder patients before and during successful ART. In particular, we calculated the correlations among apoptosis and viral load (VL) levels versus the quantitative expression of genes associated with death receptors or to Bcl-2 pathways. Nonparametric bivariate Spearman's analysis of significant correlations showed that apoptosis was directly correlated with mRNA levels for caspase-8, FasL, and TRAIL. Conversely, apoptosis levels were inversely correlated with mRNA levels for Bcl-xl, Bcl-2, and Mcl-1, respectively. In addition, while VL was directly correlated with the expression of caspase 8, it was inversely correlated with mRNA levels for Bcl-2 and Mcl-1. These results, although worthy of further investigation, show that variations of apoptosis levels in PBMCs of HIV-1+ patients during ART are strictly related to the modulation of a complex network of signaling involving both death and survival of lymphocytes.

Single or multiple HPV types in cervical cancer and associated metastases.

Almost all cervical cancers are human papillomavirus (HPV)-positive. Some aspects of HPV carcinogenesis, such as factors involved in the transformation process and the mono- or polyclonal origin of the carcinogenic process, need to be defined. The latter aspect is addressed in our study. Cervical samples were collected from 102 patients with squamous cell carcinoma. The HPV positivity was established by PCR analysis performed using consensus and specific primers for the L1 and E6/E7 regions, respectively. Eighty-seven samples were positive for the L1 gene and 5 for the E6/E7 genes. Overall, 92 samples contained segments of HPV-DNA (90.2%). HPV-16 was most frequently found either alone or associated with other genotypes (63%). All genotypes identified as a single infection, except HPV-73, belonged to the high-risk HPV group. Among multiple infections, the HPV-31+54 couple was the most frequent. The presence of two genotypes in a primary tumor raises the question of their distribution in a single tumor cell. We attempted to answer this question by comparing the HPV patterns in primary tumors and metastases, considering that metastases derive from cell clones released from the primary tumor. The HPV patterns of primary tumors and metastases overlapped in most patients, even when primary tumors contained a double genotype, thus suggesting that single tumor cells may contain multiple HPV genotypes.

Detection and expression of human papillomavirus oncogenes in non-small cell lung cancer.

Human papillomavirus (HPV) has been found in lung cancer cases with variable frequency. In the present study, we analysed a series of 38 patients with non-small cell lung cancer (NSCLC) (21 paraffin-embedded archival samples and 17 fresh surgical specimens) for the presence of E6 and E7 oncogenes of HPV16, 18 and 31. Eight of the tumours were positive (21%): six HPV16, one HPV16+18, and one HPV31. The normal tissue surrounding the HPV-positive tumour was negative for the presence of the virus. Sequencing analysis of URR, of HPV16, which was the most frequently found HPV type in our cases, showed an adenosine deletion at nucleotide 7861 (E2-binding site) in four out of six patients. Sequencing of the entire E6 and E7 genes of HPV16 showed a T to G transition at nucleotide position 350 of E6, in all examined cases. This mutation is associated to the European variant of HPV16. Analysis of E6 and E7 transcripts was performed on the six fresh surgical specimens infected by HPV16. Our study showed that all of the tumours investigated, except one, contained E6 and E7 transcripts. Only in one case could we identify an unspliced form of the E6 transcript. Our results strengthen the relationship between HPV and NSCLC and support the hypothesis that HPV infection could play a role in bronchial carcinogenesis.