RNA extraction method is crucial for human papillomavirus E6/E7 oncogenes detection.

BACKGROUND: Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients' lesions and progression. METHODS: This study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method. RESULTS: E6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%). CONCLUSIONS: Extraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.

Assessment of human papillomavirus E6/E7 oncogene expression as cervical disease biomarker.

BACKGROUND: The aims of this study were to detect HPV E6/E7 mRNA expression in women with high-risk genotypes (HPV-16, -18, -31, -33 and -45) analysing its relationship with tissue pathology and 2) 2-year follow-up of E6/E7 mRNA tested group. METHODS: Our samples were genotyped and classified by pathologists according to Bethesda system. After RNA extraction, E6/E7 oncogene mRNA detection was performed by NucliSens® EasyQ® HPV v1 Test (bioMérieux). RESULTS: The results of the present study showed that E6/E7 mRNA positivity rate was 68.29 % in women tested once and 69.56 % in women tested twice. According to tissue pathology, all samples with high-grade lesions were positive for mRNA. Among women with low-grade lesions varied over the years from 89.28 to 84 % in women tested once and from 77.77 to 70 % in tested twice. Among women without lesion, positivity rate maintained in women tested once (from 50 to 41.38 %) and decreased in tested twice, from 63.63 to 44.44 %. Regarding lesion evolution, mRNA positivity was higher in women with lesion progression (53.13 %) and in women with positive results in two tested samples (83.33 %). CONCLUSION: HPV E6/E7 mRNA detection may be an effective screening test and biomarker for cervical cancer in women infected with these five genotypes. Nonetheless, further studies are needed to standardize as routine triage test.

Distribution of genital human papillomavirus genotypes in benign clinical manifestations among men from Northern Spain.

BACKGROUND: In the literature, data on the prevalence of human papillomavirus (HPV) infection in men vary significantly and the exact distribution of specific genotypes is still unclear. As infections usually occur without symptoms, men might only attend their hospital clinic when they have a specific concern, being in most cases genital warts (condylomas), which are often caused by low-risk HPV genotypes. The aim of this study was to assess HPV genotype distribution and prevalence among men attending hospital for HPV-associated conditions and to evaluate infection-associated factors. METHODS: Samples from men with clinical manifestations of HPV-related infections seen during 2007-2012 at the Clinical Microbiology and Infectious Control Department at Basurto University Hospital were genotyped using Linear Array HPV Genotyping Test kit (Roche Molecular Diagnostics, Germany). Data on probable risk factors were collected and investigated for possible association. RESULTS: Of 184 anogenital samples, 138 (75 %) were tested as positive for HPV; 57 (41.3 %) single HPV infections and 81 (58.7 %) multiple infections. Only 45.6 % of HPV-positive samples presented low-risk genotypes 6 or 11, whereas 71/138 (51.4 %) had at least one oncogenic (high-risk) genotype. Oncogenic genotypes and multiple HPV infections were both associated with a higher number of lifetime sexual partners and their incidence appeared to increase with patient age. CONCLUSIONS: Although it is accepted that HPV 6 and 11 genotypes are main causes of condylomas, our findings show a high incidence of multiple infections and high-risk genotypes in men with benign HPV manifestations. The fact that the condyloma is a skin lesion facilitates the entry of virus into cells and thus cancer progression; therefore, monitoring for HPV is important, especially in those patients with high-risk genotypes (regardless of whether they cause condyloma).

Human Papillomavirus 16 Variants May Be Identified by E6 Gene Analysis.

AIMS: The aims of the study were (1) to characterize the genetic variability of human papillomavirus (HPV) genotype 16 in the E6 region when this genotype is present in multiple infection samples, (2) to assess the prevalence of variants in our region and (3) to analyze the relationship between variants, patients' ages and pathology. METHODS: The Clinical Microbiology and Infection Control Department analyzed samples which were positive for genotype 16 and other genotypes from 2007 to 2013. Variants were assigned to European, Euro-German, Asian, Asian-American or African lineage by sequence analysis. The relationship among variants, age and different types of lesion was studied. RESULTS: In HPV-16 sequence analysis, the European variant was detected in 85.10% of samples, the Asian-American in 7.80%, the African in 4.25% and the Euro-German in 2.83% of specimens. Sequence genetic variability showed 16 nucleotide substitutions. Moreover, non-European variants were mainly found in old women and in isolates from high-grade squamous intraepithelial lesions since European variants were mainly detected in negative cytologies. CONCLUSION: Multiple infections may take effect on nucleotide substitution and the appearance of recombinant samples. Single gene analysis makes it impossible to detect recombination which has a great influence on drug response and vaccine strategies. Thus, E6 gene analysis would be enough to identify HPV-16 intratypic variants but not to confirm the results.

Comparison of INNO-LIPA and TRUGENE assays for genotyping and drug-resistance mutations in chronic hepatitis B virus infection.

OBJECTIVE: Hepatitis B virus (HBV) genotyping and detection of resistance to drugs have become essential in epidemiological and clinical diagnosis. Our main objective was to determine the prevalence of HBV genotypes and drug-resistance mutations in chronic asymptomatic carriers and chronic HBV sufferers comparing 2 detection assays. METHODS: Serum samples from 28 chronic HBV patients and 22 chronic asymptomatic carriers were analyzed. For HBV genotyping, the INNO-LIPA and TRUGENE™ HBV genotyping kits were evaluated. For drug-resistance mutations, INNO-LIPA DR v2 and INNO-LIPA DR v3 prototype and the TRUGENE™ HBV genotyping kit were evaluated. RESULTS: In HBV genotyping, concordant results were 98% and both assays were able to detect more than one genotype. Different genotypes were detected, the most prevalent being D (46%) and A (26%). In relation to drug-resistance mutations, the sensitivity of the line probe assay was lower than TRUGENE because INNO-LIPA could not detect two mutations (S202G and V214A). CONCLUSIONS: Both assays are easy and suitable for detecting HBV genotype and drug-resistance mutations and for routine laboratory use. However, TRUGENE presented better sensitivity in both analyses and it is possible to conduct both on the same sample. This assay is also able to detect primary and secondary mutations.

Ultrafast detection of ß-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of ß-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for ß-lactamase resistance genes. Multilocus sequence typing and ß-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant ß-lactamase resistance genes in less than 1 h. Multilocus sequence typing and ß-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

Klebsiella pneumoniae is a bacterium that can cause infections in the blood. These infections can be severe, especially if K. pneumoniae is not susceptible to antibiotics ('antibiotic resistant'). Tools that can detect this resistance are important. In this study, we tested one such tool called MinION™ with blood samples. In 1 h, we were able to identify the bacteria within the sample and their resistance. This type of testing would help clinicians to give the best treatment to patients. More studies are needed to prove the usefulness of MinION for processing samples from real patients.

Ten Issues for Updating in Community-Acquired Pneumonia: An Expert Review.

Community-acquired pneumonia represents the third-highest cause of mortality in industrialized countries and the first due to infection. Although guidelines for the approach to this infection model are widely implemented in international health schemes, information continually emerges that generates controversy or requires updating its management. This paper reviews the most important issues in the approach to this process, such as an aetiologic update using new molecular platforms or imaging techniques, including the diagnostic stewardship in different clinical settings. It also reviews both the Intensive Care Unit admission criteria and those of clinical stability to discharge. An update in antibiotic, in oxygen, or steroidal therapy is presented. It also analyzes the management out-of-hospital in CAP requiring hospitalization, the main factors for readmission, and an approach to therapeutic failure or rescue. Finally, the main strategies for prevention and vaccination in both immunocompetent and immunocompromised hosts are reviewed.

Human papillomavirus (HPV) genotype 18 variants in patients with clinical manifestations of HPV related infections in Bilbao, Spain.

BACKGROUND: Human papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential. OBJECTIVES: We aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion. METHODS: From 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied. RESULTS: Genetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions. CONCLUSIONS: Multiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies.Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly.

Evolution of hepatitis B virus during long-term therapy in patients with chronic hepatitis B.

BACKGROUND: Long-term lamivudine (LAM), adefovir (ADV) and entecavir (ETV) treatment induce the emergence of drug-resistant hepatitis B virus (HBV) in patients with chronic hepatitis B infection. AIM: To evaluate the LAM, ADV and ETV resistance mutations detected in our patient group. MATERIALS AND METHODS: Twenty patients who had received at least two years of treatment with nucleoside/tide analogues were enrolled in this study. Patients with detectable HBV DNA were analyzed in order to detect resistance mutations and in this group of patients treatment was change. RESULTS: Three patients developed LAM resistance mutations (2 presented rtM204I and one rtL180M+rtM204V/I) and one patient showed rtN236T ADV resistance mutation. During ADV and LAM treatment, one patient developed ADV plus LAM resistance mutations (rtI163V+rtL180M+rtA181V+rtN236T), in this case, HBV strains harbouring polymerase mutations did not develop LAM associated rtM204V/I primary mutation. In addition, ETV resistance mutations (rtL180M+rtT184A+rtS202G+rtM204V) were detected in one patient. CONCLUSIONS: These findings suggest that monotherapy resulted in a limited virological response and combination strategies including potent antiviral agents should be recommended for patients with resistant mutations.

Ultra-fast direct method for identifying microorganisms from BACTEC lytic/10 anaerobic/F flasks.

Background: Fast diagnosis of bloodstream infections remains the most important challenge for clinical microbiologists. The introduction of the mass-spectrometry represents a breakthrough, although several methods are already commonly used for the direct identification from positive blood cultures we present a faster method (ultra fast) for Lytic anaerobic flasks. Methods: We compare the ultra-fast (UF) method with the extensively employed differential centrifugation method (DC) and both to routine identification after 18-24 h of incubation. UF and DC method correlation rates to the gold standard were calculated, and statistical significance was proved with the Z test. Results: UF performed better overall than DC, with this difference being statistically significant. This tendency was observed in every subanalysis.

GEHEP 010 study: Prevalence and distribution of hepatitis B virus genotypes in Spain (2000-2016).

OBJECTIVE: To study the prevalence and distribution of HBV genotypes in Spain for the period 2000-2016. METHODS: Retrospective study recruiting 2559 patients from 17 hospitals. Distribution of HBV genotypes, as well as sex, age, geographical origin, mode of transmission, HDV-, HIV- and/or HCV-coinfection, and treatment were recorded. RESULTS: 1924 chronically HBV native Spanish patients have been recruited. Median age was 54 years (IQR: 41-62), 69.6% male, 6.3% HIV-coinfected, 3.1% were HCV-coinfected, 1.7% HDV-co/superinfected. Genotype distribution was: 55.9% D, 33.5% A, 5.6% F, 0.8% G, and 1.9% other genotypes (E, B, H and C). HBV genotype A was closely associated with male sex, sexual transmission, and HIV-coinfection. In contrast, HBV genotype D was associated with female sex and vertical transmission. Different patterns of genotype distribution and diversity were found between different geographical regions. In addition, HBV epidemiological patterns are evolving in Spain, mainly because of immigration. Finally, similar overall rates of treatment success across all HBV genotypes were found. CONCLUSIONS: We present here the most recent data on molecular epidemiology of HBV in Spain (GEHEP010 Study). This study confirms that the HBV genotype distribution in Spain varies based on age, sex, origin, HIV-coinfection, geographical regions and epidemiological groups.

Monitoring of therapy in patients with chronic hepatitis B virus.

OBJECTIVE: The aims of this study were to evaluate therapy with lamivudine (LAM) and adefovir dipivoxil (ADV) monotherapy in chronic hepatitis B virus (HBV)-infected patients with frequent measurements of DNA levels, to characterize HBV genotypes, and to determine the emergence of nucleos(t)ide analogue mutants before and during the therapy by direct-sequencing the reverse transcriptase region and by INNO-LiPA HBV DR v3. MATERIALS AND METHODS: A total of 15 chronic HBV patients were analysed: 11 were treated with ADV and four were treated with LAM. RESULTS: Viral genotype was determined, showing the presence of genotype D (73%) in 11 patients and genotype A (27%) in four patients. In the viral response to treatment, three patients developed substitutions at rtM204I associated with LAM resistance and one of these patients presented rtM204V/I plus rtL180M mutation. In contrast, of the 11 patients treated with ADV, three patients developed mutations (rtN236T; rtA181V; rtA181V plus rtN236T). With regard to this case, the same results were observed by INNO-LiPA HBV DR v3 and direct sequencing, but by direct sequencing we detected an extra mutation rtQ215S that was present in two patients: one patient who was on treatment with LAM had an rtQ215S mutation in addition to an rtM204I, and the second patient treated with ADV had rtA181V. CONCLUSION: Direct sequence analysis is an essential tool to optimize therapeutic management of HBV chronic infection in clinical practice to choose the appropriate nucleos(t)ide analogues and to detect extra mutations that are not included in the commercial kit.

HGV/GB virus C transmission by blood components in patients undergoing open-heart surgery.

BACKGROUND: To establish the rate of HGV/GB virus C (GBV-C) transmission by blood components in open-heart surgery patients. STUDY DESIGN AND METHODS: From 55 patients receiving blood components, sera were collected before and 2, 4, 6, 8, 10, 12, 16, 20, 26, and 32 weeks after heart surgery. Serum samples from patients and implicated blood donations were tested for HGV/GBV-C RNA by PCR. Recipients of RNA-positive blood components were also tested for the presence of E2 antibodies (E2Ab) by ELISA. RESULTS: Of 55 recipients, 18 received RNA-positive blood components. Of 14 recipients of RNA-positive blood components, who were negative for RNA or E2Ab before transfusion, 8 became RNA positive and one developed E2Ab after transfusion. Three recipients of RNA-positive blood components had E2Ab before transfusion, and none of these became RNA positive after transfusion. One of 18 recipients was RNA positive before and after transfusion. Of 55 recipients, 37 received RNA-negative blood components: 34 were RNA negative before and after transfusion. Of 37 recipients, 3 were RNA positive before and after transfusion. CONCLUSION: Of susceptible patients, 64 percent became infected with HGV/GVC-C when transfused with RNA-positive blood components. E2Ab-positive patients were protected against HGV/GBV-C infection.