Genital ulcers caused by herpes simplex virus. / Úlceras genitales por virus herpes simplex.

Genital herpes is a sexually transmitted disease caused by herpes simplex virus type1 (HSV-1) and type2 (HSV-2) belonging to the alphaherpesvirus family, that includes the varicella zoster virus. HSV infection continues to be the most common cause of vulvar ulcers among the sexually active population. Its incidence increases every year. This review summarises the microbiology of the virus, pathogenesis and infection in genitalia, clinical manifestations and correct identification, the different laboratory diagnostic methods, and choice of the correct treatment according to the first infection, recurrence or special cases. Finally, the cost of routine herpes simplex virus infection is analysed.

Hepatitis C virus deep sequencing for sub-genotype identification in mixed infections: A real-life experience.

BACKGROUND: The effectiveness of the new generation of hepatitis C treatments named direct-acting antiviral agents (DAAs) depends on the genotype, subtype, and resistance-associated substitutions present in individual patients. The aim of this study was to evaluate a massive sequencing platform for the analysis of genotypes and subtypes of hepatitis C virus (HCV) in order to optimize therapy. METHODS: A total of 84 patients with hepatitis C were analyzed. The routine genotyping methodology for HCV used at the study institution (Versant HCV Assay, LiPA) was compared with a deep sequencing platform (454/GS-Junior and Illumina MiSeq). RESULTS: The mean viral load in these HCV patients was 6.89×106±7.02×105. Viral genotypes analyzed by LiPA were distributed as follows: 26% genotype 1a (22/84), 55% genotype 1b (46/84), 1% genotype 1 (1/84), 2.5% genotype 3 (2/84), 6% genotype 3a (5/84), 6% genotype 4a/c/d (5/84). When analyzed by deep sequencing, the samples were distributed as follows: 27% genotype 1a (23/84), 56% genotype 1b (47/84), 8% genotype 3a (7/84), 5% genotype 4d (4/84), 2.5% genotype 4f (2/84). Six of the 84 patients (7%) were infected with more than one subtype. Among these, 33% (2/6) failed DAA-based triple therapy. CONCLUSIONS: The detection of mixed infection could explain some treatment failures. Accurate determination of viral genotypes and subtypes would allow optimal patient management and improve the effectiveness of DAA therapy.

Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients.

Testing for Candida albicans germ-tube antibody IFA IgG assay (CAGTA) is used to detect invasive candidiasis infection. However, most suitable assays lack automation and rapid single-sample testing. The CAGTA assay was adapted in an automatic monotest system (invasive candidiasis [CAGTA] VirClia® IgG monotest (VirClia®), a chemiluminescence assay with ready-to-use reagents that provides a rapid objective result. CAGTA assay was compared with the monotest automatic VirClia® assay in order to establish the diagnostic reliability, accuracy, and usefulness of this method. A prospective study with 361 samples from 179 non-neutropenic critically ill adults patients was conducted, including 21 patients with candidemia, 18 with intra-abdominal candidiasis, 84 with Candida spp. colonization, and 56 with culture-negative samples, as well as samples from ten healthy subjects. Overall agreement between the two assays (CAGTA and VirCLIA) was 85.3%. These assays were compared with the gold-standard method to determine the sensitivity, specificity as well as positive and negative predictive values. In patients with candidemia, values for CAGTA and VirCLIA assays were 76.2 versus 85.7%, 80.3 versus 75.8%, 55.2 versus 52.9%, and 91.4 versus 94.3%, respectively. The corresponding values in patients with intra-abdominal candidiasis were 61.1 versus 66.7%, 80.3 versus 75.8%, 45.8 versus 42.9%, and 88.3 versus 89.3%, respectively. No differences were found according to the species of Candida isolated in culture, except for Candida albicans and C. parapsilosis, for which VirClia® was better than CAGTA. According to these results, the automated VirClia® assay was a reliable, rapid, and very easy to perform technique as tool for the diagnosis invasive candidiasis.

Evaluation of a dilution method for non-evaluable results in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the Cobas 4800 platform. / Valoración de un método de dilución para la resolución de resultados no valorables en la detección de Chlamydia trachomatis y Neisseria gonorrhoeae con la plataforma cobas 4800.

INTRODUCTION: A variable percentage of samples analysed using the Cobas 4800 assay can give an invalid result by PCR inhibition or erroneous due to incorrect DNA extraction with the Cobas 4800 CT/NG test. METHOD: An analysis was performed using the vortex agitation and dilution protocol on the original sample (swab or urine) for a total of 116 samples. In order to analyse the sensitivity of this method, 100 samples (swabs and urine) with known results were retested. RESULTS: A total of 98.3% (114/116) of the samples analysed were resolved with this protocol with 100% agreement after reviewing clinical data, Gram stain, and other samples analysed in parallel from the same patient. DISCUSSION: The data indicate no loss of sensitivity with this protocol; thus Cobas 4800 users could use this method without the need for alternative methods.

Clinical and epidemiological characterisation of lymphogranuloma venereum in southwest Spain, 2013-2015.

OBJECTIVES: Lymphogranuloma venereum (LGV) infections caused by Chlamydia trachomatis L serovars have emerged in 2003 in Europe among HIV-positive men having sex with men (MSM). Our aim was to evaluate LGV prevalence and predictors in a high-risk population attending to two STI clinics in the southwest of Spain between December 2013 and April 2015. METHODS: Screening of C. trachomatis using commercial kits was carried out, followed by real-time pmpH-PCR discriminating LGV strains, and finally ompA gene was sequenced for phylogenetic reconstruction. RESULTS: A total of 6398 samples were tested, of which, 594 (9.3%) were C. trachomatis-positive specimens and successfully typed by pmpH PCR. Five hundred and eighty-one samples contained non-LGV and 13 (2.2%; 95% CI 1.3% to 3.7%) samples had LGV. One hundred and sixty-six (27.9%; 95% CI 24.5% to 31.7%) CT-positive results were found in MSM. All C. trachomatis LGV types were found in rectal samples from MSM (13/166, 7.8%; 95% CI 4.5% to 13.0%). Of these, five (38.5%; 95% CI 17.7% to 64.5%) patients were asymptomatic and 11 (84.6%; 95% CI 57.8% to 95.7%; p<0.001) were also HIV positive. Successful treatment of LGV was achieved in all patients including 11/13 (84.6%) who received single-dose azithromycin. All of the L types were confirmed to be genotype L2b with ompA PCR and sequencing. CONCLUSIONS: This analysis shows that LGV infections are occurring in MSM in southwest Spain, where no data about LGV have been described before, reinforcing the need for screening and genotyping for LGV. LGV should be taken into account when considering treatment and management of rectal C. trachomatis infections, including in asymptomatic HIV-positive MSM. Larger studies on appropriate treatment for asymptomatic LGV infection are needed.

Performance of the HSV OligoGen kit for the diagnosis of herpes simplex virus type 1 and 2.

PCR methods are nowadays between the most rapid and sensitive methods for screening and diagnosing herpes simplex virus (HSV) type 1 and 2. The aim of this study was to analyze the reliability, accuracy, and usefulness of the new assay HSV OligoGen kit in comparison with the Roche LightCycler HSV ½ Qual Kit assay for the detection of HSV in clinical samples. For this analysis, a prospective study was designed for detection of HSV-1 and HSV-2 including 110 ulcer specimens, 48 urine, 48 endocervical, 43 cerebral spinal fluids, 4 urethral and 3 pharyngeal swabs that were sent from a regional STI clinic or an Intensive Clinical Unit, both in Seville, Spain. In comparison to the Roche LightCycler HSV ½ Qual Kit assay, sensitivity, specificity, positive and negative predicative values, and kappa value for HSV detection using the HSV OligoGen kit were 96.2%, 100%, 100%, 98.3%, and 0.97 for HSV-1, respectively. For HSV-2, the corresponding values were 98.3%, 100%, 100%, 99.5%, and 0.98, respectively. Statistical data obtained in this study confirms the usefulness and reliable results of this new assay.

Comparison of the CT OligoGen kit with cobas 4800 assay for detection of Chlamydia trachomatis.

INTRODUCTION: A prospective study was designed to assess the performance of the new CT OligoGen kit and the cobas 4800 assay for detection of Chlamydia trachomatis. METHODS: A set of samples that included urine samples (n=212), endocervical (n=167), rectal (n=53), pharyngeal (n=7) and urethral swabs (n=3). The samples were sent from a regional sexually transmitted diseases (STD) clinic in Seville, Spain, and were collected from 261 men and 181 women. Discordant results were re-analyzed and clinical data and other tests were reviewed in order to resolve them. RESULTS: Sensitivity, specificity, positive predicative value (PPV), negative predictive value (NPV) and kappa value for C. trachomatis detection using the CT OligoGen kit were 98.5%, 100%, 100%, 95.4% and 0.97, respectively. CONCLUSIONS: This new kit had a high sensitivity, specificity, PPV and NPV for C. trachomatis, therefore the performance profile confirms the usefulness and reliable results of this new assay.

High prevalence of antibodies against hepatitis E virus in HIV-infected patients with unexplained liver disease.

OBJECTIVE: To look for evidence of hepatitis E virus (HEV) exposure in HIV-infected patients with unexplained elevations of liver stiffness (LS). METHODS: Case-control study conducted in 31 HIV-infected patients with unexplained elevations of LS and in 31 HIV-controls with normal LS, matched by age, sex and CD4 cell-counts. Serum HEV antibodies were tested by two ELISA procedures and by Immunoblot. We defined exposure to HEV as the detection of serum HEV antibodies by at least one of the two ELISA assays, provided that it was confirmed by Immunoblot. A real-time PCR RNA assay was conducted in all plasma samples to identify subjects with active HEV infection. RESULTS: Exposure to HEV was demonstrated, according to the criteria used in this study, in 9 (29%) of the cases, whereas it was shown in 5 (16%) of the controls (p=.3). Serum HEV RNA was detected in none of the controls and in only in one case. This patient had a documented chronic hepatitis E with progression to cirrhosis. CONCLUSIONS: HEV antibodies are frequently found in HIV-infected patients with unexplained liver disease.

Hepatitis C virus genotype 4 in Southern and Central Spain does not originate from recent foreign migration waves.

Hepatitis C virus genotype 4 (HCV-4) is highly prevalent in Spain, but the information on the molecular characterization of HCV-4 in this region is scarce. Due to this, the molecular characteristics and the evolution of HCV-4 infection in Seville were analyzed (Southern Spain) and compared them with samples from Madrid. HCV genotype was determined by LIPA 2.0 assay and confirmed by sequence analysis of NS5B. Phylogenetic tree was estimated by MEGA 5.10. Bayesian coalescent-based methods were used to estimate the substitution rate and the age of the most recent common ancestor (MRCA). In the phylogenetic analysis of 50 NS5B HCV-4 from Seville and 11 from Madrid, 2 clusters were distinguished: The first cluster (HCV-4a) included 48% of the sequences from Seville and 9% of sequences from Madrid. The second cluster included the remaining sequences belonging to HCV-4d. The mean estimated substitution rate was 2.39 × 10(-3) for HCV-4a and 1.81 × 10(-3) for HCV-4d for Seville and 2.32 × 10(-3) for HCV-4d from Madrid. The date for MRCA was estimated to be around 1981-1984 for HCV-4 from Seville. The dates for MRCA were dated before the recent flow of immigration in Spain. Therefore, the results presented in this study argues against the possibility of a foreign introduction of the HCV-4 from other regions with high prevalence, at least during the last two, decades in which there was a great flow of immigrants. Additionally, an unusual high prevalence of subtype 4a was observed in Seville.

Interobserver concordance in controlled attenuation parameter measurement, a novel tool for the assessment of hepatic steatosis on the basis of transient elastography.

BACKGROUND: The combination of transient elastometry with a controlled attenuation parameter (CAP) is available to evaluate hepatic steatosis (HS) along with liver stiffness. AIMS: To assess the concordance of CAP measurements between two independent observers in patients infected by HIV and/or hepatitis virus, as well as to determine the concordance of classification of the grade of HS using two cut-off values. MATERIALS AND METHODS: In a cross-sectional prospective study, CAP-enabled transient elastometry acquisitions were performed by two independent observers in patients with HIV or hepatitis virus infection. The interobserver concordance between the CAP examinations was assessed using the intraclass correlation coefficient and the concordance in the classification of patients in the grades of HS was characterized using the κ index. RESULTS: A total of 118 patients were included. Twenty (17%) patients were HIV monoinfected, 44 (37.3%) were hepatitis C virus monoinfected, and 52 (44%) had HIV/hepatitis C virus coinfection. The median (Q1-Q3) of the absolute difference of CAP values between the two observers was 20 (10-41) dB/m. The overall intraclass correlation coefficient was 0.84 (95% confidence interval: 0.77-0.88). The corresponding figures for liver stiffness measurements were 0.9 (0.4-2.6) kPa and 0.96 (95% confidence interval: 0.94-0.97). The κ indexes for the concordance of classification for the presence of HS, cut-off of 215 dB/m, and significant HS, cut-off of 252 dB/m, were 0.53 and 0.62, respectively. CONCLUSION: The determination of HS by means of CAP in HIV and/or hepatitis virus infection represents an observer-independent and easily performable method. However, the use of cut-off values for the classification of patients is suboptimal.

Evaluation of the cobas 4800 CT/NG Test for detecting Chlamydia trachomatis and Neisseria gonorrhoeae DNA in urogenital swabs and urine specimens.

We have evaluated 696 samples (488 swabs and 208 urine specimens) with the cobas 4800 (c4800) CT/NG Test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae DNA in swab and urine specimens. c4800 results were compared with those obtained from COBAS AMPLICOR (CAM) CT/NG Test. Discordant results were reanalyzed with the MultiNA system and compared with clinical data. For C. trachomatis detection by both methods, we obtained 93.8%, 100%, 100%, and 99.1% for sensitivity, specificity, and positive and negative predictive values, respectively. For urine specimens analyzed in c4800, our results were 96.6%, 100%, 100%, and 99.4%, respectively. For N. gonorrhoeae detection, swab results were:88.0%, 100%, 100%, and 99.4%. For urine specimen, results obtained were 100%, 100%, 100%, and 100%. Reanalyses were all concordant between both methods. c4800 results were comparable with those obtained with the CAM system. We had an excellent correlation between swab and urine specimens analyzed by c4800.

HIV-coinfection leads to a modest increase in plasma HCV-RNA load in patients with chronic HCV infection.

The influence of HIV coinfection on plasma hepatitis C virus (HCV) RNA load has not been reliably evaluated. We analyzed plasma HCV RNA load in 396 HCV-monoinfected and 467 HIV/HCV-coinfected patients. Median HCV RNA concentrations (interquartile range) in HCV-monoinfected patients were 5.88 (5.3-6.2) log(10)IU/mL versus 5.96 (5.6-6.5) log(10)IU/mL in HIV/HCV-coinfected individuals (p=0.033) as determined with the Cobas Amplicor Test and 6.06 (5.4-5.7) log(10)IU/mL versus 6.3 (5.5-6.9) log(10)IU/mL (p=0.026) using the Cobas TaqMan System. The plasma HCV RNA load in patients with HIV infection and undetectable plasmatic HIV RNA was similar to that observed in HCV-monoinfected individuals [6.02 (5.45-6.61) log(10)IU/mL versus 6.01 (5.36-6.59) log(10)IU/mL, respectively (p=1.0)]. In conclusion, HIV coinfection tends to be associated with higher plasma HCV RNA load, however, the magnitude of the differences is small and this effect can be counterbalanced with antiviral therapy.

No influence of antiretroviral therapy on the mutation rate of the HCV NS5B polymerase in HIV/HCV-coinfected patients.

OBJECTIVES: To assess the impact of antiretroviral treatment (ART), including nucleoside analogues retrotranscriptase inhibitors (NRTIs), on the mutation rate of hepatitis C virus (HCV) NS5B polymerase and on the ratio of substitution at synonymous and nonsynonymous sites (dN/dS) this polymerase in HIV/HCV-coinfected patients. PATIENTS AND METHODS: Sixty-one patients on defined ART were included in this study. The NS5B polymerase of HCV was sequenced at baseline and after at least two years of ART. The mutation rate and the dN/dS were calculated at both times. RESULTS: The NS5B gene from forty-nine (80.3%) patients including; 19 HCV-1a (38.8%), 13 HCV-1b (26.5%), 8 HCV-3a (16.3%) and 9 HCV-4d (18.4%), could be sequenced. Thirty-two (65.3%) patients received non-nucleoside analogues and 41 (83.7%) received protease inhibitor. The mean estimated substitution rates at baseline and at the end of follow-up were from 1.38 to 3.5×10(-3)substitution/site/year (s/s/y) and from 1.39 to 3.18×10(-3)s/s/y, respectively, varying according to HCV genotype. All HCV genotypes at baseline and the end time point had values of dN/dS <1. At the end of follow-up, most of sites experienced negative selection and positive selection occurred only in a few sites. CONCLUSION: The mutation rate of NS5B in HIV/HCV-coinfected patients is within the range previously reported in studies in HCV-monoinfected patients. Additionally, the use of ART, including NRTIs, in these patients does not affect neither mutation rate nor the dN/dS of the HCV NS5B protein, suggesting that its use would not generate new resistance mutants to the polymerase inhibitors of HCV.