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.

Comparison of urine and cervical samples for detecting human papillomavirus (HPV) with the Cobas 4800 HPV test.

BACKGROUND: Human papillomavirus (HPV) is the main cause of cervical cancer. The development of non-invasive self-sample collection methods would have the potential advantage of increasing the acceptance of the screening procedures. OBJECTIVES: To compare human papillomavirus (HPV) DNA detection and genotyping with the Cobas 4800 HPV test (Roche Diagnostic, Spain) on paired cervical and first voided urine. STUDY DESIGN: Paired urine and cervical samples were collected from 125 women referred for evaluation of abnormal Pap smear results. RESULTS: The overall percent agreement between HPV detection in urine and cervical samples was 88%. A substantial concordance rate of HPV DNA detection in both samples was observed (κ=0.76; 95% IC: 64-87). In this high prevalence population the sensitivity, specificity, NPV and PPV for detection of HPV DNA from urine versus cervical samples were 90.5% (95% IC: 80-95%), 85%, (95% IC: 74-92%), 89.8% (95% IC: 79.5-95.3) and 86.4% (95% IC: 76.1-92.7) respectively. Compared to histologically confirmed CIN 2/3 disease, the clinical sensitivity and specificity for the detection of high-risk HPV in urine samples were 95% (95% IC: 76-97%) and 52.4% (95% IC: 40-64%) respectively. CONCLUSIONS: These results suggest that urine samples processed with Cobas 4800 HPV test may be useful for clinical management of HPV infection.

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.

Low prevalence of occult HBV infection among HIV-infected patients in southern Spain

Introduction: The aim of this study was to assess the prevalence of occult HBV infection in HIV-positive patients in a centre in Southern Spain. Methods: The HBV serological markers were investigated in all the patients and the presence of HBV-DNA was tested by PCR in patients with isolated anti-HBc. Results: An isolated anti-HBc pattern was detected in 144/520 (27.7%) patients. HBV-DNA was detected in one of these patients (0.7%).Conclusions: In Southern Spain, there is a low prevalence of occult HBV infection among HIV-infected patients, despite increasing immigration from endemic countries (AU)

Introducción: El objetivo de este estudio fue evaluar la prevalencia de infección oculta por VHB en pacientes infectados por VIH del sur de España. Métodos: Se investigaron los marcadores serológicos para VHB y se analizó la presencia de ADN-VHB en los anti-HBc "solo". Resultados: El patrón anti-HBc "solo" fue detectado en 144/520 (27,7%) pacientes. El ADN-VHB fue detectado en un paciente (0,7%).Conclusiones: La prevalencia de infección oculta por VHB es baja entre los pacientes infectados por VIH a pesar del aumento en la inmigración desde países endémicos (AU)

Low prevalence of occult HBV infection among HIV-infected patients in Southern Spain.

INTRODUCTION: The aim of this study was to assess the prevalence of occult HBV infection in HIV-positive patients in a centre in Southern Spain. METHODS: The HBV serological markers were investigated in all the patients and the presence of HBV-DNA was tested by PCR in patients with isolated anti-HBc. RESULTS: An isolated anti-HBc pattern was detected in 144/520 (27.7%) patients. HBV-DNA was detected in one of these patients (0.7%). CONCLUSIONS: In Southern Spain, there is a low prevalence of occult HBV infection among HIV-infected patients, despite increasing immigration from endemic countries.

Molecular diagnosis of Aspergillus fumigatus endocarditis.

A 66-year-old male with ischaemic cardiomyopathy and chronic lymphocytic leukemia developed signs of severe systemic inflammatory response syndrome. Serial blood cultures were negative and a SeptiFast test detected the presence of Aspergillus fumigatus DNA. Afterwards, detection of galactomannan and 1,3-ß-D-glucan showed a positive result. Autopsy revealed the presence of branched fungal structures suggestive of Aspergillus.

Prediction of response to pegylated interferon plus ribavirin by IL28B gene variation in patients coinfected with HIV and hepatitis C virus.

BACKGROUND: Variation in the IL28B gene is associated with sustained virologic response (SVR) to pegylated interferon plus ribavirin in hepatitis C virus (HCV)-monoinfected patients with genotype 1. Data on other genotypes and on patients coinfected with human immunodeficiency virus (HIV) and HCV are more limited. We aimed to assess the predictive ability of variations in the single-nucleotide polymorphism rs12979860 for SVR in HIV/HCV-coinfected patients, regardless of HCV genotype. METHODS: The rs12979860 genotype was determined by polymerase chain reaction in 154 patients who had received therapy against HCV with pegylated interferon plus ribavirin. RESULTS: rs12979860 genotype was TT in 20 patients (13%), TC in 66 patients (43%), and CC in 68 patients (44%). Rates of SVR in patients with genotype CC and in those with genotype TC or TT, according to HCV genotype, were, respectively, 50% and 17% (P < .001) in patients with genotype 1, 80% and 25% (P = .027) in patients with genotype 4, and 93% and 77% (P = .115) in patients with genotype 3. The median (interquartile range) low-density lipoprotein cholesterol level in patients with rs12979860 CC was 89 mg/dL (73-120 mg/dL) versus 75 mg/dL (55-91 mg/dL) (P = .001) in those with TC or TT. Independent predictors of SVR were HCV genotype 2-3 (odds ratio [OR], 13.98; 95% confidence interval [CI], 4.87-40.1; P < .001), rs12979860 CC (OR, 5.05; 95% CI, 2.04-12.5; P < .001), baseline plasma HCV RNA load of < or =600,000 IU/mL (OR, 1.99; 95% CI, 1.18- 3.34; P = .009), and female sex (OR, 4.28; 95% CI, 1.08-16.96; P = .039). CONCLUSIONS: IL28B gene variations independently predict SVR in HIV/HCV-coinfected patients with HCV genotype 1 and non-genotype 1 HCV infection. The association between rs12979860 and plasma low-density lipoprotein cholesterol suggests that the system low-density lipoprotein ligand/receptor might be involved in the effect of this genotype.

Impact of observer experience on the reproducibility of transient elastometry in HIV/HCV co-infected patients.

BACKGROUND: Data on the influence of observer experience on transient elastometry (TE) in hepatitis C virus (HCV) infection are scarce, and there are no data on HCV/HIV co-infected patients. METHODS: TE determination was conducted by an experienced and an inexperienced observer in 93 patients who were divided into three groups according to the chronological order of their attendance. The interobserver variability of TE results was analyzed by the intraclass correlation index (ICC) and the interobserver agreement of the classification of significant fibrosis and cirrhosis by the kappa index. RESULTS: The overall ICC was 0.970 (95% confidence interval [CI] 0.954-0.980). For groups 1, 2, and 3, the ICCs were 0.952 (95% CI 0.901-0.977), 0.966 (95% CI 0.931-0.984), and 0.986 (95% CI 0.971-0.993), respectively. The kappa index for the classification of cirrhosis (cutoff value >or=14.6 kPa) for the three groups was 0.53, 1.00, and 0.91. The use of two cutoff points to exclude (<6 kPa) or diagnose significant fibrosis (>9 kPa) yielded a kappa index of 0.72, 1.00, and 1.00, respectively. CONCLUSIONS: In HIV/HCV co-infected patients, the concordance between an inexperienced and an experienced observer is acceptable after a short instruction period. To maximize this concordance, a training period of 60 measurements should be recommended.

Transient rebounds of HIV plasma viremia are associated with the emergence of drug resistance mutations in patients on highly active antiretroviral therapy.

BACKGROUND: There are very few and contradictory data about the consequences of 'blips', transient rebounds of HIV viremia. We assessed the emergence of new drug resistance mutations during blips among HIV-infected patients on HAART from a cohort in which we found no association between blips and virological failure. METHODS: Seventeen patients with blips were selected from 330 patients under HAART for over 48 weeks according to these criteria: (1) presence of only one blip, viremia < or =1000copies/ml preceded by two consecutive visits and followed by one visit showing undetectable viremia; (2) therapy adherence > or =95%; (3) availability of frozen sera. RESULTS: HIV RNA could be extracted and amplified from five patients. In another two patients only the protease region could be amplified. Drug mutations in either the retrotranscriptase or protease genes, not detected at baseline, were observed in six patients at the blip. None of the patients showed detectable viremia during a median (range) follow-up after the blip of 120 (36-156) weeks. CONCLUSIONS: Transient rebounds of viremia in patients on HAART are associated with the emergence of new drug resistant HIV variants. In spite of it, virological failure is not observed after a median follow-up of over 2 years.

Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR.

Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram's stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.