HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Rapid virological response as a predictor of sustained response in HCV-infected patients with persistently normal alanine aminotransferase levels: A multicenter study.

Rapid virological response (RVR) is now considered the strongest predictor of sustained virological response (SVR) in patients with HCV undergoing antiviral treatment, and thus, shorter antiviral treatment for these patients has been suggested. However, no data exist on the predictive value of RVR in HCV carriers with normal ALT values. A total of 137 patients with persistently normal ALT treated with peginterferon alfa 2a and ribavirin were studied. Fifteen patients dropped out early because of side effects, and in 10 patients with HCV-1 treatment was discontinued because of lack of early virological response (EVR). RVR was observed in 68% of the patients (42% patients with HCV-1, 90% HCV-2 and 64% HCV-3). An end-of-treatment response was observed in 86% of the patients (68% HCV-1, 100% HCV-2 and 91% HCV-3). SVR was maintained in 91 patients (46% HCV-1, 97% HCV-2 and 82% HCV-3). Overall, 92% patients with rapid response did obtain HCV eradication vs only 38% of those without rapid response. HCV-1 patients with baseline HCV RNA <400×10(3) IU/mL were more likely to achieve RVR and SVR than those with higher HCV RNA levels. We conclude that patients with genotype 1 and normal ALT who achieve HCV RNA negativity at week 4 may have a higher probability of eradicating their infection. Because of the concomitant favourable demographic and virological features often found in this particular subset of patients, the duration of therapy in these people might be shortened in the case of RVR. Persistently normal alanine aminotransferase levels patients with genotype 2 or 3 have a high chance of achieving SVR, so retesting of HCV RNA during treatment may have no additional practical value in these subjects.

Management of chronic hepatitis in drug addicts: a systematic review.

Injection drug users constitute the largest group of person at high risk for acquiring chronic hepatitis C, B and Delta. In particular viral, host and environmental factors all seem to favour rapid spread of these infections among drugs addicts. Host factors include behaviours that expose individuals to hepatitis virus such as the shared use of drug preparation, injection equipment and not protected sexual relationship with other drugs users. While in some clinical studies adherence to treatment regimens was often poor and to treat chronic hepatitis in injection drug users was stated as futile, in other controlled clinical studies adherence and sustained biological response to antiviral treatment was slightly lower or similar to that reported in other groups of patients. In this review we describe the epidemiology, diagnosis and treatment of chronic hepatitis C, B and Delta in intravenous drug users.

One-step nested PCR for detection of 2 LTR circles in PBMCs of HIV-1 infected patients with no detectable plasma HIV RNA.

A highly sensitive nested PCR was carried out in order to detect 2 LTR circles as a marker of recent and ongoing viral replication in HIV-1 infected patients with HIV plasma RNA undetectable. This "in house" two-step nested PCR is very sensitive, but it is not feasible for routine tests and presents a high risk of contamination. In order to reduce the time of reactions and crossover contamination, the possibility was explored to carry out a single step nested PCR, in which the two successive amplification rounds are carried out in the same tube. This single step nested PCR has the same sensitivity of the two-step nested, is easy to conduct and requires a short time of reaction. The two different PCR methods were compared and the clinical use of monitoring 2 LTR DNA circles in HIV-1 infected patients with undetectable plasma viral load is discussed.

Pulmonary tuberculosis in HIV-infected patients presenting with normal chest radiograph and negative sputum smear.

BACKGROUND: HIV-infected patients with pulmonary tuberculosis exhibit atypical radiological presentation and negative sputum smear more frequently than their HIV-negative counterparts. PATIENTS AND METHODS: We performed a retrospective study based on a chart review of 146 HIV-infected patients with pulmonary symptoms and culture-proven pulmonary tuberculosis. We compared clinical characteristics and the outcome in 71 patients (49%) with positive sputum smear (SS+), 62 patients (42%) with negative sputum smear/abnormal chest X-ray (SS-/CXR+) and 13 patients (9%) with negative sputum smear/normal chest X-ray (SS-/CXR-). Patients were enrolled from January 1987 to December 1998, and were followed up until December 1999. RESULTS: On hospital admission the three groups of patients examined did not differ significantly in demographic characteristics, degree of immunosuppression or Mycobacterium tuberculosis drug-susceptibility pattern. SS-/CXR- patients were significantly Less LikeLy to present with prolonged fever and dyspnea. Median survival was shorter for SS-/CXR- patients (6.4 months vs 20.2 and 18.8 months in the other two groups). In multivariate analysis, SS-/CXR-patients had a significantly increased risk of death (hazard ratio 3.0, 95% confidence interval, 1.4 to 6.4, p = 0.004) compared to SS+ patients. This increase in risk was no longer statistically significant when initiation of antituberculous therapy within 8 weeks from the collection date of the first specimen yielding M. tuberculosis was included in the multivariate model. CONCLUSION: Decreased survival was observed in HIV-infected patients with pulmonary tuberculosis and with both negative sputum smear and normaL chest X-ray presentation. This may primarily be a resuLt of delayed tuberculosis diagnosis and initiation of antituberculous therapy. The latter delay may also lead to a faster progression of HIV infection in SS-/CXR patients, in whom diagnostic oversight may be common.

Long-term efficacy of interferon alpha-2b and lamivudine in combination compared to lamivudine monotherapy in patients with chronic hepatitis B. An Italian multicenter, randomized trial.

BACKGROUND/AIMS: To evaluate the therapeutic efficacy of interferon alpha-2b and lamivudine in combination compared to lamivudine monotherapy in patients with chronic hepatitis B. METHODS: One hundred and fifty-one patients were randomly assigned to receive either recombinant interferon alpha-2b (nine million units three times per week) and lamivudine (100 mg/daily per os) for 24 weeks or lamivudine alone (100 mg/daily per os) for 52 weeks. Patients were followed up for a further 48 weeks. RESULTS: Sustained HBeAg seroconversion with undetectable serum levels of HBV DNA was observed in 25 of 76 patients (33%) treated with the combination therapy and in 11 of 75 patients (15%) treated with monotherapy (P=0.014). Histological improvement defined as a reduction of at least two points in the inflammation score as compared with pretreatment score was observed in 35 of 76 patients (46%) treated with combination therapy and in 20 of 75 patients (27%) treated with monotherapy (P=0.021). Both therapeutic regimens were well tolerated. CONCLUSIONS: Six-month treatment with interferon alpha-2b and lamivudine in combination appeared to increase the rate of sustained HBeAg seroconversion compared to 1-year lamivudine monotherapy. However, the potential benefit of combining lamivudine and interferon should be investigated further in studies with different regimens of combination therapy.

Endocarditis caused by Aspergillus species in injection drug users.

Aspergillus endocarditis is a rare event; a very few cases have been reported in injection drug users (IDUs) and patients infected with human immunodeficiency virus (HIV). We report 2 proven cases and 1 highly suggestive case of Aspergillus endocarditis in IDUs, 2 of whom were infected with HIV, and discuss some related clinical and pathogenic aspects.

Primary pulmonary hypertension in HIV patients: a systematic review.

The relationship between grade of pulmonary hypertension and factors associated with human immunodeficiency virus among patients with HIV infection is poorly documented. This report documents the most extensive attempt made thus far to determine whether a relationship exists between degree of pulmonary hypertension and the following conditions: HIV risk factor, degree of immunosuppression, presence or absence of AIDS, and presence or absence of liver cirrhosis. A retrospective study involving a search of the published literature on primary pulmonary hypertension among HIV cases from 1987 to 1998, using the Medline and Aidsline databases was conducted. Patients for whom secondary causes of pulmonary hypertension could be excluded were selected, and the following information for each was recorded: age, gender, risk factors for HIV infection, HIV disease stage according to the Centers for Disease Control, previous opportunistic and neoplastic diseases, CD4+ cell count (cells/L), presence or absence of liver cirrhosis, pulmonary systolic artery pressure level, and lung pathology specimens. Information about the patient's survival time was also recorded. Seventy-six patients were judged to have primary pulmonary hypertension and were included in the study. While no correlation was found between pulmonary systolic artery pressure level and CD4+ cell counts, a statistically significant difference was found between HIV-positive patients with and without AIDS as determined by the Centers for Disease Control criteria with regard to the degree of pulmonary hypertension, expressed as pulmonary systolic artery pressure level (85.4 +/- 17 mm Hg vs 71.8 +/- 15 mm Hg, p < 0.013). Although a higher PAPS was present in HIV cirrhotic patients, a statistically significant difference was not found between degree of pulmonary hypertension and evidence of hepatic cirrhosis (85 +/- 21 mm Hg vs 73.1 +/- 15 mm Hg, p < 0.062). Patients with AIDS and primary pulmonary hypertension present a higher degree of pulmonary hypertension than non-AIDS patients. Pulmonary hypertension associated with HIV seems to be related to a cytokine-related stimulation and proliferation of endothelium. High levels of cytokines present in AIDS patients can favor pulmonary hypertension, but the role of a host response to HIV--determined by one or more HLA subtypes--is suspected to enhance high cytokine production levels.

Clinical manifestation of HIV-related pulmonary hypertension.

In recent years, much more thought has been given to the pathogenic role of HIV and to the clinical manifestations of HIV-related pulmonary hypertension (HRPH), which currently represents one of the most severe events during HIV disease. HRPH occurs in early and late stages of HIV infection and does not seem to be related to the degree of immune deficiency. Many of the symptoms in HRPH result from right ventricular dysfunction: the first clinical manifestation is effort intolerance and exertional dyspnea that will progress to the point of breathlessness at rest. The diagnosis of HRPH can be made only after all etiologies for pulmonary hypertension have been excluded. Echocardiography has been proven to be an extremely useful tool for diagnosing HRPH, and Doppler echocardiography can be used to estimate systolic pulmonary artery pressure and to monitor the effects of therapy. Assessment of hemodynamic measures by catheterization remains, however, the best test for evaluating response to therapy. Cardiac catheterization is mandatory to characterize the disease and exclude an underlying cardiac shunt as etiology. Vasodilators have been extensively used in the treatment of pulmonary hypertension, since vasoconstriction is a determinant characteristic of this disease. However, HRPH remains a progressive disease for which treatment is often unsatisfactory and there is no cure. As new, more efficient antiretroviral treatment are introduced, clinicians should expect to encounter an increasing number of cases of pulmonary hypertension in HIV+ patients in the future.

Pathogenesis of HIV-related pulmonary hypertension.

Human immunodeficiency virus (HIV)-related pulmonary hypertension (HRPR) is a cardiovascular complication of HIV infection that has been recognized in the last years with increasing frequency. The etiology of HRPH is unknown. All the attempts to isolate HIV on pulmonary vessels in HRPH patients failed, and an indirect role for HIV in this disease has been hypothesized. Current theories on the pathogenesis focus on abnormalities of endothelial and smooth muscle cells of pulmonary vasculature. Endothelial and smooth muscle cell injury could be due to a high production or to a reduced clearance of cytokines in these patients. In fact, in several studies high levels of ET-1, IL-1alpha, IL-6 and PDGF in primary pulmonary hypertension (PPH) and in HRPH have been found. HIV gp 120 could induce the production of these cytokines by a stimulation of monocytes/macrophages. A high alpha1-adrenoreceptors stimulation of pulmonary vessels could be also implicated in the pathogenesis of HRPH. Chronic hypoxia is observed with increased frequency in HIV patients, and this could induce a chronic stimulation of alpha1-receptors of pulmonary vasculature with typical pathological changes. However, only a small percentage of HIV- patients develop HRPH. This observation suggests the existence of an idiosyncratic susceptibility to the development of vascular disease. This susceptibility could have a genetic basis, and might be determined by particular major histocompatibility complex alleles.

Evaluation of long-term efficacy of interferon alpha-2b and ribavirin in combination in naive patients with chronic hepatitis C: an Italian multicenter experience. Ribavirin-Interferon in Chronic Hepatitis Italian Group Investigators.

BACKGROUND/AIMS: A combination of interferon alpha and ribavirin has been suggested to reach a higher rate of sustained virological response in patients with chronic hepatitis C than monotherapy. In this study we assessed the long-term efficacy of this combination therapy in the treatment of selected Italian naive chronic hepatitis C patients compared to interferon alpha monotherapy. METHODS: We enrolled 428 naive patients who were randomly assigned to receive either recombinant interferon alpha-2b and ribavirin for 24 weeks or interferon alpha-2b alone for 48 weeks. The primary end-point of the study was the rate of sustained virological response. Serum HCV RNA levels were determined before treatment; during treatment at weeks 12 and 24 in the patients receiving the combination therapy; at weeks 12, 24, 36 and 48 in the patients receiving monotherapy; and after therapy at weeks 12, 24 and 48 in the patients in both study groups. RESULTS: Sustained virological response was observed in 43% of the patients treated with combination therapy and in 14% of the patients treated with monotherapy. Logistic regression analysis showed that sustained response was associated with the combination therapy, with HCV genotype other than 1b, with an HCV viral load of 3x10(6) copies/ml or less, with an inflammation score of 7 or less, and with an estimated duration of disease of 10 years or less. CONCLUSIONS: A 24-week treatment course with interferon alpha-2b and ribavirin offers a greater chance of sustained virological response compared to treatment with interferon alpha-2b alone for 48 weeks, and may be indicated as initial therapy in such patients.

Clinical course of cardiomyopathy in HIV-infected patients with or without encephalopathy related to the myocardial expression of tumour necrosis factor-alpha and nitric oxide synthase.

OBJECTIVE: To define whether the development of encephalopathy influences the clinical course of HIV-associated cardiomyopathy (HIV-DCM) in relation to the myocardial expression of tumour necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). DESIGN: Prospective study. SETTING: University hospitals and AIDS centres. METHODS: 115 HIV-infected patients with echocardiographic diagnosis of HIV-associated cardiomyopathy (34 with encephalopathy and 81 without encephalopathy) were followed for a mean of 24 +/- 3.2 months. All patients underwent endomyocardial biopsy for determination of myocardial immunostaining intensity of TNF-alpha and iNOS. Cerebrospinal fluid (CSF) from patients with encephalopathy was examined for the presence of viruses. Patients underwent clinical examination every 3 months and echocardiographic examination every 6 months. The intensity of TNF-alpha and iNOS immunostaining was also evaluated on postmortem cerebral tissue of patients who died of congestive heart failure (CHF). RESULTS: A greater impairment of echocardiographic parameters was observed in patients with HIV-associated cardiomyopathy after development of encephalopathy. These parameters tended to worsen progressively during the follow-up period and were inversely correlated with HIV-1 viral load, CD4 cell count, mini mental status score and the intensity of myocardial and cerebral TNF-alpha and iNOS staining. CSF specimens were available in 29 patients with encephalopathy. HIV-1 sequences were detected in CSF of all these patients with cytomegalovirus sequences in two. The mortality rate for CHF was greater among patients with encephalopathy (73% versus 12%). CONCLUSIONS: The development of encephalopathy has an adverse effect on the clinical course of HIV-associated cardiomyopathy. In the relationship between cardiomyopathy and encephalopathy, the activation of iNOS by TNF-alpha may have a significant pathogenetic role in HIV disease.

Intravenous recombinant interferon-beta versus interferon-alpha-2b and ribavirin in combination for short-term treatment of chronic hepatitis C patients not responding to interferon-alpha. Multicenter Interferon Beta Italian Group Investigators.

BACKGROUND: Little is known about the therapeutic role of intravenous interferon-beta in chronic hepatitis C patients unresponsive to a previous treatment with interferon-alpha. METHODS: Two hundred interferon-alpha non-responders were randomized to receive either intravenous recombinant interferon-beta or interferon-alpha-2b and ribavirin for 12 weeks. The responders in both groups were followed up for a further 48 weeks. RESULTS: At week 12 a biochemical and virologic response was documented in 42% of the patients treated with interferon-beta and in 22% of the patients treated with combination therapy. A sustained response was observed in 21% of the patients treated with interferon-beta and in 13% of those treated with combination therapy, with similar differences on intention-to-treat analysis. CONCLUSIONS: Short-term treatment with intravenous interferon-beta seems to offer a chance for sustained response in a subset of interferon-alpha non-responders. The role of long-term therapy in these patients still remains to be explored.

Intensity of myocardial expression of inducible nitric oxide synthase influences the clinical course of human immunodeficiency virus-associated cardiomyopathy. Gruppo Italiano per lo Studio Cardiologico dei pazienti affetti da AIDS (GISCA).

BACKGROUND: Increased levels of tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) have been reported in patients with dilated cardiomyopathy. We investigated the myocardial expression of TNF-alpha and iNOS in patients with HIV-associated cardiomyopathy (HIV-DCM) compared with patients with idiopathic dilated cardiomyopathy (IDCM). METHODS AND RESULTS: Endomyocardial biopsy specimens from 82 HIV-DCM and 80 IDCM patients were processed for determination of the immunostaining intensity of TNF-alpha and iNOS and for virological examination. Negative controls were derived from autopsy myocardium specimens from 32 HIV-negative patients without known heart disease. The mortality rate for congestive heart failure between groups according to the intensity of iNOS staining was also evaluated. The mean intensity of both TNF-alpha and iNOS staining was greater in patients with HIV-DCM (0.81 and 1.007, respectively) than in patients with IDCM (0.44 and 0.49, respectively) and controls (0.025 and 0.027, respectively). The staining intensity of both TNF-alpha and iNOS was inversely correlated with CD4 count. The staining intensity of iNOS was greater in HIV-DCM patients with HIV/coxsackievirus B3 (CVB3) or with HIV/cytomegalovirus coinfection than in IDCM patients showing infection with CVB3 and adenovirus alone. The staining intensity of iNOS correlated to mortality rate, because it was higher in HIV-DCM patients and, in particular, in those with an optical density unit >1. CONCLUSIONS: Cytokine activation seems to play a significant pathogenetic role in both HIV-DCM and IDCM. In HIV-DCM patients, the state of immunodeficiency may favor the selection of viral variants of increased pathogenicity, influencing the clinical course of cardiomyopathy by enhancement of the inflammatory process.

Influence of thrombolytic therapy on inferior acute myocardial infarction with concomitant anterior ST segment depression.

The purposes of this study were to analyze the prognostic significance of precordial ST segment depression and to determine whether thrombolytic therapy is effective for all patients with inferior acute myocardial infarction (AMI) or whether there is a different effectiveness for patients with concomitant anterior ST segment depression persisting for 24 hours or longer. Medical charts of 176 patients were studied. On the basis of ECG the patients were subclassified into three groups according to the presence, persistence, or absence of significant ST segment depression: Group 1: anterior ST segment depression persisting for less than 24 hours (45.4%); Group 2: anterior ST segment depression persisting for more than 24 hours (17.6%); Group 3: no anterior ST segment depression (37%). Age, Killip class, peak creatine kinase, hospital deaths, left ventricular ejection fraction, regional wall motion score, postinfarction angina, and ventricular/supraventricular arrhythmia of all patients were studied. Parameters of the three groups were compared: worse results were found in group 1 and the worst in group 2. This result is independent of thrombolytic therapy. Finally, the same parameters of thrombolyzed and nonthrombolyzed groups were compared: no statistically significant difference was observed. Among thrombolyzed patients the number of those with ST depression lasting more than 24 hours is lower than in nonthrombolyzed patients. It can be assumed that thrombolytic therapy in inferior AMI determines a shifting of patients from a worse prognosis group (ST segment depression persisting for more than 24 hours) to a better prognosis group (ST segment depression persisting for less than 24 hours).