Eligibility and feasibility of the treatment of chronic hepatitis C in a Cohort of Italian HIV-positive patients at a single HIV reference center.

Chronic hepatitis C is frequent and aggressive among HIV-positive patients; evaluation for anti-hepatitis C virus (HCV)-specific therapy is mandatory, but it has many limitations, due to efficacy, tolerability but also applicability. The objective of our retrospective analysis was to evaluate the eligibility and feasibility of anti-HCV therapy in HIV/HCV-coinfected patients followed at the II Department of Infectious Diseases, L. Sacco Hospital, Milan, Italy, from 2000 to March 2010. In our database, 545 HIV/HCV-coinfected patients were present, representing 40% of our whole HIV population, and 421 included in the analysis. One hundred twenty-four patients were excluded because of loss to follow-up (81) or deceased (43). Forty-eight patients spontaneously cleared HCV during follow-up (11%). Ninety-nine patients received anti-HCV therapy (26%), while the majority was excluded for several reasons (mainly concomitant diseases and low CD4(+) cell count). Globally, we found that in at least one third of untreated patients modifiable barriers to treatment were present. The access to therapy was significantly associated with the absence of history of intravenous drug use (p=0.01), a higher CD4(+) cells count at nadir (p=0.01), the presence of more than 6 HAART regimens (p=0.04), higher alanine aminotransferase (ALT) levels (p<0.0001), HCV genotype 2 or 3 (p=0.005). In a multivariate analysis, the same factors remained significantly associated with anti-HCV therapy. In conclusion, the feasibility of anti-HCV therapy in HIV/HCV-coinfected patients, in our highly specialized center, is approximately 26%. Relative contraindications, such as substance abuses, mild and controlled concomitant conditions, and low compliance are common and modifiable in order to reconsider patients as suitable for therapy.

Predictability of sustained virological response to pegylated interferon alpha-2b Plus ribavirin therapy by week-8 viral response in HIV-positive patients with chronic hepatitis C virus infection.

Chronic hepatitis C is frequent and aggressive in HIV-positive patients. Identification of early predictors of response to anti-HCV therapy is needed for a lower rate of response and higher discontinuations, compared to HCV mono-infected subjects. The aim of our study was to evaluate the predictive value of virological response (VR) at week 4-8-12 of Pegylated interferon alpha-2b (PEG-IFN) plus ribavirin (RBV) on sustained virological response (SVR) in HIV-HCV co-infected patients. 100 patients were treated with PEG-IFN (1.5 mcg/Kg/w) plus RBV (> or =10.6 mg/kg/d) and randomized for 24-48 or 48-72 weeks, respectively for genotype 2-3 and 1-4, in case of response (HCV-RNA PCR negativity) at the end of standard therapy (24 weeks for genotype 2-3, 48 weeks for genotype 1-4). Transcription-Mediated Amplification (TMA) assay for HCV-RNA was also applied. 27 patients reached end-of-treatment response (9 genotype 1-4, 18 genotype 2-3), 21 achieved SVR (8 genotype 1-4, 13 genotype 2-3). 35 patients dropped, 15 due to side-effects. SVR was statistically related to lower baseline HCV-RNA and to VR at week 4-8-12, with PPV 64%, 53% and 58%, and NPV 81%, 96% and 88%, respectively. In 27 patients, TMA was performed and confirmed standard PCR, except in two cases of relapse, who were PCR negative but TMA positive at week-12. In conclusion, VR at week 8 showed the highest NPV on SVR (96%). The study of viral kinetics requires further investigations in HIV-positive patients to guarantee a cost-effective therapy and to guide individually the duration of treatment. In this setting, TMA might be useful.

Risk factors for fibrosis progression in HIV/HCV coinfected patients from a retrospective analysis of liver biopsies in 1985-2002.

OBJECTIVES: To identify predictive factors for moderate/severe liver fibrosis and to analyse fibrosis progression in paired liver biopsies from HIV-positive patients with chronic hepatitis C virus (HCV) infection. METHODS: HIV/HCV coinfected patients followed at the 2nd Department of Infectious Diseases of L. Sacco Hospital in Milan, Italy, with at least one liver biopsy specimen were retrospectively evaluated. RESULTS: A total of 110 patients were enrolled in the study. In a univariate analysis, predictive factors of Ishak-Knodell stage > or =3 were a history of alcohol abuse [odds ratio (OR) 3.6, P=0.004], alanine aminotransferase level >100 IU/L at biopsy (OR 2.4, P=0.05), necro-inflammatory grade > or =9 (OR 37.14, P<0.0001) and CD4 count <350 cells/microL at nadir (OR 5.3, P=0.05). In a multivariate analysis, age >35 years (OR 3.19, P=0.04) and alcohol abuse (OR 4.36, P=0.002) remained independently associated with Ishak-Knodell stage. Paired liver biopsies were available in 36 patients; 18 showed an increase of at least one stage in the subsequent liver biopsy. Either in a univariate or in a multivariate analysis, a decrease of CD4 cell count of more than 10% between two biopsies (OR 6.85, P=0.002) was significantly associated with liver fibrosis progression. CONCLUSION: Our findings highlight the relevance of encouraging a withdrawal of alcohol consumption in people with chronic HCV infection and of carrying out close follow-up of patients, especially if they are more than 35 years old. It is therefore mandatory to evaluate HIV/HCV coinfected patients for anti-HCV treatment and to increase CD4 cell count through antiretroviral therapy in order to reduce the risk of fibrosis progression and to slow the evolution of liver disease.

Pneumocystis carinii diagnosis: an update.

From 1994 to date we have been using the internal transcribed spacers (ITSs) nested polymerase chain reaction (PCR) to investigate the possibility of diagnosing Pneumocystis carinii pneumonia on non-invasive samples collected from HIV-positive patients with pulmonary involvement. The objectives were: (1) to test the sensitivity, specificity and prognostic value of PCR in diagnosis and follow up of PCP; (2) to investigate the eventual occurrence and role of asymptomatic carriers of P. carinii; (3) to evaluate the prognostic significance of blood PCR positivity versus respiratory samples; (4) to verify the occurrence of exogenous infections or endogenous reactivations in cases of recurrent P. carinii pneumonia; and (5) to study the possible correlation between P. carinii genotype identified and capability of blood dissemination, prior prophylactic treatments, clinical parameters and outcome of the patients.

In vitro activity of human immunodeficiency virus protease inhibitors against Pneumocystis carinii.

Since 1996, the introduction of protease inhibitors (PIs) has led to a dramatic decrease of human immunodeficiency virus-related Pneumocystis carinii pneumonia. This effect is clearly due, in large part, to the induction of immune reconstitution by highly active antiretroviral therapy (HAART). However, it is conceivable that PIs had other beneficial effects, including direct activity against Pneumocystis. In this study, the occurrence of specific aspartyl proteases in Pneumocystis is described. These protease targets seemed to be affected in vitro by antiretroviral PIs. These data suggest intriguing implications for the possible antipneumocystis benefit of receiving indinavir, ritonavir, nelfinavir, or saquinavir during HAART.

Detection of rat Pneumocystis carinii proteinases and elastase and antipneumocystis activity of proteinase inhibitors in vitro.

Proteinases play an important role in survival of microorganisms and in pathogenicity of diseases. By using a modified SDS-gelatin-polyacrylamide gel system, proteinases of rat-P.carinii were detected as bands of proteolytic digestion after electrophoresis. P.carinii organisms obtained from dexamethasone immunosuppressed transtracheally infected rats were cultured in spinner flask suspension cultures to minimize host cell contamination. At pH 8.3, seven Pc-specific proteolytic bands were detected in three clusters of different molecular weights clearly different from host cell patterns. By using a range of pH, various preparations of organisms and both infected and uninfected culture media, proteolytic activities have been partially characterized. Elastase secretion has been assessed based on elastin digestion model. Proteinase inhibitors have been tested for their ability to inhibit P.carinii growth in HEL299 short-term monolayer cultures. Results indicate that proteolytic activities are involved in the proliferation of microorganisms since leupeptin exerted in vitro antipneumocystis activity while aprotinin enhanced P.carinii growth.

In vitro drug assays and statistical analysis.

The development of in vitro drug tests to assess the efficacy of drugs against Pneumocystis carinii has been hindered by the lack of efficient methods for continuous cultivation of the microorganism. However, different short-term culture systems have been proposed by many teams. In the present contribution an in vitro microplate drug assay and two statistical programs allowing the analysis of results are presented.

Combined use of blood and oropharyngeal samples for noninvasive diagnosis of Pneumocystis carinii pneumonia using the polymerase chain reaction.

To evaluate the clinical use of a polymerase chain reaction (PCR) assay for diagnosis of Pneumocystis carinii pneumonia (PCP) using samples collected noninvasively, the Internal Transcribed Spacers (ITSs) nested PCR was performed on 148 samples from 40 subjects. Bronchoalveolar lavage (BAL) fluid sera, gargled oropharyngeal washes, and peripheral blood mononuclear cells (PBMC) from 14 AIDS patients (mean age, 35.6 years; mean CD4+ cell count, 49.2 cells/mm3) with proven PCP and from 13 HIV-seropositive controls (mean age, 34.6 years; mean CD4+ cell count, 107.3 cells/mm3) with other AIDS-related opportunistic infections were evaluated. Sera and oropharyngeal samples were also collected from 13 HIV-seronegative health care personnel working in an infectious disease ward for use as negative controls. The ITSs nested PCR confirmed the morphological diagnosis of PCP in all patients when BAL fluid was tested (100% sensitivity). This technique also detected Pneumocystis carinii DNA in oropharyngeal samples from 78.6% of patients, in sera from 71.4% of patients, in PBMC from 35.7% of patients. When all results obtained after ITSs nested PCR were considered together for the same patient, the sensitivity for PCP diagnosis was 100% for blood and oropharyngeal samples (gargled saline), as confirmed by subsequent BAL. All samples collected noninvasively from 26 of 26 controls were negative using ITSs nested PCR (100% specificity).

Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome.

OBJECTIVES: To verify the clinical value of two different polymerase chain reactions (PCRs) for noninvasive diagnosis and follow-up during Pneumocystis carinii f. sp. hominis pneumonia (PCP) and to analyze the P. carinii f. sp. hominis genotypes involved. METHODS: Internal transcribed spacers (ITSs) nested PCR was applied to 630 samples (bronchoalveolar lavage, sera, peripheral blood mononuclear cells, and oropharyngeal samples) from 122 patients with acquired immunodeficiency syndrome and pneumonia and 40 control samples from 20 subjects seronegative for human immunodeficiency virus. One hundred and eighty samples also were examined by mt-rRNA PCR. Bronchoalveolar lavage samples and 33 sera were analyzed by type-specific oligonucleotide hybridization. RESULTS: On bronchoalveolar lavage samples, the two PCRs consistently confirmed the morphologic diagnosis of PCP. The sensitivity of ITSs nested PCR versus mt-rRNA PCR was 57.3% versus 14.3% on sera, 32.3% versus 22. 8% on peripheral blood mononuclear cells, and 69.1% versus 48.6% on oropharyngeal samples (garglings). Both PCRs had 100% specificity. Type-specific oligonucleotide hybridization revealed in 72.2% of bronchoalveolar lavage samples a single P. carinii f. sp. hominis genotype, whereas in 27.8% co-infection with more than one strain was detected. CONCLUSION: On noninvasive samples, ITSs nested PCR was more sensitive than mt-rRNA PCR, and it confirmed the diagnosis in all patients with PCP. For each patient with PCP at least one noninvasive sample was positive for P. carinii f. sp. hominis DNA.

ITSs genotypes and blood dissemination during acute PCP.

The presence of P. carinii DNA in serum and in Peripheral Blood Mononuclear Cells (PBMC) during acute phase of PCP in AIDS patients was previously demonstrated by several authors using different specific primers. Amplification by ITSs nested PCR followed by TSO hybridization of P. carinii isolates derived from BAL and blood samples allows to compare genotypes involved in the disease and genotype-related dynamics of Pc-DNA clearance from blood during therapy. Different virulence characteristics among P. carinii genotypes could explain the various spectrum of clinical presentation (pulmonary and extrapulmonary) and susceptibility to classic antipneumocystic drugs during PCP.