Natural NS5A inhibitor resistance associated substitutions in hepatitis C virus genotype 1 infected patients from Italy.

OBJECTIVES: Genetic variability in NS5A is associated with different levels of resistance to the currently licensed NS5A inhibitors. The aim of this study was to detect NS5A inhibitor resistance associated substitutions (RASs) in hepatitis C virus (HCV) genotype 1 (GT1) patients who are naive to direct-acting HCV antivirals. METHODS: Amplification, Sanger sequencing and phylogenetic analysis of the HCV NS5A region were performed on plasma obtained from 122 consecutive patients with HCV chronic infection attending four different clinics in Italy. RESULTS: NS5A inhibitor RASs were detected in 14/61 (23.0%) HCV GT1b and 3/61 (4.9%) HCV GT1a infected patients (p 0.007). The pan-genotypic RAS Y93H was detected in 1 (1.6%) GT1a and 4 (6.6%) GT1b patients. GT1a sequences clustered into two different clades with RASs detected in 1/34 (2.9%) clade I and 2/27 (7.4%) clade II sequences. CONCLUSIONS: Although the impact of naturally occurring NS5A RASs might be limited with upcoming pan-genotypic treatment regimens, this information is still useful to map naturally occurring HCV variants in different geographic areas in the context of current HCV therapy.

Antibody testing and RT-PCR results in hepatitis C virus (HCV) infection: HCV-RNA detection in PBMC of plasma viremia-negative HCV-seropositive persons.

To evaluate the concordance between viremia and antibody testing in hepatitis C virus (HCV) diagnosis, 682 serum or plasma samples collected from patients with known or suspected HCV infection were tested. An overall concordance of 77% between serological and PCR results was found, 5% was RNA positive/antibody negative and 18% antibody positive/RNA negative. The relationship between HCV infection, risk group and clinical diagnosis was studied in 116 patients: the presence of anti-HCV antibody without viremia was shown in 72.7% of asymptomatic subjects and 17.6% of chronic hepatitis subjects without interferon treatment. However, the detection of HCV-RNA in peripheral blood mononuclear cells (PBMC) in four out of 38 plasma viremia-negative HCV-seropositive subjects (10.5%), showed that HCV-RNA could persist in PBMC and could begin the viral replication again at different times. The detection of HCV-RNA in PBMC in anti-HCV-positive subjects without viremia could reduce false-negative results of HCV-RNA testing by RT-PCR in serum or plasma.

Evaluation of the presence of 2-LTR HIV-1 unintegrated DNA as a simple molecular predictor of disease progression.

In a preliminary cross-sectional analysis of 109 human immunodeficiency virus type 1 (HIV-1)-infected subjects the presence of 2-long terminal repeat (LTR) unintegrated circular HIV-1 DNA in peripheral blood mononuclear cells (PBMC) was found to be associated with both symptomatic infection (P = 0.0037) and low CD4 counts (P = 0.0004). To investigate the prognostic significance of the presence of 2-LTR HIV-1 DNA, a subset of 23 2-LTR-negative and 25 2-LTR-positive asymptomatic individuals were followed up for 12-24 months. The two groups did not differ in terms of baseline CD4 counts, zidovudine (ZDV) therapy, and duration of HIV-1 infection. Longitudinal analysis of CD4 values did not indicate a significantly different CD4 outcome between the two groups. However, when only ZDV-treated subjects were considered, a significant (P = 0.042) decrease in CD4 counts was found at month 24 with respect to baseline in 2-LTR-positive (n = 12) but not in 2-LTR-negative (n = 11) patients. Moreover, when > 40% CD4 loss from baseline and/or development of CDC stage B or C symptoms were considered as indicators of disease progression, there was a significantly higher number of events in the whole 2-LTR-positive group than in the whole 2-LTR-negative group (P = 0.0197 at month 12, P = 0.0299 at month 18, P = 0.0373 at month 24). Thus, the presence of 2-LTR HIV-1 DNA in PBMC merits further investigation as a simple, qualitative, molecular predictor of disease progression and decreased response to antiretroviral therapy.

The potential role of hepatitis C virus in the pathogenesis of the neurological syndrome in chronic hepatitis C.

A 72-year-old man developed chronic sensory neuropathy (CSN) during chronic hepatitis C (HCV) infection. Neurological symptoms began one year after acute HCV hepatitis and slowly worsened over three years. No conventional cause for CSN was found. Circulating antinervous tissue antibodies (including anti-Hu) and inflammatory infiltrates in sural nerve biopsy specimens were absent. However, the presence of anti-HCV antibody and HCV-RNA in cerebrospinal fluid indicated that HCV had reached the intrathecal compartment, suggesting the direct viral involvement in the pathogenesis of CSN.

Zidovudine resistance mutations and human immunodeficiency virus type 1 DNA burden: longitudinal evaluation of six patients under treatment.

Zidovudine (ZDV) is by far the most widely used drug to counteract human immunodeficiency virus type 1 (HIV-1) infection, both in monotherapy and in combination therapy regimens. However, the majority of patients under prolonged ZDV therapy have been shown to harbour HIV-1 mutant genomes displaying reduced sensitivity to the drug in vitro. In order to investigate the pathogenic role of in vitro resistance to ZDV, six HIV-1-infected ZDV-treated subjects were evaluated longitudinally (mean follow-up 28.5 months, range 12-39 months) for HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) and for the presence of HIV-1 pol gene mutations responsible for ZDV resistance. Quantitation of HIV-1 DNA was performed by competitive polymerase chain reaction (cPCR) and the pol genotype was determined by direct sequencing of PCR products. All of the six patients developed one or more of the HIV-1 pol mutations known to confer resistance to ZDV in vitro (Met41-->Leu, Asp67-->Asn, Lys70-->Arg, Thr215-->Phe/Tyr, Lys219-->Gln/Glu). A temporal association was found between HIV-1 DNA burden and the level of ZDV resistance, as predicted on the basis of the pol genotype (genotypic resistance). Both virus load and ZDV resistance were inversely correlated with CD4+ cell counts. These results are compatible with a direct in vivo pathogenetic role for pol gene mutations shown to be involved in resistance to ZDV in vitro. Monitoring the degree of genotypic resistance to ZDV and to other antiretroviral drugs should be considered in designing protocols for the management of treated patients.

Concordance between polymerase chain reaction and antibody detection in the diagnosis of human immunodeficiency virus type 1 infection.

A highly sensitive nested polymerase chain reaction (PCR) protocol was used to detect human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells from 271 HIV-1-seropositive patients, 240 HIV-1-seronegative subjects at increased risk for HIV-1 infection, 51 serologically indeterminate individuals, and 120 healthy blood donors. PCR was carried out in a multiplex nested configuration with pol and env region primer sets. HIV-1 DNA was detected in all of the HIV-1 seropositive patients. In contrast, HIV-1 DNA was not detected in any of the either seronegative or serologically indeterminate subjects. Only one of 37 seronegative regular sexual partners of HIV-1-infected patients who were followed longitudinally was found to seroconvert to HIV-1. However, HIV-1 DNA and antibody results were concordant in the four samples obtained from this subject prior to and after seroconversion. These results show an excellent concordance between HIV-1 DNA and antibody detection for diagnosis of HIV-1 infection and suggest that long-term HIV-1 infection in the absence of detectable antibody is likely to occur at a very low frequency.

Low human immunodeficiency virus type 1 (HIV-1) DNA burden as a major cause for failure to detect HIV-1 DNA in clinical specimens by PCR.

To determine the sensitivity of a nested PCR procedure for detecting human immunodeficiency virus type 1 DNA in clinical specimens, 553 peripheral blood mononuclear cell samples obtained from 268 human immunodeficiency virus type 1-seropositive subjects were assayed by use of two independent primer sets for each sample. Overall, 1,088 of 1,106 (98.37%) reactions were positive. Investigation of the negative reactions showed that a low viral burden in some infected subjects, rather than primer-template mismatches, was the primary cause for the false-negative PCR results.

Intrafamilial hepatitis A outbreak: projection on the community in an area of Tuscany.

In 1988, 3 intrafamilial cases of hepatitis A occurred in a little town in the nearby of Siena: shellfishes were probably responsible for the index case, close contacts for the two secondary cases, characterized by a very long incubation period (above 60 days). An inapparent infection was detected in one household contact. No other cases of hepatitis A have been observed in that area since the intrafamilial outbreak until now. A seroepidemiological survey was carried out in a group of persons living in the same area, and attending the same school or class-room of one of the secondary intrafamilial cases. 210 sera belonging to 105 teenagers were available; an IgG anti A test was performed on a double serum sample (before and after the outbreak): no seroconversion was observed. Our report underlines the hypoendemicity of hepatitis A infection in our country.

Impact of hepatitis A in Siena: a "traveller's disease"?

Over a period of seven years (from 1980 to 1987) 58 acute viral hepatitis A cases were admitted to the Departments for Infectious Diseases. This number represents 10.76% of all viral hepatitis patients. Most of the patients had travelled to foreign countries or to areas in Italy with high hepatitis A virus (HAV) circulation. An assessment of specific immunization against HAV in normal healthy people aged 1-60, performed by ELISA, showed a very low number of sero-positive subjects. The disappearance of HAV in Italy, which is similar to other western European countries, is probably due to the improved socio-economical conditions; the "imported cases" seem to have little impact on public health.

Live-cell immunofluorescence for detection of human immunodeficiency virus type 1 (HIV-1)-related antigens in peripheral blood mononuclear cells from HIV-1 seropositive individuals.

Live-cell indirect immunofluorescence was used to detect HIV-associated antigens in fresh uncultured peripheral blood mononuclear cells from 29 HIV-seropositive subjects. Conventional fixed-cell immunofluorescence and live-cell immunofluorescence were run in parallel on each sample using monoclonal antibodies directed against HIV gag and env gene products. Sera from the same patients were also tested for HIV antigen by a sandwich enzyme immunoassay. Results strongly indicate that nondenaturing live-cell immunofluorescence is much more sensitive for detection of HIV-associated antigens and may be useful in the diagnosis of HIV infection as well as in the study of pathogenesis.

[Epidemiology of hepatitis B virus infection in the personnel of a psychiatric hospital]. / Epidemiologia dell'infezione da virus dell'epatite B nel personale di un ex ospedale psichiatrico.

The prevalence of Hepatitis B Virus (HBV) markers was studied in 350 staff members from a Psychiatric Hospital, where a high prevalence of HBV markers was observed among patients. The prevalence of HBsAg, anti-HBs plus anti-HBc, anti-HBc alone and anti-HBs alone were determined to be: 2%, 15.1%, 4% and 0.3%, respectively. The prevalence of HBV markers was greater (although not significant) among women (24.7%) as compared to men (17.8%) (p greater than 0.05), and in the personnel with more than 20 years of employment (24.1%), as compared to those with less than 20 years (20% (p greater than 0.05). The highest prevalence of infection was observed in the department in which were admitted children and young adults. The overall prevalence of HBV markers was higher among staff members than in the blood donors of our area. Hospital personnel must know such epidemiologic conditions: active immunization with the Hepatitis B vaccine must be offered to hospital employees, firstly in those institutions in which a high prevalence of HBV infection has been documented.

[Viral hepatitis. Epidemiologic evaluation of a personal case load in the area of Casale Monferrato in the years 1981-1982]. / Epatite virale. Valutazione epidemiologica di una casistica personale nell'area di Casale Monferrato negli anni 1981-1982.

The results are reported of an epidemiological assessment of 94 patients hospitalised with diagnosed acute viral hepatitis in the Infectious Disease Division of the S. Spirito Hospital, Casale Monferrato in the period march 1 1981, april 30 1982. Hepatitis was found to occur most frequently among young (under 30) males. There were more positive than negative HBsAg cases, most of the latter arising in heroin addicts.

[Epidemiologico-clinical considerations on a case file of acute hepatitis observed 1980-1983]. / Considerazioni epidemiologico-cliniche su una casistica di epatite acuta osservata negli anni 1980-1983.

Between January 1980 and December 1983, 332 consecutive cases of acute hepatitis were observed in adult patients admitted to the Department of Infectious Disease and Gastroenterology of Siena. Sex and age of the patients, the presence of jaundice, the maximum value of the serum-glutamate-pyruvate-transaminase (SGPT) were considered. Serum specimens were tested for hepatitis B surface antigen (HBsAg), antibody against hepatitis B core antigen (anti-HBc) of the IgM class, antibody against hepatitis A virus (anti-HAV) of the IgM class, antibody against cytomegalovirus (CMV) of the IgM class, Paul Bunnel Davidshon reaction. Hepatitis A was diagnosed in 25 cases (7.5%). Hepatitis B in 167 (50.3%). Hepatitis due to CMV in 2 cases (0.6%). And, by exclusion, hepatitis non A, non B in 138 cases (41.6%). Male patients were affected with significantly higher frequency than female (p less than 0.01); the same was seen for young patients (14-30 years) compared to the older ones (31-50 years, and over 50 years) (p less than 0.01 in both). Biochemical investigation showed that hepatitis A and B had a significantly higher, maximum SGPT value than hepatitis non A non B (p less than 0.01 in both). Icteric patients were significantly more frequently observed among hepatitis A and B cases than hepatitis non A non B cases (p less than 0.01 in both).

IgM antibody against hepatitis B core antigen (IgM anti-HBc): diagnostic and prognostic significance in acute HBsAg positive hepatitis.

IgM antibody against hepatitis B core antigen (IgM anti-HBc), a marker of recent hepatitis B virus infection, was sought by radioimmunoassay in sera diluted 1/4000 from 376 patients presenting to four centres in Italy with acute, apparently type B hepatitis (hepatitis B surface antigen (HBsAg) positive). In 320 patients (85%) a positive IgM anti-HBc test result confirmed that hepatitis was due to primary infection with hepatitis B virus. In the remaining 56 patients absence of the IgM marker indicated that they were previously unrecognised long term carriers of HBsAg. Further serum analysis often showed delta infection and occasionally hepatitis A or cytomegalovirus infection as the true cause of their illness. After six to eight months circulating HBsAg persisted in 38 of 45 patients (84%) without IgM anti-HBc but in only six of 150 patients (4%) with the IgM antibody (p less than 0.0001). A negative IgM anti-HBc test result in patients with acute HBsAg positive hepatitis points to a factor other than hepatitis B virus as the cause of the liver damage and predicts the carriage of HBsAg.

Complexes of hepatitis B surface antigen and immunoglobulin M in the sera of patients with hepatitis B virus infection.

Hepatitis B surface antigen (HBsAg) bound to immunoglobulin M (IgM) was detected in sera of HBsAg carriers by a radioimmunoassay based on selective absorption of the immunoglobulin on a solid phase coated with antiserum to human IgM. Isopycnic banding and rate-zonal sedimentation have shown that the reaction is related to particulate forms of the HBsAg complexed with IgM. The binding of IgM possibly occurred because of a selective affinity of these molecules to the surface of HBsAg particles. HBsAg/IgM was found transiently in 24 of 25 (96%) patients with acute self-limited hepatitis B and persistently in 6 of 25 patients whose acute hepatitis B progressed to chronicity. It was also found in 20 of 39 (51%) chronic HBsAg carriers with inactive and asymptomatic infection. The HBsAg/IgM phenomenon is not dependent on replication of hepatitis B virions; its persistence in patients with acute hepatitis B may provide complementary evidence of transition of the infection to chronicity.

HBsAg/IgM complexes in serum of HBsAg carriers: partial characterization and clinical significance.

HBsAg bound to IgM was detected in serum of HBsAg carriers with a radioimmunoassay based on selective absorption of the immunoglobulin on a solid phase coated with antiserum to human IgM. High titers of HBsAg/IgM were found in sera with the highest HBsAg binding capacity of polymerized human serum albumin (poly-HSA) and of C1q. These findings and the inhibition of HBsAg/IgM reaction by addition of purified poly-HSA suggest that the IgM component of the complex might bind to poly-HSA fixed on to HBsAg particles and possibly represent antibody to the modified plasma protein. HBsAg/IgM was detected in 95 (87%) patients with acute HBsAg positive hepatitis during the acute phase of infection and persisted after the fourth week only in patients who developed chronic liver disease. HBsAg/IgM were detected in one out of 15 carriers of the HBsAg with superimposed Non B hepatitis. HBsAg/IgM were also present in 76% to 100% of sera from chronic carriers without any relation to the extent of viral replication and to presence of severity of liver disease. Persistence of HBsAg/IgM in patients with acute hepatitis B may provide a useful tool to predict transition of HBV infection to chronicity.