Giulia Morsica

Polymerase chain reaction for Toxoplasma gondii DNA in the cerebrospinal fluid of AIDS patients with focal brain lesions

ObjectiveTo study the accuracy of polymerase chain reaction (PCR) for Toxoplasma gondii DNA in the cerebrospinal fluid (CSF) of AIDS patients for the diagnosis of T. gondii encephalitis. PatientsEighty-two AIDS patients with brain lesions. At autopsy, 19 patients (group A) had toxoplasmic encephalitis and 33 (group B) primary brain lymphoma or other infections. Brain histology was not available for 30 patients; cerebral lesions improved after anti-Toxoplasma therapy in 16 (group C), but there was no improvement in 14 patients (group D). MethodsT. gondii RH strain was serially diluted in microplate wells. After heat denaturation, nested PCR was performed on diluted tachyzoites and on 10 μl CSF with primers flanking the B1 repetitive region of T. gondii genome. ResultsDNA from one to five tachyzoites was detected in each experiment. PCR was positive in eight (42.1 %) out of 19 group A samples, none of the group B samples, 10 (62.5%) out of 16 group C samples and none of the group D samples. Among group A and C patients, PCR was positive in all 11, and in seven out of 24 (29.1%; P < 0.04) patients who had received anti-Toxoplasma therapy for less or more than 1 week at the time of rachicentesis, respectively. ConclusionsNested PCR for T. gondii in CSF may improve early differential diagnosis of AIDS-associated focal brain lesions. Higher diagnostic accuracy was achieved when lumbar puncture was performed in the first week of anti-Toxoplasma therapy.

Occult hepatitis B virus infection in a Cohort of HIV-positive patients: Correlation with hepatitis C virus coinfection, virological and immunological features

Background:An evaluation of the prevalence of occult hepatitis B virus (HBV) infection in HIV-positive individuals is important as HBV infection may have an impact on the outcome of the liver disease in these patients.Materials and Methods:Of the 1,593 HIV-positive subjects enrolled in the Italian Cohort Naïve Antiretroviral (ICONA) program, 175 (10.9%) were selected for inclusion in the study on the basis of hepatitis B surface antigen (HBsAg) negativity and antibody to hepatitis B core antigen (anti- HBc) positivity; 101/175 (58%) were also anti-hepatitis C virus (HCV) positive. HBV-DNA was detected in plasma using a highly sensitive PCR assay (detection limit: 2.6 copies/ml). Two different genomic regions were assayed. Quantification was performed by real-time PCR. The HBV genotype was determined in 20 cases with occult HBV infection. Data on the antiretroviral therapy (ART) regimen was obtained in 169 individuals: 53 (31.4%) patients were ART-naive, 46 (27.2%) were under ART without lamivudine or tenofovir, and the remaining 70 (41.4%) were under ART including lamivudine or tenofovir.Results:27/175 (15%) patients had detectable HBV-DNA in their plasma: 21/101 (21%) were anti-HCV positive and 6/74 (8%) were anti-HCV negative. Genotype D was invariably found in the 20 cases analyzed. Occult HBV infection was significantly higher in HCV-coinfected subjects: adjusted OR 5.02, 95% CI 1.31–19.26, p = 0.02. The value was not associated with immune status, HIV load, or ART regimen.Conclusions:In relation to the high prevalence of occult HBV infection, particularly in HIV/HCV-coinfected individuals, it is necessary to clarify the clinical impact of this cryptic infection by monitoring HBV-DNA in plasma using the correct approach. Similarly to HBsAg-positive individuals of the Mediterranean area, HBV genotype D is invariably detected in this cohort of HIV-infected patients with occult HBV infection.

Detection of hepatitis C virus genomic sequences in the cerebrospinal fluid of HIV‐infected patients

To assess the presence of hepatitis C virus (HCV) in the central nervous system (CNS), HCV‐RNA was sought in paired serum and cerebrospinal fluid (CSF) samples of 21 HIV/HCV‐positive patients: HCV‐RNA was detected in the serum of 19/21 patients (90.4%), and in the CSF of five of the 19 serum‐positive patients. The presence of HCV‐RNA was confirmed in follow‐up CSF samples available for three of these five patients. An identical HCV genotype was found in the paired serum/CSF samples. No correlation was found between the different genotypes and the presence of HCV in CSF of the individual patients.

Hepatitis C virus populations in the plasma, peripheral blood mononuclear cells and cerebrospinal fluid of HIV/hepatitis C virus-co-infected patients

Background:Chronic hepatitis C virus (HCV) infection has occasionally been associated with diseases of the central nervous system, thus suggesting that the virus has a direct effect on cerebral function. Objective:To investigate the presence and heterogeneity of HCV populations in the cerebrospinal fluid (CSF), plasma and peripheral blood mononuclear cells (PBMC) of HIV-infected and anti-HCV-positive patients. Methods:Reverse transcriptase–polymerase chain reaction was used to detect HCV RNA in 21 paired CSF/plasma samples and 20 PBMC samples from HIV/HCV-positive patients. HCV was genotyped by means of a hybridization assay, and its compartmental heterogeneity was analysed by cloning the related amplicons. Results:All of the plasma samples, 14 out of 20 PBMC samples and five out of 21 CSF samples were positive for HCV RNA. Of the five patients with HCV-positive CSF, two had the same genotype in the plasma/CSF/PBMC samples, two had a different genotype in the CSF from that in plasma and PBMC, and one had the same genotype in paired CSF/plasma samples. An analysis of the HCV populations showed that two patients seemingly infected with the same genotype in different compartments harbored mixed infection in the CSF but not in the plasma and PBMC samples. Conclusions:These findings of different HCV genetic diversification in different compartments suggest that CSF is an independent site of viral replication and persistence.

Viral interference between hepatitis B, C, and D viruses in dual and triple infections in HIV-positive patients.

Objective:To investigate the reciprocal inhibitory effects of hepatitis B virus (HBV)/hepatitis C virus (HCV)/hepatitis D virus (HDV) infections in naive and previously antiretroviral-experienced HIV-positive patients. Design:This retrospective study involved 72 consecutive patients of the Italian Cohort Naive Antiretroviral cohort: 21 coinfected with HBV/HCV (group 1BC), 18 infected with HBV (group 2B), and 33 infected with HCV (group 3C). Methods:Viral interference between HBV and HCV was assessed by means of the qualitative detection, quantification, and genotyping of each virus; HDV infection was assessed by means of genomic amplification. Results:Univariate analysis showed that HBV DNA was less frequently detected in group 1BC than in group 2B (16 of 21 vs 18 of 18; P = 0.02), their HBV load was significantly lower (median 3.9 vs 5.4 log10 HBV DNA copies/mL; P = 0.002), and they more frequently carried HBV genotype D (12 of 13 vs 4 of 11; P = 0.0071). HCV RNA was less frequently detected in group 1BC than in group 3C (12 of 21 vs 33 of 33; P < 0.0001), and HDV RNA was more frequently detected in group 1BC than in group 2B (9 of 21 vs 2 of 18; P = 0.028). Multivariate analysis of the HBV-infected subjects showed that the risk of HCV coinfection was associated with older age [relative risk 0.28, 95% confidence interval (CI): 0.09 to 0.90; P = 0.033 for every 10 years older] and intravenous drug use (relative risk 73, 95% CI: 2.4 to >999.999; P = 0.013). The only predictor of HBV coinfection in HCV-infected individuals was a lower HCV load (relative risk 0.30, 95% CI: 0.11 to 0.79 for every additional log10 HCV RNA; P = 0.015). Conclusion:HBV and HCV showed alternative dominant replication in the I.Co.N.A. cohort, with HBV having a more unfavorable effect on HCV replication.

Prevalence of wild-type in NS5A-PKR protein kinase binding domain in HCV-related hepatocellular carcinoma

BACKGROUND/AIMS Experimental studies have demonstrated that the wild-type PKR-NS5A strain of hepatitis C virus (HCV) may have oncogenic potential through the binding and functional repression of PKR protein kinase. To assess whether the NS5A-PKR-binding domain may be involved in HCV-related liver carcinogenesis, its sequence was analyzed in the sera of 85 patients with hepatocellular carcinoma (HCC) and in 51 patients with chronic active hepatitis (CAH). In 13 HCC cases sequence analysis was also performed in tumor and nontumor liver tissues. METHODS The nucleotide sequences of the PKR-binding domain were inferred by direct sequencing of the amplified HCV products and deduced amino acids were compared with the sequence of HCV-J. RESULTS A wild-type or single mutated strain which retains PKR-binding activity was found in 88% of HCC and 69% of CAH cases (P=0.0096). All but three HCC cases showed no divergences in amino acid changes between serum and liver tissues. The wild-type strains were equally distributed between the HCC with or without cirrhosis. CONCLUSIONS The prevalance of the wild-type NS5A-PKR strain is significantly higher in HCC than in CAH. These data suggest that inhibition of PKR activity by HCV might represent a potential mechanism of HCV-related carcinogenesis.

Mother-to-child transmission of TT virus: sequence analysis of non-coding region of TT virus in infected mother-infant pairs ∗

Summary. To investigate vertical transmission of TT virus, TTV-DNA was looked for in serum samples taken from 22 mothers and their 22 infants at birth and during nine months of follow-up. Sixteen mothers at delivery and six infants within nine months of age had TTV-DNA detected by the amplification of the non coding (NC) region. Two of these newborns had positive viremia at birth. Sequence analysis of the NC region of five mother-infant pairs revealed that the TTV strains detected at three and six months of age in two of the infants were closely related to that of their mothers, whereas two that became TTV-DNA positive at three moths had a different nucleotide sequence from that of their mothers. One of the two infants with detectable viremia at birth also had a different nucleotide sequence from her mother. These findings suggest that both in utero and perinatal transmission of TT virus may occur, and that the strain detected in the infants was not invariably dominant in the mothers at delivery.

Acute self-limiting hepatitis C after possible sexual exposure: Sequence analysis of the E-2 region of the infected patient and sexual partner

We describe a case of symptomatic acute infection with HCV in a woman whose sexual partner had chronic hepatitis C. The patient cleared HCV RNA 8 weeks after the onset of acute hepatitis and was found to be persistently HCV-RNA negative during 90 weeks of follow-up. Part of the E-2 region of HCV was directly sequenced in the patient and her sexual partner. Four local controls with subtype-1a infection and 9 1a isolates obtained from GenBank were analyzed. The average nucleotide divergence between the sequences of the infected patient and her sexual partner was 5.1%, compared with an average nucleotide divergence of 19.4% (range 16.6-21.8%) between the sequences of the patient and those of controls. Comparison of the phylogenetic trees in the partial E-2 region showed that the sequence of the patient was closely related to that of her sexual partner. Our findings suggest that the infection was transmitted to the patient from her sexual partner. The resolution of acute hepatitis C in this case was probably related to the host rather than to intrinsic characteristics of the HCV genome.We describe a case of symptomatic acute infection with HCV in a woman whose sexual partner had chronic hepatitis C. The patient cleared HCV RNA 8 weeks after the onset of acute hepatitis and was found to be persistently HCV-RNA negative during 90 weeks of follow-up. Part of the E-2 region of HCV was directly sequenced in the patient and her sexual partner. Four local controls with subtype-1a infection and 9 1a isolates obtained from GenBank were analyzed. The average nucleotide divergence between the sequences of the infected patient and her sexual partner was 5.1%, compared with an average nucleotide divergence of 19.4% (range 16.6-21.8%) between the sequences of the patient and those of controls. Comparison of the phylogenetic trees in the partial E-2 region showed that the sequence of the patient was closely related to that of her sexual partner. Our findings suggest that the infection was transmitted to the patient from her sexual partner. The resolution of acute hepatitis C in this case was probably related to the host rather than to intrinsic characteristics of the HCV genome.

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma.

Summary.  We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre‐C/C) domain in hepatocellular carcinoma (HCC). Thirty‐eight consecutive patients with HCC were included in the study – 18 of them with HBV/HCV co‐infection and 20 with HBV single infection. Twenty‐three additional patients with co‐infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre‐C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co‐infection in comparison with single‐infected HCC, whereas two different HBV viraemia profiles were detected in co‐infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre‐C/C region was found in co‐infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co‐infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre‐C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre‐C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co‐infection could be linked with accelerated HBV/HCV‐related disease progression.

Frequency of natural resistance within NS5a replication complex domain in hepatitis C genotypes 1a, 1b: Possible implication of subtype-specific resistance selection in multiple direct acting antivirals drugs combination treatment

Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs) were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV) or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV) was higher in G1b compared to G1a (37/1552 (2.4%) in 1b sequences and 15/1209 (1.2%) in 1a isolates, p = 0.040). Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1%) G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5%) harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001). Finally, 28 (2.3%) G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001). In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs) targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs.