Loredana Sarmati

Haploidentical, unmanipulated, G-CSF–primed bone marrow transplantation for patients with high-risk hematologic malignancies

UNLABELLED Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF–primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n =45) or in > second CR or active disease (high-risk: n =35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% 0.2% and 5% 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% 0.1%. The 1-year CI of treatment-related mortality was 36% 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% 8% and 33% 9% and 44% 8% and 30% 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF–primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted. KEY POINTS Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation. Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.

Gender differences in clinical progression of HIV-1-infected individuals during long-term highly active antiretroviral therapy

Objective: To assess gender differences in the long-term clinical, virological and immunological outcomes during highly active antiretroviral therapy (HAART). Methods: This longitudinal observational multicentre study followed 2460 HIV-infected patients who had begun a protease inhibitor-based regimen for a median period of 43 months. Outcome measures were virological suppression (< 500 copies/ml), confirmed virological rebound after suppression, and death or new AIDS-defining illness (ADI). Results: At baseline, 690 female patients (28.0%) had significantly lower age, higher prevalence of heterosexual contact and lower prevalence of intravenous drug use as risk factors for HIV infection compared with males. Furthermore, females had a lower number of AIDS-defining illnesses, higher CD4 cell counts and lower viral loads. No gender differences were reported in terms of proportion of patients achieving viral suppression or exhibiting rebound after achieving viral suppression. Female patients experienced reduced clinical progression during follow-up compared with males (P = 0.008) by Kaplan–Meier analysis; however this difference was not significant in an adjusted analysis. In a multivariate model, the interaction between gender and risk factor for HIV or viral load showed that female drug users and female patients with a baseline HIV RNA viral load of 104–105 copies/ml had a favourable clinical outcome compared with males (P = 0.035 and P = 0.015, respectively). Conclusion: No differences were found between genders in terms of virological and immunological outcomes during long-term HAART. Nevertheless, a lower risk of clinical progression was reported among female patients with intermediate baseline viral load than in males.

Prevalence and risk factors for human herpesvirus 8 infection in northern Cameroon.

Background: The modes of transmission of HHV‐8 are still unclear. Goal: To evaluate the distribution and transmission of HHV‐8 infection. Design: Serosurvey conducted in a Cameroon hospital among 292 persons, including children (5‐10 years), adolescents (15‐20 years), and adults (30‐40 years). Antibodies against lytic and latent antigens to HHV‐8 were detected by immunofluorescence assay; antibodies against Epstein‐Barr virus viral antigens were detected by enzyme‐linked immunoabsorbent assay. Results: The prevalence of HHV‐8 antilytic antibodies remained stable and was 39.8% among children, 51.5% among adolescents, and 61.8% among adults. Epstein‐Barr virus seroprevalence was high among children, and remained stable among adolescents and adults. A history of sexually transmitted diseases was an independent determinant of HHV‐8 infection (adjusted odds ratio 2.47; 95% CI 1.09‐4.91). Conclusion: The high prevalence of HHV‐8 infection among children indicates nonsexual modes of transmission in Cameroon, with sexual transmission occurring among adolescents and adults.

A seroprevalence study of human herpesvirus type 8 (HHV8) in eastern and Central Africa and in the Mediterranean area

Human herpes virus type 8 (HHV8) is the major determinant of Kaposis sarcoma (KS), a neoplasm with wide geographic variations in incidence rates. To assess the prevalence of HHV8 infection among populations with differing rates of KS, we used sera from 1402 persons (Central Africa: Cameroon, n = 293, age range: 5–40; eastern Africa: Uganda, n = 315, age range: 1–64: Mediterranean area: Egypt, n = 236, age range: 13–19: Italy, blood donors n = 134, age range: 20–67: Italy. HIV seroconverters n = 424, age range: 16–65). Serum samples were tested for antibodies to lytic and latent antigens of HHV8 using two immunofluorescence assays. HHV8 prevalence was evaluated according to geographic area, gender and age groups. Overall, the highest prevalence of HHV8 lytic antigens (47.5%) was recorded among children and adults in Africa. Approximately 40% of children and adolescents from Egypt and of Italian HIV-positive persons (39.9%) were HHV8 seropositive. In eastern and Central Africa and in Egypt, no differences emerged between males and females for both types of HHV8 antibodies. Conversely, Italian females were at lower HHV8 risk than their male counterparts. Moreover the prevalence of HHV8 infection tended to increase with age. This investigation partially confirms that HHV8 infection mirrors incidence rates of KS. The high prevalence of HHV8 infection in newborns, children and adolescents in Egypt, in eastern and in Central Africa strongly suggests the existence of transmission modes other than sexual.

Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

ABSTRACT Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 106 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.

18-Fluoro-2-deoxyglucose positron emission tomography-computed tomography: an additional tool in the diagnosis of prosthetic valve endocarditis

OBJECTIVES To evaluate the role of 18-fluoro-2-deoxyglucose positron emission tomography-computed tomography ((18)F-FDG-PET-CT) in the diagnosis of infectious endocarditis (IE). METHODS We retrospectively examined 27 consecutive patients who were admitted to the Infectious Diseases Department of Tor Vergata University Hospital between 2009 and 2013 with a suspicion of IE. The final IE diagnosis was defined according to the modified Duke criteria, and the microbiological and diagnostic results were collected for each patient. RESULTS Twenty out of 27 patients had a suspected prosthetic valve endocarditis (PVE) and seven had a suspected native valve endocarditis (NVE). Twenty-five out of 27 patients (92%) had a confirmed diagnosis of IE (18/25 PVE and 7/25 NVE); 16 had a positive echocardiography evaluation and 16 had positive (18)F-FDG-PET-CT findings. Echocardiography showed a higher sensitivity as a diagnostic tool for the detection of IE compared to (18)F-FDG-PET-CT (80% vs. 55%). However, a greater number of PVE had positive (18)F-FDG-PET-CT results compared to those with positive echocardiography findings (11/13 vs. 9/13), and overall 89% (16/18) of confirmed PVE resulted (18)F-FDG-PET-CT positive. Analyzing only the cases who underwent transoesophageal echocardiography, (18)F-FDG-PET-CT showed a sensitivity of 85% in PVE (vs. 69% for echocardiography and 77% for the Duke criteria). All seven patients with NVE had a positive echocardiography and negative (18)F-FDG-PET-CT findings (p<0.001). CONCLUSIONS The results of this study further highlight the limitations of echocardiography in the diagnosis of PVE and the potential advantages of (18)F-FDG-PET-CT in these cases.

Hepatitis B surface antigen genetic elements critical for immune escape correlate with hepatitis B virus reactivation upon immunosuppression

Hepatitis B virus (HBV) reactivation during immunosuppression can lead to severe acute hepatitis, fulminant liver failure, and death. Here, we investigated hepatitis B surface antigen (HBsAg) genetic features underlying this phenomenon by analyzing 93 patients: 29 developing HBV reactivation and 64 consecutive patients with chronic HBV infection (as control). HBsAg genetic diversity was analyzed by population‐based and ultradeep sequencing (UDS). Before HBV reactivation, 51.7% of patients were isolated hepatitis B core antibody (anti‐HBc) positive, 31.0% inactive carriers, 6.9% anti‐HBc/anti‐HBs (hepatitis B surface antibody) positive, 6.9% isolated anti‐HBs positive, and 3.4% had an overt HBV infection. Of HBV‐reactivated patients, 51.7% were treated with rituximab, 34.5% with different chemotherapeutics, and 13.8% with corticosteroids only for inflammatory diseases. In total, 75.9% of HBV‐reactivated patients (vs. 3.1% of control patients; P < 0.001) carried HBsAg mutations localized in immune‐active HBsAg regions. Of the 13 HBsAg mutations found in these patients, 8 of 13 (M103I‐L109I‐T118K‐P120A‐Y134H‐S143L‐D144E‐S171F) reside in a major hydrophilic loop (target of neutralizing antibodies [Abs]); some of them are already known to hamper HBsAg recognition by humoral response. The remaining five (C48G‐V96A‐L175S‐G185E‐V190A) are localized in class I/II–restricted T‐cell epitopes, suggesting a role in HBV escape from T‐cell‐mediated responses. By UDS, these mutations occurred in HBV‐reactivated patients with a median intrapatient prevalence of 73.3% (range, 27.6%‐100%) supporting their fixation in the viral population as a predominant species. In control patients carrying such mutations, their median intrapatient prevalence was 4.6% (range, 2.5%‐11.3%; P < 0.001). Finally, additional N‐linked glycosylation (NLG) sites within the major hydrophilic loop were found in 24.1% of HBV‐reactivated patients (vs. 0% of chronic patients; P < 0.001); 5 of 7 patients carrying these sites remained HBsAg negative despite HBV reactivation. NLG can mask immunogenic epitopes, abrogating HBsAg recognition by Abs. Conclusion: HBV reactivation occurs in a wide variety of clinical settings requiring immune‐suppressive therapy, and correlates with HBsAg mutations endowed with enhanced capability to evade immune response. This highlights the need for careful patient monitoring in all immunosuppressive settings at reactivation risk and of establishing a prompt therapy to prevent HBV‐related clinical complications. (Hepatology 2015;61:823–833)

Secondary Integrase Resistance Mutations Found in HIV-1 Minority Quasispecies in Integrase Therapy-Naive Patients Have Little or No Effect on Susceptibility to Integrase Inhibitors

ABSTRACT The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

Cellular proviral HIV-DNA decline and viral isolation in naïve subjects with 500 × 106/l CD4 cells treated with highly active antiretroviral therapy

ObjectiveTo evaluate the decay rate of cellular proviral HIV-DNA and viral replication in patients receiving highly active antiretroviral therapy (HAART) in the very early phase of infection.MethodsThirty-four patients treated with HAART and retrospectively selected for progressive decline of plasma

Incidence of Kaposi's sarcoma and HHV-8 seroprevalence among homosexual men with known dates of HIV seroconversion

ObjectivesTo evaluate temporal trends of Kaposis sarcoma (KS) and of the KS-related human herpesvirus (HHV-8) among homosexual men who seroconverted for HIV between 1984 and 1997. MethodsThe study participants were 387 homosexual men. Changes over a period of time were assessed by estimating KS incidence rates per 1000 person–years for the periods 1984–1989, 1990–1992, 1993–1995, and 1996–1997. The proportional incidence of KS as the AIDS-defining disease for the same periods was also calculated. To evaluate a cohort effect of calendar period, Kaplan–Meier curves were used to estimate the risk of KS by period of HIV seroconversion [i.e. before 1990 (median year of seroconversion) versus later]. Relative hazards for the four periods were estimated using competitive-risks models. We also estimated HHV-8 seroprevalence over the study period. ResultsForty-eight participants developed KS. Between 1984 and 1995, the incidence rate of KS per 1000 person–years increased from 3.9 to 32.8, whereas the proportional incidence decreased from 33.3 to 24.3%. The risk of developing KS after HIV seroconversion did not change when comparing the seroconversion periods (i.e. before 1990 versus later). HHV-8 seroprevalence also remained stable. The rates of KS and the relative hazards dramatically decreased after 1995. ConclusionsAlthough KS incidence rates increased up to 1995, the proportional incidence decreased, due to the higher increase in rates of other AIDS-defining diseases. The finding that the risk of developing KS after HIV seroconversion remained stable over time is consistent with the stable trend of HHV-8 seroprevalence. The dramatic decrease in KS incidence rates after 1995 coincides with combined antiretroviral therapy.