Zipi Kra-Oz

Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a β cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.

Sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus?

Background: Acute bronchiolitis (AB) is a common disease of young children with peak incidence during the winter season. Respiratory syncytial virus (RSV) is a major causative organism, yet recent relatively small sized studies have suggested an increased role of other organisms as sole or codetected organisms. The aim of this study was to assess the prevalence of sole- and mixed-organisms infections in hospitalized children with AB, using combined antigen-based and polymerase chain reaction assays (PCR). Methods: Sputum or nasal wash specimens obtained from 490 previously healthy children ≤2 years of age hospitalized with AB between December 1, 2005 and March 31, 2006 were tested: (1) For RSV, by rapid antigen detection test; (2) For RSV, influenza A, B, Parainfluenza 1 to 3, and adenovirus antigens by direct fluorescent assay; (3) For influenza A and B, RSV, Parainfluenza 1 to 3 viruses RNA by reverse transcription (RT) PCR assay; (4) For human metapneumovirus and rhinovirus RNA by RT real-time PCR assay; (5) For adenovirus, and Bordetella pertussis DNA by conventional PCR assays; (6) For human bocavirus DNA by real-tine PCR assays. Results: At least 1 organism was detected in 465 (91%) children. In 283 (61%), 117 (25%), and 23 (5%) children, 1, 2, and 3/4 organisms were detected, respectively. The most commonly detected organism was RSV, detected in 76%, and as a sole organism in 49%. Rhinovirus, human metapneumovirus, influenza virus A, bocavirus, Bordetella pertussis, and adenovirus were detected as a sole organism in 7%, 2.1%, 1%, 0.6%, 0.6%, and 0.2% of the children, respectively. Conclusions: Respiratory organisms were detected in the majority of the children, of whom about one third suffered from mixed organism infection. RSV was the most prevalent sole detected organism. The relevance of all other organisms may be much less than previously suggested.

Absence of APOL1 Risk Variants Protects against HIV-Associated Nephropathy in the Ethiopian Population

Background: Susceptibility to end-stage kidney disease (ESKD) among HIV-infected Americans of African ancestral heritage has been attributed to APOL1 genetic variation. We determined the frequency of the APOL1 G1 and G2 risk variants together with the prevalence of HIV-associated nephropathy (HIVAN) among individuals of Ethiopian ancestry to determine whether the kidney disease genetic risk is PanAfrican or restricted to West Africa, and can explain the previously reported low risk of HIVAN among Ethiopians. Methods: We studied a cohort of 338 HIV-infected individuals of Ethiopian ancestry treated in one Israeli and one Ethiopian center. We sought clinical evidence for HIVAN (serum creatinine >1.4 mg/dl or proteinuria >30 mg/dl in a spot urine sample). Genetic analyses included the genotyping of the APOL1 G1 and G2 variants, and a panel of 33 genomic ancestry-informative markers. Statistical analysis compared clinical and genetic indices for HIV-infected individuals of Ethiopian ancestry and overall Ethiopians to those reported for HIV-infected African-Americans, overall African-Americans, West Africans and non-Africans. Findings: Three (0.8%) of 338 HIV-infected patients of Ethiopian ancestry showed clinical criteria compatible with renal impairment. Two of these 3 patients also have severe poorly controlled diabetes mellitus. The third nondiabetic patient underwent renal biopsy which ruled out HIVAN. This absence of clinically apparent HIVAN was significantly different from that reported for African-Americans. The APOL1 G1 and G2 risk variants were found, respectively, in 0 and 2 (heterozygote state) of the 338 HIV-infected individuals. Global ancestry and the frequencies of the APOL1 G1 and G2 variants are not statistically different from their frequencies in the general Ethiopian population, but are significantly and dramatically lower than those observed among HIV-infected African-Americans, African-Americans and West Africans. Interpretation: The coinciding absence of HIVAN and the APOL1 risk variants among HIV-infected individuals of Ethiopian ancestry support a Western rather than Pan-African ancestry risk for ESKD, and can readily explain the lack of HIVAN among individuals of Ethiopian ancestry.

Human parechovirus type 3 central nervous system infections in Israeli infants

INTRODUCTION Human parechoviruses (HPeV) have been recognized as the causative agents of central nervous system (CNS) infection of infants and young children in different parts of the world. The role of HPeV in CNS infection of Israeli infants and children is unknown. OBJECTIVES To assess the detection rate of HPeV in enterovirus RT-PCR-negative cerebrospinal fluid (CSF) samples obtained during the years 2007-2009 from children 0-5 years old with suspected CNS infection or from very young infants with unexplained fever in four medical centers in Israel. STUDY DESIGN A total of 367 CSF samples were retrospectively tested for the presence of HPeV RNA using nested RT-PCR assay. Positive samples were further typed on the basis of molecular sequencing. Retrospective analysis of the medical charts was performed. RESULTS HPeV3 RNA was detected in CSF obtained between May and September 2008 in 13 patients, all of whom were <3 months old (3.5% of all CSFs; 11.3% of all infants<3 months in 2008). The HPeV-positive CSF samples were without pleocytosis. All HPeV3-positive patients recovered without obvious short term sequelae. CONCLUSION HPeV infection could play an important role in summertime febrile/CNS illness in young infants during specific years with high HPeV activity. PCR detection of parechoviral RNA in CSF should be included in the diagnostic evaluation of fever or CNS infection of neonates and very young infants. The rapid identification of HPeV in CSF could curtail unnecessary empirical antibiotic treatment and shorten hospital stay in selected patients.

Insight into the intrinsic sensitivity of the PCR assay used to detect CMV infection in amniotic fluid specimens

BACKGROUND PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. OBJECTIVES To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. STUDY DESIGN Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. RESULTS The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3 x 10(6) viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2 x 10(4) copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8 x 10(3) copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. CONCLUSIONS AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.

Detection, control, and management of a respiratory syncytial virus outbreak in a pediatric hematology-oncology department.

Background: Immunocompromised patients are at increased risk for severe respiratory syncytial virus (RSV) infection. Palivizumab is approved for prevention of RSV in specific populations but not for treatment. Few studies demonstrated the safety and successful treatment with intravenous (IV) palivizumab. We describe our experience with IV palivizumab treatment for RSV in a pediatric hematology-oncology department during an outbreak. Methods: During a short period of renovations, oncology patients were placed in a general pediatric ward. After a case of severe fatal RSV pneumonia in a 2-year-old male patient with acute myeloid leukemia, all patients were actively screened twice weekly regardless of symptoms. Respiratory samples were tested for RSV using rapid immunochromatography detection, immunofluorescence, or reverse transcriptase polymerase chain reaction. A single dose of palivizumab (15 mg/kg) was given to children below 3 years of age who tested positive for RSV. Results: Over a 6-week period, 12 patients tested positive for RSV. Seven patients were treated with palivizumab. Five patients had respiratory symptoms, and 2 were asymptomatic. No adverse events were attributed to IV palivizumab treatment. Early-treated patients had no complications attributed to RSV. Conclusions: Containment of RSV outbreak in high-risk children is difficult. Screening with reverse transcriptase polymerase chain reaction and the early use of IV palivizumab is safe and may prevent complications of RSV infection among these patients.

Reactivation of Varicella Presenting as Pseudotumor Cerebri: Three Cases and a Review of the Literature

The possible association of pseudotumor cerebri and varicella infection was previously mentioned in a few case reports. In those cases, the history and clinical features of active varicella were obvious, and signs were directly related to the varicella infection. We describe three immunocompetent children with pseudotumor cerebri as the only manifestation of Varicella zoster virus reactivation, with a review of the literature. We suggest considering Varicella zoster virus in children with pseudotumor cerebri, even in the absence of a history of recent varicella infection.

A Population-Structured HIV Epidemic in Israel: Roles of Risk and Ethnicity.

Background HIV in Israel started with a subtype-B epidemic among men who have sex with men, followed in the 1980s and 1990s by introductions of subtype C from Ethiopia (predominantly acquired by heterosexual transmission) and subtype A from the former Soviet Union (FSU, most often acquired by intravenous drug use). The epidemic matured over the last 15 years without additional large influx of exogenous infections. Between 2005 and 2013 the number of infected men who have sex with men (MSM) increased 2.9-fold, compared to 1.6-fold and 1.3-fold for intravenous drug users (IVDU) and Ethiopian-origin residents. Understanding contemporary spread is essential for effective public health planning. Methods We analyzed demographic and virologic data from 1,427 HIV-infected individuals diagnosed with HIV-I during 1998–2012. HIV phylogenies were reconstructed with maximum-likelihood and Bayesian methods. Results Subtype-B viruses, but not A or C, demonstrated a striking number of large clusters with common ancestors having posterior probability ≥0.95, including some suggesting presence of transmission networks. Transmitted drug resistance was highest in subtype B (13%). MSM represented a frequent risk factor in cross-ethnic transmission, demonstrated by the presence of Israeli-born with non-B virus infections and FSU immigrants with non-A subtypes. Conclusions Reconstructed phylogenetic trees demonstrated substantial grouping in subtype B, but not in non-MSM subtype-A or in subtype-C, reflecting differences in transmission dynamics linked to HIV transmission categories. Cross-ethnic spread occurred through multiple independent introductions, with MSM playing a prevalent role in the transmission of the virus. Such data provide a baseline to track epidemic trends and will be useful in informing and quantifying efforts to reduce HIV transmission.

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment. Methods Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS. Results 607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16). Conclusions Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.

HCV genotype-1 subtypes and resistance-associated substitutions in drug-naive and in direct-acting antiviral treatment failure patients.

BACKGROUND Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. METHODS In a multicentre cohort study (n=308), NS3 or NS5B sequencing (n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. RESULTS GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. CONCLUSIONS NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.