Transmission Dynamics of HIV Subtype A in Tel Aviv, Israel: Implications for HIV Spread and Eradication.

OBJECTIVE: Subtype-A HIV was introduced into Israel in the mid-1990s, predominantly by immigrants from the former Soviet Union (FSU) infected via intravenous drug use (IVDU). HIV subsequently spread beyond the FSU-IVDU community. In 2012, a mini-HIV outbreak, associated with injection of amphetamine cathinone derivatives, started in Tel Aviv, prompting public health response. To assess current trends and the impact of the outbreak and control measures, we conducted a phyloepidemiologic analysis. METHOD: Demographic and clinical records and HIV sequences were compiled from 312 subtype-A HIV-infected individuals attending the Tel-Aviv Sourasky Medical Center between 2005-2016, where >40% of all subtype-A HIV-infected individuals in Israel are undergoing care. Molecular evolutionary genetics analysis (MEGA) and ayesian evolutionary analysis sampling trees (BEAST) programs were implemented in a phylogenetic analysis of pol sequences. Reconstructed phylogenies were assessed in the context of demographic information and drug-resistance profiles. Clusters were identified as sequence populations with posterior probability ≥0.95 of having a recent common ancestor. RESULTS: After 2010, the subtype-A epidemic acquired substantial phylogenetic structure, having been unrecognized in studies covering the earlier period. Nearly 50% of all sequences were present in 11 distinct clusters consisting of 4-43 individuals. Cluster composition reflected transmission across ethnic groups, with men who have sex with men (MSM) playing an increasing role. The cathinone-associated cluster was larger than previously documented, containing variants that continued to spread within and beyond the IVDU community. CONCLUSIONS: Phyloepidemiologic analysis revealed diverse clusters of HIV infection with MSM having a central role in transmission across ethic groups. A mini outbreak was reduced by public health measures, but molecular evidence of ongoing transmission suggests additional measures are necessary.

Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef.

The role of HIV-specific CD8 T cell activity in the course of HIV infection and the way it affects the virus that resides in the latent reservoir resting memory cells is debated. The PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. CD4 T cells and CD8 T cells procured from PBMC of HIV-infected patients were co-incubated and analyzed: Formation of CD8 T cells and HIV-infected CD4 T cell conjugates and apoptosis of these CD4 T cells were observed by fluorescence microscopy with in situ PCR of HIV LTR DNA. Furthermore, conjugation of CD8 T cells with CD4 T cells and apoptosis of CD4 T cells was observed and quantified by imaging flow cytometry using anti-human activated caspase 3 antibody and TUNEL assay. The conjugation activity and apoptosis were found to be much higher in patients with acute HIV infection or AIDS compared to patients in chronic infection on antiretroviral therapy (ART) or not. Patients on ART had low grade conjugation and apoptosis of isolated CD69, CD25, and HLA-DR-negative CD4 T cells (latent reservoir cells) by CD8 T cells. Using in situ PCR The latent reservoir CD4 T cells were shown to contain most of the HIV DNA. We demonstrate in HIV-infected patients, that CD8 T cells conjugate with and kill HIV-infected CD4 T cells, including HIV-infected resting memory CD4 T cells, throughout the course of HIV infection. We propose that in HIV-infected patients CD4 T cell annihilation is caused in part by ongoing activity of HIV-specific CD8 T cells. HIV Nef protein interacts with ASK 1 and inhibits its pro-apoptotic death signaling by Fas/FasL, thus protecting HIV-infected cells from CD8 T cells killing. A peptide that interrupts Nef-ASK1 interaction that had been delivered into CD4 T cells procured from patients on ART resulted in the increase of their apoptosis inflicted by autologous CD8 T cells. We suggest that elimination of the HIV-infected latent reservoir CD4 T cells can be achieved by Nef inhibition.

Evaluation of the virtues and pitfalls in an HIV screening algorithm based on two fourth generation assays - A step towards an improved national algorithm.

BACKGROUND: Fourth-generation immunoassays used for HIV screening, simultaneously detect anti-HIV antibodies and HIV-1 P24 antigen, but are prone to false-positive results. Usually, they are followed by highly specific third-generation assay, able to differentiate between HIV-1/2 infections. In Israel, screening algorithm is based on consecutive testing by two fourth-generation assays and confirmation by a third-generation test. OBJECTIVES: To evaluate the performance of this algorithm. STUDY DESIGN: Architect HIV1/2 Combo (Combo) reactive results were tested by Vidas HIV Duo Ultra (VD). Confirmation was by INNO-LIA HIV 1/2 or Geenius assays. Five-year results were retrospectively analyzed. HIV true positives (TPs), acute infected (AI), false-positives (FPs) and HIV negatives, were as defined by the algorithm. RESULTS: 501,338 individuals were screened, of which 956 were TPs, 64 AI and 30 F Ps. Specificity was almost 100% and positive predictive value 97%. VD was negative in 94% of confirmed Combo false-reactive individuals. The Combo results in the first tested sample differed substantially between TPs, AI and FPs, enabling the determination of a cutoff value that distinguished 94% of TPs and AI from FPs. CONCLUSIONS: An algorithm is suggested that will use a single sample collection. HIV negative diagnosis will be based on Combo unreactive or Combo reactive/VD negative results. HIV positive diagnosis will be based on Combo reactive/ VD positive results, given a Combo value above a designated cutoff. Below this cutoff samples will be tested by a molecular assay. Since HIV-2 rarely occurs in Israel, the use of a third-generation confirmation assay should be discussed.

Comparison between Roche and Xpert in HIV-1 RNA quantitation: A high concordance between the two techniques except for a CRF02_AG subtype variant with high viral load titters detected by Roche but undetected by Xpert.

BACKGROUND: HIV-1 viral load (VL) testing is important to predict viral progression and to monitor the response to antiretroviral therapy. New HIV-1 VL tests are continuously introduced to the market. Their performance is usually compared to Abbott and/or Roche HIV-1 VL assays, as reference. The Xpert HIV-1 VL test was recently introduced, but its performance compared to Roche has not been sufficiently studied. OBJECTIVES: To compare the Xpert assay with Roche and to assess its use in the HIV clinical laboratory. STUDY DESIGN: A total of 383 plasma samples of HIV-1 infected patients previously tested by Roche, were retrospectively tested by Xpert to determine concordance between the two assays. Samples included a diversity of HIV-1 subtypes and a wide range of VLs. RESULTS: There was a high concordance between the two assays, except for a CRF02_AG subtype variant with high VL titters, that was detected by Roche but undetected by Xpert. The 5' long terminal repeat gene region of this virus, targeted by the Xpert assay, was amplified and sequenced. A 25 nucleotide insert was identified, but was unmatched to any known sequences of HIV-1. This particular insert, however could not explain the false-negativity by the Xpert assay. CONCLUSIONS: This study underlines the challenge to routine VL testing due to the high genetic diversity of HIV-1. Clinicians should, therefore be advised that a negative VL in cases where the clinical picture does not match the laboratory report, might in fact be, a false-negative result of the VL assay.

Validation of two commercial real-time PCR assays for rapid screening of the HLA-B*57:01 allele in the HIV clinical laboratory.

The pharmacogenetics approach to screen for the presence of the HLA-B*57:01 allele in HIV-1 infected patients is mandatory to prevent the potential development of hypersensitivity reaction to abacavir treatment. Given the limitations of current genotype methodologies, commercial real-time PCR assays were specifically developed for this purpose, but have not been sufficiently validated and are still not widely used. Here, in the context of the HIV laboratory, we assessed the ability of two commercial kits, the LightSNiP rs2395029 HPC5 assay (TIB Molbiol) and the DuplicαReal-TimeHLA-B*5701 Genotyping kit (Euroclone), to retrospectively detect HLA-B*57:01 positive and negative samples of Israeli HIV-1 infected patients. The LightSNiP rs2395029 HPC5 assay had false-positive results, whereas the DuplicαReal-Time HLA-B*5701 Genotyping kit was highly accurate and could be readily implemented into clinical practice. It is hoped that this study will facilitate the assessment of additional commercial kits for HLA-B*57:01 detection and expand their use in the clinical laboratory. Such studies can likely help the use of abacavir treatment in HIV-1 infected patients.

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.

Peripheral blood mononuclear cells of HIV-infected patients contain CD8 T cells that form conjugates with and kill HIV-infected autologous CD4 T cells.

Peripheral blood mononuclear cells (PBMC) of untreated, HIV-infected patients contain HIV-specific CD8 T cells as well as their corresponding targets, HIV-infected CD4 T cells. To determine if CD4 T-cell depletion in HIV-infected patients may result from autologous CD8-CD4 T-cell interaction, CD8 and CD4 T cells procured from PBMC of acute and chronic untreated HIV-infected patients were sorted and co-incubated. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy. Apoptosis of CD4 T cells in conjugation was recorded by digitized images and was further observed and measured by FACS using Annexin staining. Perforin expression in the CD8 T cells was measured using intracellular monoclonal perforin antibody staining. HIV DNA in the conjugated CD4 T cells was detected by in situ PCR. We found that 6·1 ± 0·5% of CD4 T cells from acute HIV-infected patients and 3·0 ± 0·5% from chronic HIV-infected patients formed CD8-CD4 T-cell conjugates. Annexin binding and cell morphology typical of apoptosis were observed in the conjugated CD4 T cells. The majority of CD8 T cells that had conjugated to CD4 T cells expressed perforin. The conjugated CD4 T cells exhibited nuclear HIV DNA. CD8 T cells and HIV-infected CD4 T cells, both procured from the PBMC of untreated HIV-infected patients, form conjugates. Apoptotic lytic activity has been observed in the conjugated CD4 T cells. We propose that CD4 T-cell annihilation in HIV-infected patients results, at least in part, from the interactions of perforin-rich CD8 T cells with autologous, HIV-infected CD4 T cells.

[Relevance of HIV resistance testing for treatment decisions].

Resistance testing is part of the routine checkup for HIV carriers in Israel and in most of the developed world. Viruses with mutations which confer resistance to antiretroviral therapy in treated patients and in new HIV carriers are identified. The results of these tests form the basis for updating the HIV treatment guidelines and contribute to the epidemiological and phylogenetic understanding of the HIV epidemic. The viral reverse transcriptase and protease are the targets for most of the antiretroviral drugs in use today and are included in the standard resistance testing. Recently, genotypic examination of the integrase and tropism test to verify use of the HIV CCR5 co-receptor have been introduced to better support treatment decisions and to enable effective use of all available drug combinations. New and more sensitive molecular tests, such as ultra-deep sequencing, are expected to broaden our knowledge of rare mutations not detected by the currently used methodologies. Consequently, we will be able to improve treatment strategy and life quality and increase Life expectancy of HIV carriers.

Are the human papillomavirus genotypes different in cervical cancer and intraepithelial neoplasia in Jewish Israeli women, a low-risk population?

OBJECTIVES: Israel is traditionally considered to have the lowest prevalence of cervical cancer compared with that in other countries of the Western world. The aim of the present study was to establish the human papillomavirus (HPV) genotypes distribution among Israeli Jewish women with premalignant and cervical cancer. METHODS: Fifty-two specimens with invasive cervical cancer and 50 specimens with high-grade cervical intraepithelial neoplasia (CIN2/3) were identified. Human papillomavirus genotyping in paraffin-embedded specimens was performed by deparaffinization of the tissue sections and DNA extraction, followed by HPV genotype detection using a validated polymerase chain reaction (PCR)-based HPV GenoArray test kit, to simultaneously identify 21 HPV genotypes. RESULTS: Forty-eight (48/52; 92.3%) cervical cancer samples demonstrated PCR-amplifiable DNA (non-HPV DNA). Forty (83.3%) of 48 samples were high-risk (HR) HPV positive. Six (12.5%) of 48 patients showed multiple HR HPV infections. Human papillomavirus types 16 and 18 dominated covering 28 (58.3%) and 14 (29.16%) of 48 samples. Human papillomavirus types 16 and 18 coinfected all 6 cases of multiple HR HPV infections. In CIN2/3 samples, 37 (78.7%) of 47 samples demonstrated PCR-amplifiable DNA (non-HPV DNA), and 20 (54.0%) of these 37 samples were infected by HPV. Human papillomavirus type 16 was found in 19 (95.0%) of 20 cases. Human papillomavirus type 18 was found in 3 (15.0%) of 20 cases; hence, HPV16 and HPV18 contributed to 100% of the cases. CONCLUSIONS: Human papillomavirus types 16 and 18 were responsible for the vast majority of invasive cervical cancer and CIN2/3 specimens (81.2% and 100%, respectively). Therefore, it is essential to include the HPV vaccine in the vaccine schedule of the Israeli population.

Transmission patterns of HIV-subtypes A/AE versus B: inferring risk-behavior trends and treatment-efficacy limitations from viral genotypic data obtained prior to and during antiretroviral therapy.

BACKGROUND: HIV subtypes A and CRF01_AE (A/AE) became prevalent in Israel, first through immigration of infected people, mostly intravenous-drug users (IVDU), from Former Soviet-Union (FSU) countries and then also by local spreading. We retrospectively studied virus-transmission patterns of these subtypes in comparison to the longer-established subtype B, evaluating in particular risk-group related differences. We also examined to what extent distinct drug-resistance patterns in subtypes A/AE versus B reflected differences in patient behavior and drug-treatment history. METHODS: Reverse-transcriptase (RT) and protease sequences were retrospectively analyzed along with clinical and epidemiological data. MEGA, ClusalX, and Beast programs were used in a phylogenetic analysis to identify transmission networks. RESULTS: 318 drug-naive individuals with A/AE or patients failing combination antiretroviral therapy (cART) were identified. 61% were IVDU. Compared to infected homosexuals, IVDU transmitted HIV infrequently and, typically, only to a single partner. 6.8% of drug-naive patients had drug resistance. Treatment-failing, regimen-stratified subtype-A/AE- and B-patients differed from each other significantly in the frequencies of the major resistance-conferring mutations T215FY, K219QE and several secondary mutations. Notably, failing boosted protease-inhibitors (PI) treatment was not significantly associated with protease or RT mutations in either subtype. CONCLUSIONS: While sizable transmission networks occur in infected homosexuals, continued HIV transmission among IVDU in Israel is largely sporadic and the rate is relatively modest, as is that of drug-resistance transmission. Deviation of drug-naive A/AE sequences from subtype-B consensus sequence, documented here, may subtly affect drug-resistance pathways. Conspicuous differences in overall drug-resistance that are manifest before regimen stratification can be largely explained in terms of treatment history, by the different efficacy/adherence limitations of older versus newer regimens. The phenomenon of treatment failure in boosted-PI-including regimens in the apparent absence of drug-resistance to any of the drugs, and its relation to adherence, require further investigation.

Evaluation of a benchtop HIV ultradeep pyrosequencing drug resistance assay in the clinical laboratory.

Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had low-abundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

Syphilis outbreak among men who have sex with men, Tel Aviv, Israel, 2008-2009.

BACKGROUND: Outbreaks of syphilis have been described among men who have sex with men (MSM) in many western urban communities in the last few years. OBJECTIVES: To describe the first reported outbreak of syphilis among MSM in Israel within a decade of a constant increase in human immunodeficiency virus (HIV) prevalence. METHODS: All patients diagnosed with syphilis were contacted and asked about their sexual behavior, substance use and previous infections. All were tested for HIV and a phylogenetic analysis was performed. RESULTS: A total of 23 (59%) of all 39 male patients diagnosed with primary or secondary syphilis between August 2008 and August 2009 were interviewed. All were MSM and performed anal intercourse, while 13 (55%) reported unprotected anal intercourse. Most participants (21, 91%) practiced unprotected oral intercourse. Nine participants (39%) reported unprotected oral intercourse while using condoms during anal intercourse. Ten participants (43%) reported sexual contacts while traveling abroad in the previous few months. Most participants (96%) were co-infected with HIV, and 15 (68%) were already aware of their HIV infection. Fifteen (66%) reported the use of recreational drugs, alcohol, or both before or during sex. No common source or core transmitters were identified. CONCLUSIONS: This syphilis outbreak included MSM who were co-infected with HIV and were characterized by risky sexual behavior including multiple partners, unprotected anal intercourse and substance use. Future targeted interventions should focus on HIV-infected MSM for secondary prevention.

Serum immunoglobulin G avidity test as a tool for improving diagnosis of cat scratch disease.

Cat scratch disease (CSD) is a common cause of subacute infectious regional lymphadenitis, caused by Bartonella henselae. Presently, detection of anti-B. henselae antibodies by immunofluorescence antibody assay or enzyme immunoassay (EIA) is the most widely used diagnostic test for CSD, but both are limited in establishing the timing of infection with B. henselae. In the present work we developed an avidity test for anti-B. henselae immunoglobulin G (IgG) based on EIA to distinguish recent from past CSD. We used 101 serum samples from 79 CSD patients with positive anti-B. henselae IgG as verified by EIA, and systematically developed an avidity assay using various detergent (urea) concentrations and incubation settings to optimize the test conditions to differentiate early CSD (less than 12 weeks) from late CSD (12 weeks or more). After serial experiments, the optimal conditions for performing the avidity test included incubation for 10 min at room temperature with 8 M urea at pH 7.4, and these parameters were used in the study. Our experiments showed that while the avidity indexes (AIs) of the early CSD samples were widely distributed, all the late CSD sera samples had AIs above 43, indicating that an AI <43 can serve as evidence of early CSD. The results of this study indicate that the avidity test can be useful in the serodiagnosis of CSD, particularly when anti-B. henselae IgM antibodies are not detected.

Cat scratch disease during pregnancy.

OBJECTIVE: To describe the pregnancy outcome, including long-term follow-up of the offspring, of pregnant women with cat scratch disease. METHODS: A surveillance study performed over 19 years identified eight pregnant women with cat scratch disease. A case of cat scratch disease was defined as a patient with a history of cat contact with regional lymphadenitis, other manifestations, or a combination of these consistent with the disease and one or more confirmatory laboratory tests. The clinical and laboratory manifestations and pregnancy outcome of all women diagnosed with cat scratch disease during pregnancy are described. RESULTS: Five of the eight pregnant women had typical disease with regional lymphadenitis; two had regional lymphadenitis with arthralgia, myalgia, and erythema nodosum; and one had neuroretinitis. Delayed diagnosis was common, although all women had a history of recent cat exposure. One woman who presented with clinical cat scratch disease during the first month of pregnancy had a spontaneous abortion. Another elected to terminate the pregnancy because of concerns related to radiation associated with abdominal computed tomography scan performed as part of an evaluation for suspected malignancy. The other six women gave birth to healthy newborns without congenital anomalies. No sequelae were recorded in mothers or children during a median follow-up of 4.5 years (range 0.5-9.5 years). CONCLUSION: With the exception of one early spontaneous abortion in which causality to cat scratch disease could not be established, neither deleterious effects of cat scratch disease on newborns nor reports of long-term sequelae were found. Physicians, especially family physicians and obstetrician-gynecologists need to be more familiar with the clinical manifestations of cat scratch disease. Close monitoring of infected women during pregnancy is advisable until more data are available to determine the optimal diagnostic and therapeutic approach.

Incidence of cytomegalovirus-associated thrombosis and its risk factors: a case-control study.

INTRODUCTION: Cytomegalovirus (CMV)-associated thrombosis has been reported sporadically in the medical literature until now. However, thrombosis incidence and its risk factors have never been studied in a cohort of patients with acute CMV infection. MATERIALS AND METHODS: A retrospective case-control study. Medical charts and imaging study reports of all consecutive patients diagnosed with acute CMV infection during the years 2005-2006 in a tertiary medical center were reviewed for the presence of arterial and/or venous thromboses, and their acquired as well as inherited predispositions. The control group included age-matched and sex-matched consecutive patients, in whom acute CMV infection was excluded. Laboratory tests used for acute CMV infection diagnosis/exclusion were also matched, including serology, antigenemia, and PCR. RESULTS: Included were 140 patients with acute CMV infection (study group) and 140 consecutive matched patients in whom acute CMV infection was excluded (control group). Among the control group, none of the patients had thrombosis, while among the study group, nine (6.4%; p=0.003) patients had thrombosis: five (3.6%; p=0.025) patients had arterial thrombosis and four (2.9%; p=0.045) patients had venous thrombosis. Binary logistic regression analysis showed that acute CMV infection was independently associated with thrombosis among the whole cohort (p=0.004), while use of oral contraceptives/hormones or pregnancy were independently associated with thrombosis among patients with acute CMV infection (p=0.043). CONCLUSIONS: Thrombosis in patients with acute CMV infection is not rare. Acute CMV infection is associated with thrombosis independent of other risk factors for thrombosis. We hope to raise physician's awareness to the association between acute CMV infection and thrombosis.

Molecular detection of Bartonella henselae and Bartonella koehlerae from aortic valves of Boxer dogs with infective endocarditis.

Cardiac aortic valves from five dogs that died from acquired infective endocarditis were retrospectively molecularly screened for Bartonella infection. Identification was carried out using PCR targeting four gene fragments (rpoB, ribC, 16S rRNA and gltA), and the 16S-23S intergenic spacer (ITS). Bartonella henselae DNA was detected in aortic valve tissue from one Boxer dog with moderate subaortic stenosis (SAS). Bartonella koehlerae DNA was detected from the aortic valve of another Boxer dog with severe SAS. The latter dog was both a littermate and a housemate of the dog with the B. henselae infection. Other animals residing at the same household were also screened for Bartonella infection. B. henselae was molecularly detected in a spleen aspirate from the dogs' mother, and isolated and molecularly characterized from another housemate cat. This is the first molecular identification of B. henselae and B. koehlerae, two zoonotic Bartonella species, from valves of dogs with canine infective endocarditis, suggesting their role in the pathogenesis of this disease. Moreover, this is the first report describing the detection of B. koehlerae from dogs.

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR.

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S-23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.

Prevalence of the K65R resistance reverse transcriptase mutation in different HIV-1 subtypes in Israel.

The K65R mutation in HIV-1 reverse transcriptase (RT) can be selected by the RT inhibitors tenofovir (TDF), abacavir (ABC), and didanosine (DDI). Recently, in vitro studies have shown that K65R is selected in tissue culture more rapidly with subtype C than subtype B viruses. The prevalence of K65R in viruses sequenced at the Tel-Aviv AIDS Center was evaluated. This study analyzed retrospectively sequences from 1999 to 2007 in patients treated with TDF, ABC, and/or DDI and compared rates of mutational prevalence between subtypes. Fisher's exact test was used to determine statistical significance. Forty-four sequences from patients treated with the three above-cited drugs were analyzed. Subtypes A (n = 1), CRF01_AE (n = 4), CRF02_AG (n = 2), B (n = 21), C (n = 11), D (n = 1), F (n = 3), and G (n = 1) were represented. Seven non-B viruses had the K65R mutation, which was only found in one subtype B virus. Of these seven samples four were subtype C, one was subtype CRF01_AE, and two were subtype CRF02_AG. None of the eight viruses with K65R harbored thymidine analogue mutations. In this study, non-subtype B viruses possessed the K65R mutation at higher incidence than subtype B viruses. Subtype C viruses may be especially prone to develop this mutation. Larger studies are needed to confirm these data. Efforts should be intensified to understand better differences in drug resistance between various HIV subtypes.

Isolation and genetic characterization of a Bartonella strain closely related to Bartonella tribocorum and Bartonella elizabethae in Israeli commensal rats.

Ten Bartonella isolates were cultured from blood drawn from black rats (Rattus rattus) captured in the Tel Aviv area. Genetic characterization included amplification and sequencing of five gene fragments including the ribC, rpoB, 16S, groEL, and gltA and the 16S-23S intergenic spacer region. Sequence comparisons showed that all 10 isolates were identical in all genes studied comprising a total of 3,873 bp analyzed. The sequences of each of the partial genes analyzed indicated a high sequence similarity (97-99.8%) to B. tribocorum or B. elizabethae. The gltA sequence was 100% homologous to a genotype identified in R. rattus in Dhaka, Bangladesh, suggesting the existence of a widespread Asian Bartonella strain infecting the black rats (R. rattus). The detection of a Bartonella genotype closely related to B. elizabethae in the biggest metropolitan center in Israel warrants further study of its zoonotic potential and pathogenic characteristics.

Pilot study of prevalence of high-risk human papillomavirus genotypes in Israeli Jewish women referred for colposcopic examination.

The objective of the present study was to determine the prevalence of high-risk (HR) human papillomavirus (HPV) genotypes in a group of Israeli Jewish women referred for colposcopic examination. Scrape specimens were prospectively collected from 84 women referred for colposcopic examination. All the women underwent Papanicolaou (Pap) smears and colposcopies, and some also underwent cervical or loop electrosurgical excision procedure biopsy. HR HPV was detected in scrape specimens (Amplicor HPV test; Roche Molecular Systems), and the individual genotypes in these specimens were identified (HPV GenoArray test kit; Hybribio Ltd., Hong Kong). Forty-one (49%) specimens were positive by the Amplicor HPV test. Sixty-four samples (41 positive and 23 negative by the Amplicor HPV test) were also assayed by use of the HPV GenoArray kit. The overall level of agreement between the two assays was 93.8% (Cohen's kappa = 0.98). HR genotypes were found in 37/41 (90%) HPV-positive samples. The prevalences of the HR HPV genotypes in the 37 HPV-positive samples were 41% of patients for HPV type 16 (HPV-16), 22% for HPV-39, 19% for HPV-52, and 14% for HPV-18. Forty-one percent of these patients were infected with a single HR genotype, whereas 59% were infected with mixtures of HR genotypes. The presence of a relatively high percentage of HPV types 39 and 52 and the relatively high incidence of infections with mixtures of genotypes may be one of the reasons for the low rate of conversion from high-grade squamous intraepithelial lesions to invasive carcinoma in Israeli women. Larger and more comprehensive studies are warranted to investigate this issue in greater detail.