Drug-resistant HIV infection among drug-naive patients in Israel.

BACKGROUND: In Israel, <0.06% of the general population is infected with human immunodeficiency virus (HIV), with a much higher prevalence among specific groups. These groups are distinguished demographically by risk behavior category and by virus subtype. We investigated transmission of drug resistance within groups to assess the impact of these factors. METHODS: Plasma samples from >15% of all patients with new diagnoses of HIV infection were randomly collected between June 1999 and June 2003. Sequences from 176 drug-naive patients included 20 of subtype A, 20 of subtype AE, 2 of subtype AC, 29 of subtype B, 100 of subtype C, and 5 of subtype F. RESULTS: Major drug resistance mutations (protease: L90M; reverse transcriptase: M41L, K103N, V106M, M184V, Y181S, G190A, L210W, T215Y/F, and K219R) were detected in 1 subject with A subtype, 3 with subtype B, and 9 with subtype C. In addition, 1 subject with A subtypes, 2 with subtype B, and 10 with subtype C had secondary mutations (protease: M46I; reverse transcriptase: A98G, K101Q, and V108I). Only 1 patient had mutations associated with >1 class of drugs. Among subjects who contracted HIV infection in Israel, 16 of 56 (1 of 7 with subtypes A or AE, 4 of 17 with subtype B, and 11 of 32 with subtype C; P=.7-1.0) carried resistant virus--a significantly higher proportion (P<.001) than in subjects infected in other countries (10 of 120 infected). CONCLUSIONS: Drug-resistant virus was detected in 14.8% of patients with new diagnoses of HIV infection but in 28.6% of patients known to have been infected in Israel. The implications include a need for pretreatment resistance testing and for better programs aimed at prevention of transmission, directed particularly at patients. We did not find significant differences in transmission of resistant virus between those infected with subtypes B and C, despite the different demographic background. A conclusive analysis and interpretation should await a more extensive study.

Evaluation of ELISA-based sero-diagnosis of dengue fever in travelers.

Diagnosis of dengue virus infection in travelers is often based on commercially available ELISA-based serological assays and not on the more difficult and costly procedures of Hemagglutination inhibition (HI), virus isolation or RT-PCR. These standard assays are not quantitative and are designed to diagnose primary and secondary dengue virus infections by testing for IgG and IgM antibodies. However, cross reactivity between various flaviviruses and the fact that most travelers today are prevaccinated against Japanese encelphalitis (JE) and yellow fever (YF) create a potential problem in such diagnosis. Our study was aimed at measuring the extent of false positive diagnosis in prevaccinated travelers which we have assessed by testing for dengue IgG and IgM antibodies in a group of prevaccinated healthy travelers using the PanBio indirect IgG ELISA and IgM capture ELISA kits. The IgM test was negative in all healthy vaccinees, thus, being highly specific. However, the kit had a disadvantage, which was recognized in other travelers clinically ill with dengue fever (DF), in which the IgM response was detected only 4-8 days after onset of the clinical symptoms. The IgG test yielded 11-17 and 15-44% positives in healthy travelers vaccinated against JE and YF, respectively. We conclude that the specificity of the IgG-ELISA assay in prevaccinated travelers is much lower than in unvaccinated populations. Thus, an IgG-positive results in a vaccinated traveler and IgM negative result during the 1st week of the illness period, are both inconclusive.

Molecular epidemiology of adenovirus type 7 in Israel: identification of two new genome types, Ad7k and Ad7d2.

The molecular epidemiology of Adenovirus type 7 in Israel was investigated. Fifty-seven adenovirus isolates identified as serotypes 7 or 7a which were recovered from patients in Israel between 1968 and 1995 were analyzed by restriction enzymes digestion using BamHI for primary discrimination and identification of genome types and by six additional enzymes: BstEII, HpaI, BglI, BglII, BclI, and XbaI for confirmation and determination of genomic subtypes. Four digestion patterns were identified with BamHI; one of them was new. Using BstEII, two patterns were obtained, one of them new. Digestion with the other five enzymes yielded known patterns. The analysis revealed four different genomic types and subtypes, which circulated in Israel in different years: subtype 7a1; type 7b, a type with a new BamHI pattern which was designated type 7K, and a subtype with a new BstEII pattern which differed from type 7d by one restriction site and was designated type 7d2. Twenty-two isolates from 1968 through 1975 and from 1984 were Ad7a1. Three isolates from 1973-1974 were Ad7b. Five isolates from 1968 through 1973 were Ad7K and 27 isolates from 1992 through 1995 were Ad7d2. This demonstrates the temporal change in the circulating genome types with up to three genome types cocirculating in 1 year (1973). The two new types, Ad7k and Ad7d2 could have evolved in Israel or could have been imported by travellers and immigrants from neighboring or distant countries.

Detection of adeno-associated virus type 2 sequences in the human genital tract.

Adeno-associated virus (AAV) is a defective parvovirus with unknown pathogenicity. It requires helper functions for its normal replication in human tissue and therefore is not readily isolated from clinical specimens. We have used the PCR method to examine the following clinical samples for the presence of AAV sequences: (i) 15 nasopharyngeal aspirates from symptomatic patients, (ii) 7 swab or fluid specimens from vesicles of patients suspected of having varicella-zoster virus infections, (iii) 21 human papilloma virus-positive genital biopsy specimens, (iv) 61 genital swab specimens from women suspected of having herpes simplex virus (HSV) infection examined either directly or following propagation in tissue culture, (v) 62 samples of first-trimester aborted material, including 38 samples from spontaneous abortions and 24 samples from induced abortions, (vi) 11 samples of chorionic villi taken from women undergoing genetic prenatal diagnosis, and (vii) three lots of cultured human embryonic cells. AAV sequences were detected only in samples taken from the genital tracts of women suspected of having HSV infection and not in any of the other types of samples. Samples from 11 patients were positive for AAV: for 4 patients the original swab sample was positive, for 4 patients the cultured swab sample was positive, and for 3 patients both the original swab samples and the cultures were positive. Five of the 11 patients were infected with HSV. Our study demonstrates the presence of AAV in the female genital tract. However, in contrast to a previous report (E. Tobiasch, M. Rabreau, K. Geletneky, S. Larue-Charlus, F. Severin, N. Becker, and J. R. Schlehofer, J. Med. Virol. 44:215-222, 1994), we did not find solid evidence of its replication in maternal or embryonal tissues from the first trimester of pregnancy. The questions of a potential pathogenic etiology of AAV and the interaction with HSV remain open.

Replication of adeno-associated virus type 2 in human lymphocytic cells and interaction with HIV-1.

Adeno-associated virus (AAV) is a nonpathogenic parvovirus which normally requires helper adenovirus or herpes-virus for replication. We examined the growth of AAV type 2 in human lymphocytes and its possible interaction with HIV-1. Three B cell lines (CK-B, HS-2, and UC729) and four T cell lines (Molt-4, Jurkat, HUT78, and HUT78+HIV, which is persistently infected with HIV-1) were infected with AAV either in the presence or in the absence of adenovirus. AAV DNA was found in cells of all the lines following incubation with the virus, indicating absorption. AAV DNA replication occurred in most cell lines without particular preference for B or T cells, but only in the presence of helper virus, either adenovirus or Epstein-Barr virus. Expression of AAV proteins was examined by immunoblotting and ELISA, using sera specific for AAV Rep or capsid proteins. The level of AAV protein synthesis correlated with the efficiency of AAV DNA replication, and both varied between cell lines. The yield of infectious AAV was low in most cases, except in one T4 line (Jurkat), where AAV replication and protein synthesis in the presence of adenovirus were very extensive. In HUT78+HIV cells both adenovirus and AAV (in the presence of Ad2) replicated efficiently. The effects of adenovirus plus AAV coinfections on HIV-1 replication, measured by reverse-transcriptase (RT) activity, were mild. Infection with adenovirus or AAV alone resulted in a 60-70% increase in RT activity, while infection with AAV plus adenovirus resulted in a 20% decrease in RT activity. The yield of infectious AAV in this cell line was very low.

Detection of adeno-associated virus type 2 in human peripheral blood cells.

The non-pathogenic human parvovirus, adeno-associated virus (AAV) is helper virus-dependent. However, it integrates into the cellular genome in the absence of its helper viruses. Therefore it could become a useful vector for gene therapy. Previous studies and our own results have shown that 40 to 80% of adults are seropositive for AAV and that seroconversion occurs during the first few years of life, but little is known about the route of natural infection with the virus. We used the polymerase chain reaction to detect the AAV-2 genome and identify AAV sequences within peripheral blood leukocytes (PBLs). We could detect AAV in PBLs of two of 55 healthy blood donors, and two of 16 haemophilic patients. AAV DNA replication and viral protein production in PBLs propagated in tissue culture were also examined. AAV DNA replicated very efficiently in the presence of helper adenovirus, but capsid proteins were produced at a lower level and the yield of infectious virus was very low. Our findings prove that in vivo infection of PBLs occurs, and that PBLs could mediate the spread of AAV infection to different body tissues.

EBV genome and immunoglobulin gene rearrangement in the differential diagnosis of nasopharyngeal carcinoma and lymphoma.

Head and neck tumors include nasopharyngeal carcinoma (NPC) and lymphoma. The differential diagnosis of these tumors is based on histology, immunocytochemical staining, and EBV serology. In rare cases it might be difficult to distinguish between NPC and lymphoma in HE section or biopsies. DNA hybridization with cloned EBV and human immunoglobulin gene fragments allows the detection of EBV-related sequences and immunoglobulin gene rearrangements. The presence of EBV genome supports the diagnosis of NPC or EBV related BL, while rearrangement of immunoglobulin genes points to B-cell lymphoma. The diagnosis in 11 patients suspected of head and neck tumors was carried out by hybridization of DNA extracted from the tumors and assayed with cloned EBV and IgHCJ DNA probes. One patient proved to have EBV-associated BL based on positive hybridization with EBV probes and immunoglobulin rearrangement, presenting a unique hybridization with cloned EBV DNA BamHI W fragment, with bands of 3.2 and 3.9 kb. BL was confirmed in this patient by demonstration of c-myc rearrangement. A second patient was negative in hybridization with EBV, and positive for immunoglobulin rearrangement, and therefore was diagnosed as having B-cell lymphoma. In seven patients NPC was confirmed by hybridization with EBV-DNA probes. In two patients, both NPC and B-cell lymphomas were excluded.

Aplastic crisis due to human B19 parvovirus infection in red cell pyrimidine-5'-nucleotidase deficiency.

Two siblings with chronic hemolytic anemia due to red cell pyrimidine-5'-nucleotidase (P-5'-N) deficiency, presented within a few days of each other with a febrile illness and pancytopenia. The cause of the aplastic crisis was an acute infection with human B19 parvovirus (B19 HPV) as proven by immunoelectron microscopy and DNA hybridization. This is the first report on the association of B19-HPV-related aplastic crisis with P-5'-N deficiency.

Foetal testicular steroidogenesis and responsiveness to LH in freemartins and their male co-twins.

Foetal gonads were obtained from 1) 8 male foetuses that were co-twin to freemartins, 2) 8 isosexual twins, 3) 59 singletons of 45-75 days of gestations, 4) 5 isosexual and 5 male co-twins of 90-120 days and 5) 5 freemartins at 70-120 days of gestation. The gonads were incubated in supplemented medium 199 for 24 h in the absence or presence or LH and the testosterone and progesterone produced measured by RIA. During the time of sexual differentiation (45-75 days) the testes of the male co-twins produced significantly less testosterone than testes from isosexual or singletons of the same age. Testes of foetuses older than 90 days were refractory to LH-stimulation, but freemartin ovaries and testes from their male co-twins continued to respond to LH with increased testosterone production. No significant differences in progesterone production were detected amount co-twins, isosexual twins or singletons at any age. We conclude that the testes of the male twin that is co-twin to a freemartin displays abnormal steroidogenesis. This may be related to reports of abnormal testes after birth in males co-twin to freemartins.