Successful control of a large outbreak of HIV infection associated with injection of cathinone derivatives in Tel Aviv, Israel.

OBJECTIVES: Outbreaks of HIV infection have been linked to injectable drug abuse, but specific triggers often remain obscure. We report on an outbreak of primary HIV infection among people who inject drugs (PWID) in Tel Aviv, associated with a local shift in drug-use practices. METHODS: A cluster of primary HIV infection cases in PWID was detected in May 2012. Retrospective and prospective multi-hospital case finding was initiated. PWID were interviewed and risk factors for primary HIV infection were identified. Starting in December 2012, a multifaceted intervention was implemented, including educational activities, increasing syringe exchange supplies, active screening, early initiation of antiretroviral therapy, and referral to drug withdrawal programmes. RESULTS: Forty-two PWID with primary HIV infection were detected between May 2012 and April 2013. Compared with the corresponding pre-outbreak period, the annual incidence of primary HIV infection in PWID increased from 0 to 20 cases/1000 population (p <0.0001). Sixty-nine per cent were hospitalized because of concomitant bacterial infections and sepsis. Phylogenetic analysis of HIV isolates from case patients showed tight clustering suggesting a single common source of infection. The outbreak was temporally related to a widespread shift from heroin to injectable cathinone-derivatives and buprenorphine, which entailed high-risk injection practices. Targeted intervention resulted in a dramatic and sustained reduction in HIV infection in the PWID population. CONCLUSIONS: Injectable amphetamines are gaining momentum among PWID worldwide. Tracing of this outbreak to cathinone use and implementing a targeted intervention programme effectively terminated the outbreak.

Emergence of an HIV-1 cluster harbouring the major protease L90M mutation among treatment-naïve patients in Tel Aviv, Israel.

OBJECTIVE: Drug resistance-associated mutations (DRMs) among HIV-1 treatment-naïve patients have increased in recent years. Their incidence and prevalence in various exposure risk categories (ERCs) were evaluated. DESIGN: Plasma samples of HIV-1 treatment-naïve patients diagnosed between 2001 and 2009 at the Tel Aviv Medical Center were screened for DRMs. METHODS: Samples obtained from patients following the HIV diagnosis were analysed retrospectively. Genotyping was carried out using the Trugene HIV-1 genotype kit (Siemens, Berkeley, CA, USA). Phylogenetic relationships among viral sequences were estimated using the maximum likelihood method. RESULTS: Thirty-eight of the 266 analysed sequences (14.3%) had DRMs, all occurring exclusively in the group of men who have sex with men (MSM). The rate of DRMs has constantly risen, reaching a peak of 21.9% in 2009. Notably, protease inhibitor (PI) DRMs became the most frequent DRMs in 2009. Phylogenetic analysis showed a tight cluster comprising 13 of 14 viruses harbouring the L90M major PI resistance mutation, suggesting a single infection source. CONCLUSION: There was an unexpectedly high rate of the major L90M PI resistance mutation in the MSM group. The clustered transmission of this mutation might be related to a high-risk sexual behaviour. Added to nonnucleoside reverse transcriptase inhibitor and nucleoside reverse transcriptase inhibitor resistance mutations, such a PI mutation may limit future therapeutic options for this particular patient population.

Enzyme immunoassay for the diagnosis of cat-scratch disease defined by polymerase chain reaction.

Whole-cell immunofluorescent antibody (IFA) tests for detection of anti-Bartonella henselae immunoglobulin (Ig) G are commonly used to diagnose cat-scratch disease (CSD). The need to cultivate B. henselae in Vero cells for antigen preparation and the absence of routinely applied IFA assays for IgM constitute the major disadvantages of this form of test. We describe the results of an enzyme immunoassay (EIA) for IgM and IgG that used N-lauroyl-sarcosine-insoluble outer membrane antigens from agar-grown B. henselae performed in 84 patients with definite CSD (regional lymphadenitis, cat contact, and > or =1 confirmatory test: polymerase chain reaction, skin test, or B. henselae culture). Although this method has been used as a diagnostic tool in several case reports, it has not previously been evaluated in a large study of definitively proven CSD cases. Results of this study indicate that the EIA described herein can play an important role in the serodiagnosis of CSD, although improvement of the sensitivity, particularly that of the IgM, would be desirable.

DNA amplification for the diagnosis of cat-scratch disease in small-quantity clinical specimens.

Diagnosis of cat-scratch disease (CSD) by polymerase chain reaction (PCR) of lymph node fineneedle aspiration (FNA) and primary lesion specimens can be difficult owing to the minute amount of available material. A PCR assay specifically suited to test these specimens was developed. First, small-quantity (10 microL) samples were prepared from 17 CSD-positive and 16 CSD-negative specimens, and DNA extraction and amplification from these samples were compared using 3 methods. Sensitivity and specificity of PCR were 100% using material collected on glass microscope slides and by using Qiagen (Hilden, Germany) columns for DNA extraction. Then, this method was used to test 11 archival glass microscope slides of FNA (7 malignant neoplasms, 4 undiagnosed lymphadenitis) and 2 primary lesion specimens. Two of the 4 lymphadenitis samples and the 2 primary lesion specimens were PCR positive. The technique presented could facilitate CSD diagnosis from a wider range of clinical samples.

Unusual eruption as a presenting symptom of cat scratch disease.

Cat scratch disease (CSD) is a common infectious cause of subacute regional lymphadenopathy. Bartonella henselae is the principal etiologic agent. About 10% of CSD patients experience atypical manifestations, including rashes. The most common cutaneous manifestation of CSD is a papule at the inoculation site. We report a case of CSD presenting with an eruption on the upper trunk, reminiscent of Sweet's syndrome, accompanied by lymphadenopathy, arthralgia, and fever. Response to systemic corticosteroids was remarkable. Histopathologic findings refuted the diagnosis of Sweet's syndrome. Identification of anti-B henselae antibodies and B henselae DNA in the affected lymph node confirmed the diagnosis of CSD. This is a first report of extensive papuloedematous eruption as a cutaneous manifestation of CSD. Accurate diagnosis is possible due to the availability of serological tests and DNA amplification techniques.

Cat scratch disease: the rare role of Afipia felis.

Since its isolation in 1988, Afipia felis has been associated with cat scratch disease (CSD) in only one report and its role in CSD has been questioned. We have cultured A. felis from a lymph node of a patient with CSD. 16S rRNA gene sequencing, DNA relatedness studies, fatty acid analysis, and PCR of the A. felis ferredoxin gene showed that the isolate is identical to the previously reported A. felis isolate. To determine the role of A. felis in CSD, PCR of the 16S rRNA gene followed by hybridizations with specific probes were performed with lymph node specimens from CSD patients. All 32 specimens tested positive for Bartonella henselae and negative for A. felis. We conclude that A. felis is a rare cause of CSD. Diagnostic tests not conducive to the identification of A. felis might cause the diagnosis of CSD due to A. felis to be missed.

The DNA sequence of the RK strain of human herpesvirus 7.

The complete DNA sequence of human herpesvirus-7 (HHV-7) strain RK was determined following direct cloning of virion DNA fragments into a sequencing vector. The sequence was compared with the previously published complete sequences of HHV-7 strain JI and human herpesvirus-6 (HHV-6) strain U1102. Despite a very close relationship between the two HHV-7 strains, differences are apparent in regions containing tandem reiterations, particularly in the "telomeric" reiterations located near the termini of the large direct repeat at the genome ends, and in a total of 179 additional positions distributed throughout the genome (i.e., about one nucleotide difference per kbp). This extent of divergence implies that the two strains arose from an ancestral virus several thousands of years ago. Differences that affect coding potential do not cluster in particular protein-coding regions, indicating that specific HHV-7 genes have not been measurably subject to unusual evolutionary pressures since divergence. Reassessments of genetic content indicated that the HHV-7 genome contains 84 different genes, whereas the HHV-6 genome contains 85. All HHV-7 genes but 1 have direct HHV-6 counterparts, and all but 2 HHV-6 genes have HHV-7 homologues. Sequence comparisons between HHV-7 and HHV-6 provided evidence that the protein-coding regions of 11 genes are expressed by splicing.

Molecular diagnosis of cat scratch disease: a two-step approach.

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.

Cardiac L-type Ca2+ channel triggers transmitter release in PC12 cells.

Among the various voltage-sensitive Ca2+ channels present in PC12 cells are the dihydropyridine (DHP)-sensitive L-channel, the omega-conotoxin (omega-CgTx)-sensitive N-channel, and an atypical omega-CgTx/DHP-insensitive Ca2+ channel. Depolarization-evoked Ca2+ entry and [3H]dopamine release is mediated by L-type Ca2+ channels determined by the use of Ca2+ channel antagonists, and a single protein of 250 kDa is recognized by L-type-specific antibodies. Screening of a PC12 cDNA library revealed two types of Ca2+ channels which were identified by partial sequencing. A pc12-L clone displayed virtually identical sequence homology to the cardiac L-type channel. The identical sequence homology of the single alternative splicing region confirmed clone pc12-L as the rbC-I transcript, a cardiac-neuronal alpha 1 subunit expressed in rat brain. Clone pc12-N displayed identical sequence homology to rbB-I, a neuronal alpha 1 subunit of the N-type Ca2+ channel expressed in rat brain; Northern blot analysis identified RNA of a size similar to that previously described for rat brain.

A radioimmunoassay for the diagnosis of malaria.

A newly developed radioimmunoassay for the diagnosis of malaria has been tested in South Africa. The radioimmunoassay is an antibody binding-inhibition assay, based on a monoclonal antibody (D5) cross-reacting with Plasmodium berghei and P. residual binding activity was tested on antigen-coated microtiter plates. A sample was considered positive if it inhibited binding of the antibodies to an extent exceeding that of the microscopically negative blood samples. Blood was collected on 3 separate occasions from a total of 530 individuals living in a malaria-endemic area and was examined by radioimmunoassay and microscopy. Group 1, consisting of 194 samples, yielded 12 samples positive by microscopy and 10 of these (83%) were also positive by radioimmunoassay. One sample in this group was "positive" in the radioimmunoassay but negative on microscopy (false positive). In the 320 samples of group 2, 13 were positive by microscopy and 6 (46%) by radioimmunoassay. Group 3, which included 16 samples preselected as positive by microscopic examination and 16 controls, was examined after 4 weeks storage at -20 degrees C. Twelve samples (75%) were positive by radioimmunoassay. Tests carried out to determine the effect of blood storage on the activity of the antigen indicated that activity was preserved with little loss over a 3-month period.

Detection of Plasmodium falciparum using a radioimmunoassay based on a crossreacting, monoclonal anti-P. berghei antibody-P. berghei antigen system.

An antibody binding-inhibition test is described, which allows the detection of P. falciparum in red blood cells (RBC) infected in vitro, using a crossreacting, monoclonal anti-P. berghei antibody and P. berghei coated microtiter plates. Experiments carried out to determine the coating efficiency of various P. berghei and P. falciparum derived antigen preparations showed that intact, saponin freed P. berghei parasites and sonicated, RBC parasitized with P. falciparum had the highest binding activity. Binding of the monoclonal antibody to the antigen coated plates was effectively inhibited by preincubation with sonicated, P. falciparum infected RBC. The minimal degree of infection detectable was about 0.008% parasitemia (400 parasitized RBC/microliters blood). The sensitivity of detection was not appreciably affected by the source of the coating antigen. We conclude that the difficulty and expense involved in the use of P. falciparum based immunodiagnostic tests for large scale screening for malaria can be obviated by making use of P. berghei based assays.

Use of a monoclonal, anti Plasmodium berghei antibody, cross reacting with P. falciparum, for the detection of P. falciparum in in vitro infected blood.

This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.