Utility of a multiplex PCR assay for detecting herpesvirus DNA in clinical samples

A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material. The susceptibility of these viruses to in vivo inhibition by at least one antiviral drug was an important consideration in their inclusion in the multiplex detection system. An aliquot of equine herpesvirus was introduced into each specimen prior to extraction and served as an indicator of potential inhibitors of the PCR and a detector of suboptimal PCR conditions. Compared to virus isolation and immunofluorescence-based antigen detection, the multiplex assay yielded higher detection rates for all viruses represented in the assay. The turnaround time for performance of the assay was markedly reduced compared to those for the other techniques used to identify these viruses. More than 21,000 tests have been performed using the assay. Overall, the multiplex PCR enabled the detection of substantially increased numbers of herpesviruses, in some cases in specimens or anatomical sites where previously they were rarely if ever identified using traditional detection methods.     

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10⁻⁶M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.     

Anti-HBs Cellular Immune Response in Kidney Recipients before and 4 Months after Transplantation

Patients with renal failure represent a population at risk for hepatitis B, since only 50 to 60% of them develop protective humoral responses after vaccination. As this could be due to an altered regulation of cellular immune responses, the objectives of the present study were to evaluate the proliferative abilities of lymphocytes from patients with chronic renal failure after stimulation in vitro with a mitogen (pokeweed mitogen [PWM]) or HBsAg. In order to differentiate between the immunodeficiency associated with renal failure and that due to immunosuppression posttransplantation, the same subjects were tested before and 4 months after kidney transplantation. The lymphoproliferation assay used was performed by flow cytometry, which is based on sequential analysis of the cell cycle and which allows analysis of cytokine production. Serologically, the group of 36 patients tested comprised 22% nonresponders, 30% poor responders, and 48% responders. Lymphocyte growth was observed for all patients after stimulation with PWM, indicating that these cells had the capacity to proliferate in vitro. The level of lymphoproliferation in response to PWM was significantly reduced after transplantation, yet both before and after transplantation, all serologic nonresponders developed cellular responses to at least two vaccines. No correlation between humoral and cellular responses was shown. Proliferating cells were lymphocytes, which mostly secreted interleukin 4 (IL-4) and IL-10 for the three serologic groups. This study suggests that even when repeated vaccination fails to induce significant antibody levels in patients with renal failure, specific HBs cellular responses develop, and these may prove to be efficient in protecting these patients against hepatitis B.     

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.     

Cost comparison of genetic and clinical screening in families with hereditary hemorrhagic telangiectasia

Endoglin (ENG) and ALK-1 mutations cause hereditary hemorrhagic telangiecstasia (HHT), an autosomal dominant disorder leading to vascular dysplasia in the form of mucocutaneous telangiectasia and visceral arteriovenous malformations (AVMs). We proposed to compare two alternative strategies for management of HHT: screening HHT families with molecular diagnostic tests followed by targeted clinical screening versus conventional clinical screening. A decision analytic model was constructed to compare screening strategies for a hypothetical HHT family. The family consists of 1 index case and 13 relatives. The clinical screening protocol in use at the Canadian HHT Center in Toronto was assumed to be the standard of care. Unit costs for clinical screening (in Canadian dollars) were obtained from the 2003 Ontario Health Insurance Schedule of Benefits. Genetic screening costs were estimated for quantitative multiplex PCR and sequencing of Endoglin (ENG) and ALK-1 genes, as performed at HHT Solutions, Toronto. The genetic screening strategy resulted in a net cost of $4,060 per individual versus $5,975 for the clinical screening strategy. The genetic screening strategy would save $1,915 per family member or $26,810 saved per family. Sensitivity analyses revealed that the genetic screening strategy was cost saving over all plausible ranges of input variables for all hypothetical families tested. We concluded that a genetic screening strategy with targeted clinical screening is more economically attractive than conventional clinical screening and results in a reduction in the number of clinical tests for family members who do not have HHT.     

Cerebro-osseous-digital syndrome: four new cases of a lethal skeletal dysplasia--distinct from Neu-Laxova Syndrome

Neu-Laxova Syndrome (NLS) is a severe disorder with intrauterine growth retardation, edema, and characteristic face (including microcephaly with receding forehead, protuberant eyes, a flattened nose, deformed ears, cleft palate, and micrognathia). Ichthyosis is often present. Limb anomalies include hypoplastic fingers and syndactyly of fingers and toes. Patients are usually stillborn or die shortly after birth. We report five unrelated patients--four with atypical NLS and one with typical NLS. All five patients were stillbirths. Clinically, the atypical NLS patients showed a large skull; rhizo-, meso-, and acromelia; and hypoplasia of the metacarpals and phalanges. The feet were similarly affected. Radiographically, the atypical patients showed interpediculate narrowing and hypoplastic vertebral bodies. The long bones were stick-like, showing diaphyseal widening that spared the metaphyses and was more pronounced in the lower extremities. The ilia had a half-moon configuration with widening of the sacrosciatic notches. The ischia were vertical and the pubic bone was absent. The typical NLS patient showed microcephaly, normal vertebral body, and long bone ossification, but a pelvic configuration similar to that of the atypical NLS patients. The common and distinguishing clinical and radiographic features are reviewed. Scott et al. [1981: Am J Med Genet 9:165-175] described two patients with NLS with radiographic and clinical findings similar to patients 1-4 reported here. Patients 1-4 of this report lack the typical findings of NLS and likely represent a distinct lethal skeletal dysplasia.     

The feasibility,demand, and effect of integrating primary care services with HIV voluntary counseling and testing: evaluation of a 15-year experience in Haiti, 1985-2000

BACKGROUND: HIV voluntary counseling and testing (VCT) may be an effective strategy to prevent transmission of HIV in developing countries. Hypothesizing that primary care services and HIV VCT have synergistic benefits, we examine the feasibility, the demand, and the effect of integrating on-site primary care services into VCT at a stand-alone VCT center in Port au Prince, Haiti. METHODS: Through a retrospective review of patient records, we describe the integration of primary care services at the Groupe Haitien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) VCT center between1985 and 2000. RESULTS: Between 1985 and 1999, services for HIV care, tuberculosis care, treatment of sexually transmitted diseases, and reproductive health were sequentially integrated into HIV VCT at GHESKIO. The number of new people seeking voluntary counseling and testing at GHESKIO increased from 142 in 1985 to 8175 in 1999, with an increasing percentage of women, adolescents, symptom-free clients, and self-referred clients. Of new adults seeking VCT in 1999, the center was able to provide AIDS care to 17%, tuberculosis treatment to 6%, sexually transmitted infection management to 18%, and family planning to 19%. HIV transmission between discordant couples was 0 infections/100 follow-up years (95% CI, 0-3.2); vertical transmission from mother to child was 11 infections/100 live births (95% CI, 4.6-21.9); These rates are significantly lower than expected rates of transmission in Haiti. CONCLUSIONS: This report demonstrates the feasibility, demand, and effective synergy of integrating on-site primary care services into HIV VCT in Haiti. VCT is a good entry point for people in need of services for communicable diseases and reproductive health, and, reciprocally, services attract more people to VCT, including populations that are at high risk for HIV infection. This program is being duplicated elsewhere in Haiti and can serve as a model for other countries.     

Standardized BACTEC Method To Measure Clarithromycin Susceptibility of Mycobacterium genavense

A standardized clarithromycin susceptibility test for Mycobacterium genavense is reported. The BACTEC radiometric broth dilution test method recommended for Mycobacterium avium complex was modified to develop a reliable and reproducible procedure. Test development involved optimization of medium pH and inoculum densities for antibiotic vials as well as growth control vials. MIC control organisms included Mycobacterium simiae, Mycobacterium avium, and Mycobacterium xenopi. Growth control vials required two to three inoculum dilutions, which varied for each species. Clarithromycin MICs and MBCs for 12 isolates and 1 colonial variant of M. genavense ranged from ≤0.06 to 0.25 μg/ml.