Comparison of Digene hybrid capture 2 and conventional culture for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in cervical specimens

Digene's Hybrid Capture 2 (HC2) CT/GC, CT-ID, and GC-ID DNA tests were evaluated by comparison to traditional culture methods for detecting Chlamydia trachomatis and Neisseria gonorrhoeae infections in 669 cervical specimens from high-risk female populations attending two sexually transmitted disease clinics. For detection of either or both infections, the HC2 CT/GC test algorithm had 93.8% sensitivity and 95.9% specificity compared to those of culture. After resolution of discrepant results by direct fluorescent-antibody (DFA) staining or PCR assay, the relative sensitivity and specificity of the HC2 CT/GC test algorithm increased to 94.8 and 99.8%, while the values for culture were 83.6% (McNemar's P value, 0.0062) and 100%, respectively. For detection of the individual pathogens, the relative sensitivities for the HC2 CT-ID and GC-ID tests were 97.2 and 92.2% and the specificities were greater than 99% compared to culture adjucated by DFA staining and PCR. Test performance varied at the two clinics: the HC2 CT/GC algorithm, CT-ID, and GC-ID tests had significantly higher sensitivities (McNemar's P value, <0.05) than that of culture for the population at one clinic as well as for the combined populations. At the other clinic, the HC2 tests performed as well as culture.     

Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever.

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF.     

Performance of a commercial, type-specific enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific antibodies in Ugandans

Two hundred forty-eight human immunodeficiency virus (HIV)-positive and 496 HIV-negative subjects in Uganda were tested by HerpeSelect herpes simplex virus type 2 enzyme-linked immunosorbent assay (ELISA) and Western blotting to optimize the ELISA for use in this population. A higher index cutoff value was required for optimal sensitivity and specificity, and overall performance of the assay was not affected by HIV status.     

Mitochondrial DNA segregation in hematopoietic lineages does not depend on MHC presentation of mitochondrially encoded peptides

Mutations in mitochondrial DNA (mtDNA) are associated with a broad spectrum of clinical disorders. The segregation pattern of pathogenic mtDNAs is an important determinant of both the onset and the severity of the disease phenotype, but the mechanisms controlling mtDNA segregation remain poorly understood. To investigate this, we previously generated heteroplasmic mice containing two different mtDNA haplotypes and showed that BALB/c mtDNA was invariably selected over NZB mtDNA in blood and spleen. Here, we have characterized this process in hematopoietic tissues and tested whether it involves the presentation of mtDNA-encoded peptides by MHC class Ib molecules. Selection against NZB mtDNA was widespread across different hematopoietic cell lineages and proportional to heteroplasmy levels. Backcrossing heteroplasmic mice with CAST/Ei, a strain in which the MHC class Ib molecule H2-M3 is silent, completely abolished selection against NZB mtDNA in the spleen. To test whether this effect depended on an intact immune system, we generated heteroplasmic mice missing functional copies of Tap1, beta2m or Rag1 to impair presentation or recognition of mtDNA-encoded peptides. The kinetics of selection against NZB mtDNA were unaltered in these mice compared with their wild-type littermates. We conclude that mtDNA selection in hematopoietic tissues is not based on an immune mechanism, but likely involves metabolic signaling.     

A manual bead assay for the determination of absolute CD4+ and CD8+ lymphocyte counts in human immunodeficiency virus-infected individuals.

CD4+ T lymphocytes are currently the most common surrogate marker indicating disease progression in individuals infected with human immunodeficiency virus (HIV). Since the cost of enumerating lymphocyte phenotypes is quite high, an inexpensive bead assay analyzed by light microscopy (cytosphere assay; Coulter Corporation, Hialeah, Fla.) was developed as an alternative method for counting CD4+ and CD8+ T lymphocytes. To evaluate the reliability of the cytosphere assay, heparinized blood was collected from 117 HIV-infected individuals and tested for both CD4+ and CD8+ lymphocytes by flow cytometry and the cytosphere assay. The Pearson correlation coefficient of the cytosphere assay compared with that of flow cytometry for CD4+ T lymphocytes was 0.93, with mean values +/- standard deviations of 534 +/- 509 by flow cytometry and 499 +/- 477 by the cytosphere assay. The correlation coefficient for CD8+ T lymphocytes was 0.86, with mean values of 831 +/- 543 by flow cytometry and 746 +/- 472 by the cytosphere assay. The sensitivity and specificity of the cytosphere assay in determining absolute CD4+ T-lymphocyte counts of less than 200/microliters were 97.6 and 94.7%, respectively. The positive predictive value was 90.9%, and the negative predictive value was 98.6%. The cytosphere assay was highly correlative to flow cytometry in determining CD4+ and CD8+ T-lymphocyte counts among HIV-infected patients. The ease and limited resources needed to perform this test make it ideal in developing countries and other areas where technology and finances are limited.     

Genital mycoplasma infection. Intrauterine infection: pathologic study of the fetus and placenta.

Mycoplasma infection was present in the fetuses from three spontaneous abortions and in one second-trimester newborn. Gross examination revealed in most cases a severely infected placenta and membranes, with a fetus of normal appearance. The fetal infection presumably followed placental involvement and appeared to have been acquired shortly prior to delivery. Genital mycoplasmas, Ureaplasma urealyticum or Mycoplasma hominis, were isolated from the placentas and the fetal tissues, and from the genital tracts of the mothers. Isolation of mycoplasmas from the liver indicated that bloodstream dissemination of these organisms occurred in the fetus. In the fetus, the pathologic changes were variable. Lesions were identified in the lung by scanning electron microscopy of the bronchial tree in two cases and were accompanied by interstitial pneumonia. An abnormally dilated left ventricle suggestive of cardiomyopathy was observed in one case.     

Mycobacterium ulcerans cytotoxicity in an adipose cell model

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.