Detection of infectious human immunodeficiency virus type 1 in female genital secretions by a short-term culture method

Infectious human immunodeficiency virus type 1 (HIV-1) is difficult to detect in female genital secretions by standard virus culture techniques. To improve detection of cell-free HIV-1 in female genital secretions, we adapted a short-term assay that uses the multinuclear-activation galactosidase indicator (MAGI) assay. When vaginal lavages from HIV-1-infected women were tested with the adapted MAGI assay, 25 (64%) of 39 lavages with detectable, cell-free HIV-1 RNA were shown to have infectious virus. No infectious virus was found in 10 vaginal lavages from HIV-1-infected women with undetectable vaginal viral loads. Significantly (P < 0.01) more lavages from HIV-1-infected women tested positive for infectious virus by the MAGI assay than by standard peripheral blood mononuclear cell (PBMC) coculture, which detected infectious virus in only 6 (17%) of 35 vaginal lavages. Lavages with viral loads of >10,000 copies per lavage yielded significantly (P < 0.01) more positive cultures than those with <10,000 copies by using the MAGI assay. Detection of infectious HIV-1 in vaginal lavages was not associated with the presence of genital tract infections or CD4(+)-T-cell counts. However, although the results were not significant (P = 0.08), the MAGI assay detected infectious virus from more vaginal lavages at a vaginal pH of >/=4.5 than at a pH of <4.5. These results indicate that the MAGI assay is more sensitive than PBMC culture methods for detecting infectious virus in female genital secretions. Accurate measurements of infectious virus in genital secretions will improve studies that evaluate sexual transmission of HIV-1.     

Serum Regulation of In Vitro Lymphocyte Responses in Early Experimental Syphilis

Sera from rabbits with early experimental syphilis were tested for their effect on in vitro lymphocyte transformation responses to related specific antigens (sonicated T. pallidum), unrelated specific antigens (sheep erythrocytes), and the T cell mitogen, concanavalin A. Results were compared with responses in preinfection sera and in sera from sham-infected rabbits. Titration experiments in which normal serum was used indicated that optimal lymphocyte responsiveness is obtained with a final serum concentration of 1%. Under these conditions, no differences in concanavalin A stimulation were observed in cultures with syphilitic sera. Responses to sonicated T. pallidum were inhibited, but only by 17 to 25% when compared with the response in preinfection sera. In cultures containing 10% serum, inhibition of lymphocyte proliferation to sonicated T. pallidum antigens was evident with sera from all syphilitic animals from day 10 (55% inhibition) through day 31 (80% inhibition) of infection. Responses to concanavalin A and sheep erythrocytes were significantly inhibited by day 10 sera; only 20% of the sera tested demonstrated substantial nonspecific inhibitory capacity. No differences were evident among sera from any of the sham-infected animals or among the preinfection sera from either group. Pooled serum with high inhibitory activity was fractionated by ammonium sulfate precipitation, DEAE ion exchange chromatography, and Sephadex G-200 gel filtration. Two separate inhibitors were identified: (i) a low-molecular-weight, ammonium sulfate-soluble, nonspecific inhibitory fraction containing albumin and alpha-globulins with the capacity to inhibit both antigen and mitogen responses and (ii) a high-molecular-weight, ammonium sulfate-precipitable, inhibitory fraction containing alpha-globulin and FTA-ABS-reactive immunoglobulin M which affected only the antigen-specific response to sonicated T. pallidum. Immunodiffusion failed to detect immunoglobulin or T. pallidum antigens in either fraction. DEAE-purified immunoglobulin G from immune serum was not inhibitory.     

Missed diagnosis in hematological patients—an autopsy study

Autopsy findings of missed diagnoses that would probably have changed management or prognosis occur in up to 29% of cases in general hospitals. Such proportions may be higher in subsets of patients with complex diseases. We reviewed 2908 consecutive autopsies performed over a period of 29 months in a large-volume hospital, analyzing 118 autopsies of patients with hematological malignancies or severe aplastic anemia. A review of macroscopic reports as well as microscopic examination of tissue samples was performed. Medical records were reviewed for clinical diagnoses. Discordances between clinical and autopsy diagnoses were classified using Goldmans criteria. Additionally, we searched for clinical parameters correlated with occurrence of class-I discrepancy using a multivariate method. Median age was 46.5 years, and 25.4% had received a hematopoietic stem-cell transplant. Overall, 11.9% (6.6–19.1%) of patients died before conclusion of the hematological diagnosis and 33% (24.6–42.3%) died with no active hematological disease. We found class-I discrepancy in 31.3% (23.1–40.5 %) of cases. The most common among these diagnoses were hematological disease, pneumonia and gastrointestinal bleeding. In a univariate analysis, being elderly (P=0.04) was positively correlated with the finding of class-I discrepancies; while, having received previous specific hematological treatment (P=0.0005) or hematopoietic stem-cell transplants (P=0.013), or being admitted to a specialized hematology unit (P=0.0006) were negatively correlated to the occurrence of such discrepancies. Multivariate analysis showed that care in a specialized hematology unit (OR 0.34, 0.12–0.93) was independently associated with lower occurrence of discrepancies. We concluded that critical diagnoses are often missed in highly complex hematological patients especially in the absence of admission to specialized hematology units.     

Proton spectroscopy study of the left dorsolateral prefrontal cortex in pediatric depressed patients

The dorsolateral prefrontal cortex (DLPFC) plays an essential role in mood regulation and integration of cognitive functions that are abnormal in major depressive disorder (MDD). Few neuroimaging studies have evaluated the still maturing DLPFC in depressed children and adolescents. We conducted single voxel proton magnetic resonance spectroscopy ((1)H MRS) of the left DLPFC in 14 depressed children and adolescents (13.3 +/- 2.3 years old, 10 males) and 22 matched healthy controls (13.6 +/- 2.8 years old, 13 males). Depressed subjects had significantly lower levels of glycerophosphocholine plus phosphocholine (GPC + PC; or choline-containing compounds) and higher myo-inositol levels in the left DLPFC compared to healthy controls. In the depressed subjects, we found significant inverse correlations between glutamate levels and both duration of illness and number of episodes. In healthy controls there was a significant direct correlation between age and glutamine levels, which was not present in the patient group. Lower GPC + PC levels in pediatric MDD may reflect lower cell membrane content per volume in the DLPFC. Increased myo-inositol levels in MDD may represent a disturbed secondary messenger system. GPC + PC and myo-inositol abnormalities further demonstrate the involvement of DLPFC in pediatric MDD.     

Association of DLG5 R30Q variant with inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal system known as the inflammatory bowel diseases (IBD). Recently, Stoll and colleagues reported a novel finding of genetic variation in the DLG5 gene that is associated with IBD (CD and UC combined). We present here a study of the genetic variation described in that report in two well-powered, independent case-control cohorts and one family-based collection, and confirm the proposed association between IBD and the R30Q variant of DLG5 in two of the three studies. We are, however, unable to replicate the other proposed association to the common haplotype described in Stoll et al and suggest that this other finding could conceivably have been partially a statistical fluctuation and partially a result of LD with the replicated R30Q association. This study provides support for the hypothesis that DLG5 constitutes a true IBD risk factor of modest effect.     

Hepatitis B virus transmission in Brazilian hemodialysis units: serological and molecular follow-up

A serological and molecular study of hepatitis B virus (HBV) infection was carried out in dialysis units in Central Brazil. Between 1995 and 1999, serum samples from all HBsAg-positive hemodialysis patients (n = 43) were tested for HBeAg/anti-HBe and subtyping by monoclonal ELISA. HBV DNA was detected by PCR and positive samples were genotyped by restriction fragment polymorphism pattern (RFLP) methodology. TheHBsAg prevalence declined in this population during the survey period (12-5.8%). HBeAg and anti-HBe were detected in 23 (53.5%) and 18 (41.9%) sera, respectively. Thirty-six samples could be HBsAg subtyped: 21 were subtype ayw(3), 14 belonged to adw(2) and one was identified as adw(4). HBV DNA was present in 30 serum samples. Of these, 20 (66.7%) were genotype D, 9 (30%) genotype A, and 1 (3.3%) genotype F. In addition, the RFLP pattern could be determined in samples from 18/20 genotype D patients: D3 (10 strains), D7 (7 strains) and D4 (1 strain); from 8/9 genotype A patients: A1 (6 strains) and A3 (2 strains); and from the patient infected with genotype F: F1. Patterns D3 and D7 were associated closely with HBV infection in the two largest hemodialysis units studied. These findings confirm the value of the RFLP method as an effective molecular epidemiological tool for elucidating HBV transmission in hemodialysis units.     

Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity tests

3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the (32)P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts.     

Fulminant disseminated Varicella Zoster virus infection without skin involvement.

BACKGROUND: Varicella Zoster virus (VZV) infection is potentially very serious in bone marrow transplant recipients, and may manifest as a disseminated visceral infection. This condition is generally accompanied by a vesicular rash. OBJECTIVES: We review here a case of fulminant fatal disseminated VZV infection, not accompanied by skin involvement, and the laboratory approaches currently available to diagnose this disease. STUDY DESIGN: Post mortem tissue samples were subjected to histopathological examination, and tested for herpesviruses by electron microscopy and PCR. RESULTS: Intranuclear inclusions were noted by histological examination in the lungs, liver, kidneys and bone marrow. Particles with a herpesvirus morphology were visualized in liver tissue. VZV DNA was detected in liver and bone marrow by PCR followed by sequencing of the amplicons. Viremia was documented by retrospective testing of the serum by PCR. CONCLUSIONS: A disseminated VZV infection which proved rapidly fatal was demonstrated in a case without skin manifestations. This rare presentation of VZV infection is potentially underdiagnosed. Testing for VZV viremia by PCR can at the very least suggest the diagnosis although whether plasma-associated viremia is truly pathognomonic of visceral disseminated infection remains to be established.