Absence of HIV-1 DNA in high-risk seronegative individuals using high-input polymerase chain reaction.

Evidence of frequent HIV-1 infections in antibody-negative, high-risk individuals (so-called 'silent' infections) remains controversial. To evaluate whether these discrepant results may be the consequence of intermittent detection of rare infected cells (low viral load) preceding seroconversion, we developed a modification of the polymerase chain reaction (PCR) technique which enabled analysis of 10-fold greater amounts of cellular DNA per reaction than standard PCR (2 x 10(6) rather than 0.2 x 10(6) input cells). This technique allowed consistent detection of HIV-1 provirus in two seropositive individuals who had repeatedly tested negative by standard-input PCR. However, results were negative when high-input PCR was applied to 51 specimens from 39 selected high-risk seronegative individuals. These results suggest that variations in viral load preceding or in the absence of seroconversion probably do not explain discrepant evidence regarding silent HIV-1 infection.

In vitro incorporation of serine into the staphylococcal cell wall.

A variant of Staphylococcus aureus 44A HJD was isolated by serial growth in Trypticase soy broth to which 2 M serine had been added (wt/vol). Amino acid analysis of hydrolysates of purified mucopeptides from the variant showed that they contained 1.266 serine and 2.156 glycine residues per glutamic acid residue, compared with 0.174 serine and 3.144 glycine residues per glutamic acid residue in the mucopeptide of the parent strain. In addition to this alteration in the chemical composition of the mucopeptide, the variant lost many of the biochemical and cultural characteristics of the parent organism. The variant was not sensitive to the lytic action of lysostaphin and was non-phage-typable. Moreover, in vitro tests indicated that the organism was coagulase negative, did not produce gelatinase or deoxyribonuclease, and did not hemolyze sheep erythrocytes. Apparently due to the change in the serine content in the cell wall of the parent S. aureus strain, the organism had become "epidermidis-like" in its properties.

In vitro modeling of acute salpingitis caused by Neisseria gonorrhoeae.

Normal human fallopian tube organ culture was used as an in vitro model to study Neisseria gonorrhoeae infections. The effects of various gonococcal colony phenotypes on morphology of the epithelium were studied by scanning electron microscopy. After 30 minutes' incubation, there was striking attachment of piliated transparent phenotypes to the epithelium; however, there was no obvious pathology. After 24 hours' incubation, there were microcolony formation, slight swelling and hyperplasia of the mucosa, and occasional focal necrosis and sloughing of ciliated cells. Tissue from acute salpingitis showed widespread destruction of mucosa, hyperplasia, and crypt formation. Duplication of these findings in vitro may require longer incubation and the addition of other host factors.

Scanning electron microscopy of attachment of Neisseria gonorrhoeae colony phenotypes to surfaces of human genital epithelia.

Attachment of different gonococcal colony phenotypes to explants of human genital tract epithelium was studied by means of scanning electron microscopy and radioisotope-labeled gonococci. Heavily piliated organisms attached in greater numbers than nonpiliated organisms. Gonococci from transparent colony phenotypes attached in higher numbers than gonococci from opaque phenotypes to all tissues studied. Transitional cells from cervical tissues showing a gradual squamocolumnar transition demonstrated more gonococci attached per surface area than either endocervical or fallopian tube epithelium. Squamous epithelium showed the fewest number of attached gonococci. In all tissues, the attachment of the gonococcus was to the tips and surfaces of the microvilli. Gonococcal colony phenotypes as well as the length and location of the cervical transition zone may influence the progression of cervical gonococcal infection to invasive disease.

Unusual kinetics of white cell clearance in transfused mice.

BACKGROUND: Donor white cells (WBCs) in blood transfusions are responsible for complications in recipients, including alloimmunization, graft-versus-host disease (GVHD), and virus transmission and reactivation. The recent use of sequence-specific polymerase chain reaction assays to monitor the kinetics of clearance of donor WBCs in transfused humans and dogs found transient recirculation of donor lymphocytes on Days 3 to 5 after transfusion; this presumably reflected an abortive GVHD reaction to major histocompatibility complex-incompatible recipient cells, after which donor WBCs were cleared to undetectable levels. STUDY DESIGN AND METHODS: This study sought to develop a murine model to further characterize the kinetics and major histocompatibility complex restriction of donor WBC clearance. A sensitive murine Y chromosome-specific polymerase chain reaction assay was developed and applied to serial blood samples collected after transfusions of allogeneic blood to naive inbred, primed inbred, and outbred mice, as well as after transfusions of gamma-radiated blood to naive inbred mice. RESULTS: In inbred mice, both naive and primed to the allogeneic blood donor, transfused WBCs were not cleared to undetectable levels for more than 1 month after transfusion. Transfused outbred mice also showed prolonged donor WBC survival, although at lower levels than inbred mice. There was no evidence of GVHD in either inbred or outbred mice, and gamma radiation had no significant impact on donor WBC persistence. CONCLUSION: These results contrast with the rapid clearance of donor WBCs observed in humans and dogs. The immunologic basis for this discrepancy remains unclear. Caution should be exercised in any extrapolation to humans of conclusions drawn from results in transfused mice.

Primary isolation of a new strain of the TATLOCK/Pittsburgh pneumonia agent (Legionella micdadei).

Pneumonia, empyema and lung abscesses developed in a patient following a neurosurgical procedure and associated short-term corticosteroid therapy. An organism identical to the TATLOCK/Pittsburgh pneumonia agent (Legionella micdadei) was the only organism isolated from multiple specimens from lung aspirates and chest tube drainage. The organism was isolated directly on charcoal yeast extract (CYE) agar and classified as identical to the TATLOCK bacterium by direct immunofluorescent staining and by gas-liquid chromatography of cellular fatty acids. The patient's pulmonary infection improved during treatment with penicillin. Serum specimens obtained from the patient during convalescence showed an indirect fluorescent antibody reciprocal titer of 16,000 to his homologous antigen, but he also had titers that were potentially diagnostic against antigens of the six serogroups of Legionella pneumophila and three other Legionella-like organisms. Legionella can be isolated from clinical specimens by the routine use of CYE agar, which should be incorporated as the primary isolation medium for chest fluids and lung specimens. It appears that a large battery of antigens will be required for serological testing to diagnose infections caused by L micadei, L pneumophila and Legionella-like organisms.