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An infectious etiology has been suggested for Reiter's syndrome (RS) because the disease has often been observed to follow episodes of urethritis or dysentery. Despite demonstrations of bacterial antigens in the synovial tissues of RS patients, it is not clear whether viable organisms are present in the synovium in any particular stage of this disease. Furthermore, it is not clear how either viable organisms or their product(s) might reach the joints. Infection with the bacterium Chlamydia trachomatis is the most common sexually transmitted disease in the United States, and as such this organism has emerged as a primary pathogen associated with RS. Previous work from our group has shown that synovial biopsy tissues from a majority of RS patients studied show significant levels of apparently intact chlamydial RNA, even when synovial or urethral cultures from the same patients are unequivocally negative for the organism. We show here that inapparent urethral infection with chlamydia occurs with high prevalence in men, and that inapparent cervical infection with the organism occurs at high prevalence in women. These data provide an important link in the relationship between initial chlamydial infection and possible subsequent genesis of RS, and they may give useful insight into mechanisms by which chlamydial infection can lead to development of this disease. Our data argue further that inapparent infection may be a significant factor in pathogenesis for all chlamydia-related diseases, and they suggest that, contrary to current ideas, C. trachomatis can generate disseminated infection.

Two cases of neonatal upper respiratory tract obstruction caused by herpes simplex virus are described. Infection of the upper respiratory tract with this virus should be included in the differential diagnosis of fever and stridor during the neonatal period.

Human cytomegalovirus (HCMV) can produce life-threatening disease in immunosuppressed AIDS patients and organ transplant recipients. Congenital HCMV is the leading infectious cause of birth defects, including blindness, deafness, mental retardation, and death. HCMV can be transmitted in utero, during delivery, or postnatally through physical contact, blood transfusion, and organ transplantation. There are few treatments for HCMV disease. However, ganciclovir (DHPG) has been beneficial in HCMV-infected transplant patients.

Infectious molecular clones of the human immunodeficiency virus (HIV) have been very important tools for the analysis of regulatory gene functions and the study of differential cell tropism. We have cloned and characterized a proviral sequence of HIVmn from mn strain infected H9 cells. This clone, called KP1, was found to be infectious for different cell lines and human peripheral blood lymphocytes (PBL). KP1 proviral DNA was detected in HUT-78 cells and human PBL by polymerase chain reaction (PCR) analysis after infection of these cells with cell-free supernatants from KP1 transfected human rhabdomyosarcoma (RD) cells. To the best of our knowledge, this is the first report of an infectious molecular clone of HIVmn which is a representative of one of the most prevalent strains of HIV-1 in North America and Europe. Biologically active clones of a broadly antigenic strain such as HIVmn will be extremely useful in therapeutic approaches for AIDS.

Purpose: Recent data suggest that oral sex behaviors are increasing among adolescents. Little is known about rates of adolescent oropharyngeal STD transmission. This study explores rates of CT and GC in the oropharynx of adolescent women attending an urban family planning clinic. Methods: Thirteen to 22 year-old women were approached between 10/05 and 5/07 to complete a self-administered survey and provide urine and throat specimens for NAAT. Exclusion criteria were non-English speaking, antibiotic use within 30 days, and sexual contact with only females. Of 266 women approached, 229 were eligible, and 190 (83%) participated. One hundred eightyeight (82%) participants with complete biologic data are included in this analysis. Data were analyzed descriptively. Results: Mean age of participants was 17.4 1.7 years, 91% were AA, and 62% were in high school. All but 4 (2%) had engaged in vaginal sex; 124 (66%) performed oral sex, 166 (88%) received oral sex, and 28 (15%) had anal sex. Eighty percent of women never used condoms with oral sex versus 19% who never used condoms with vaginal sex. There were 23 (12.2%) women infected with CT or GC. CT was detected in 18 (9.6%) urine samples. GC was found in 2 (1.1%) urine samples and both were co-infected with CT. Both women with GC in the urine and 15 (83%) women with CT in the urine received oral sex. Oropharyngeal NAAT detected 4 (2.1%) women infected with CT and 3 (1.6%) infected with GC. There were no oropharyngeal co-infections. Of the 4 women with CT in the oropharynx, only 2 (50%) had CT in the urine and would have been treated without oropharyngeal screening. Among the 3 women with oropharyngeal GC, none had GC in the urine. Without oropharyngeal screening, none would have been treated. One woman with oropharyngeal CT reported not performing oral sex, but reported receiving oral sex, and having both vaginal and anal sex. Women with an oropharyngeal STD (n 7) were more likely than women with an STD in the urine only (n 16) to have had anal sex, to report performing oral sex because it feels good, to report always using condoms with vaginal sex, to think that syphilis cannot be transmitted via oral sex, and to have had less sex in the past 3 months (p 0.05). Conclusions: Our data suggest that oral sex is common among urban adolescent women, and that condom use is less frequent with oral sex than with vaginal sex. Women have limited knowledge of oral STD transmission and need education on this topic. Although rates of oropharyngeal STDs in our sample were low, 5 of 7 (71%) infections would have been undetected with urinary screening alone. Future, larger studies should explore the effectiveness of screening high-risk populations for oropharyngeal GC and CT and determine algorithms for cost-effective screening. Sources of Support: Craig-Dalsimer Fund, Mary D. Ames Chair for Child Advocacy. 4.

Cost Effectiveness of an Infection Control Program to Reduce Nosocomial Respiratory Syncytial Virus Infection. † 875

By the standard p24 assay there was a 25 to 27% decrease in free p24 antigen in serum after storage at 4 degrees C over 14 days but no loss at -70 degrees C. There was no loss at either temperature by the immune complex dissociation (ICD) procedure. Furthermore, there was no significant loss of detectable p24 in serum by either the ICD or the standard p24 assay after 700 days of storage at -70 degrees C.

The early and accurate diagnosis of human immunodeficiency virus type 1(HIV-1) infection in infants born to HIV infected mothers is essential for timely medical and social care. Study of the utility of viral detection methods is important to determine which may be the preferred diagnostic tool in this population. The accuracy of both polymerase chain reaction (PCR) and viral culture was assessed over a period of seven years on a total of 795 blood samples from 395 HIV-exposed children (of which 123 patients were HIV infected). Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque gradient centrifugation and processed for PCR and culture. For qualitative viral isolation, patient samples were co-cultivated with PHA-stimulated donor PBMCs and monitored for 28 days using a p24 antigen-capture method. PCR amplification was performed using the gag primer pair SK38/39 and detection of DNA was done with 32P-labeled SK19 probe, gel electrophoresis and autoradiography. Of the 795 samples tested, 505 were from children 12mo, sensitivities ranged from 96-100% and specificities ranged from 99-100% for both PCR and culture. Positive and negative predictive values were 97-100% and 98-100%, respectively, for both assays. Only nine patient samples produced discordant results between PCR and culture. We conclude that both viral culture and PCR are highly sensitive and specific methods for determination of HIV-1 infection in infants born to infected mothers. PCR may be the preferred technique, given that it is a more rapid and less labor intensive test.

To determine the frequency and significance of cytomegalovirus (CMV) viremia and viruria in HIV-positive subjects with low CD4+ lymphocyte counts but with no clinical indications for culture, we studied 100 consecutive clinically stable subjects with CD4+ cells 100/microliters; CMV antibody was present in 96% of the remainder. Isolation of CMV from blood was uncommon (2 of 93 seropositive subjects) whereas viruria occurred in 51.6%; likelihood of having a positive urine culture was significantly related to the subject's absolute CD4+ lymphocyte count: 60% for those with CD4+ < or = 50/microliters, vs. 26.1% for those with CD4+ 51-100/microliters. Neither serum p24 antigen nor neopterin was predictive of CMV in urine or blood. No subjects submitting to ophthalmologic exam had unsuspected CMV retinitis. Subsequent development of retinitis correlated with CMV viruria on entry: 13.5% if urine-positive, 1.9% if negative (p = 0.029; Fisher exact test).

The in vitro activity of A-56268 (TE-031) was determined by either standard agar dilution or macrobroth tube dilution and compared with erythromycin and other antimicrobial agents against 329 clinical aerobic and anaerobic bacterial isolates. A-56268 showed good to excellent in vitro activity against methicillin-sensitive Staphylococcus aureus, Neisseria meningitidis, and most anaerobes tested with the exception of Bacteroides fragilis isolates. A-56268 had relatively poor activity against coagulase-negative staphylococci and methicillin-resistant Staphylococcus aureus. Except for Haemophilus spp., the activity of A-56268 was similar to or more potent than erythromycin against all other isolates tested. A-56268 did not have significant bactericidal activity against any of the isolates examined.

We previously reported that a number of Treponema pallidum membrane proteins appear to reside on the cell surface, since intact treponemes radiolabeled by overnight incubation in medium containing [35S]methionine bind immunoglobulin G (IgG) antibodies directed against these proteins. In the present study, it was found that freshly extracted organisms radiolabeled in vitro for only 2 h inefficiently bound IgG antibodies directed against just two proteins of molecular weights 40,000 and 34,000. An in vitro incubation period of greater than 8 h was required before IgG antibodies present in rabbit syphilitic serum could recognize additional protein antigens on the cell surface. Treatment of aged treponemes, but not freshly extracted organisms, with 0.04% sodium dodecyl sulfate selectively removed a membranous layer from the treponemal surface. Only three treponemal proteins were found associated with this structure, including the same 40,000- and 34,000-molecular-weight proteins mentioned above. These two proteins most likely represent endoflagellar subunits, since they were precipitated with rabbit antisera prepared against purified endoflagellar subunits of the cultivable treponemal strain Treponema phagedenis. Further evidence also was obtained that cells of T. pallidum actively secrete into their extracellular environment a unique class of low-molecular-weight proteins.

In the spring and summer of 1988, two separate outbreaks of an illness with a rash resembling erythema infectiosum occurred among members of the nursing staff of the Children's Hospital of Philadelphia. The sources were two adolescent patients with sickle cell disease and aplastic crisis who had unsuspected parvovirus infection. Tests for IgM and IgG antibodies to parvovirus B19 were positive in both patients, and electron microscopical examination showed parvovirus-like particles in the early serum samples. Of 40 health care workers exposed to infected patients, 12 (30 percent) were infected, 2 (5 percent) were possibly infected, 8 (20 percent) had evidence of a past infection with B19, and 18 (45 percent) remained seronegative. Attack rates among the susceptible contacts were 36 percent in the first outbreak and at least 38 percent in the second. Clinical symptoms began a mean of 12.6 days after exposure and included malaise, rash, and joint pain. We conclude that hospital workers are at risk of contracting nosocomial erythema infectiosum from patients with parvovirus-associated aplastic crisis. We recommend that all patients with hereditary hemolytic anemias who are admitted with a febrile illness be evaluated for aplasia and promptly placed in respiratory and contact isolation if aplastic crisis is suspected.

Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate (pH 4), dialyzed against phosphate-buffered saline (pH 7), and tested for bactericidal activity. Bactericidal assays consisted of mixing rat granule extract with 1 × 10 3 to 3 × 10 3 bacterial cells per ml at 37°C for 1 h in a medium suited for bacterial growth. The granule extract demonstrated a distinctive dose-dependent bactericidal activity against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT-2, independent of added hydrogen peroxide or other active oxygen derivatives. The rough bacterial mutants showed an ordered increase in sensitivity to the rat lysosomal extracts inversely related to the length of their lipopolysaccharide carbohydrate side chains. Fractionation of the rat polymorphonuclear neutrophil granule extract with Sephadex G-100 column chromatography revealed an elution profile containing three major areas (peaks) of protein. Polyacrylamide gel electrophoresis and examination of enzymatic activity showed that these peaks contained myeloperoxidase (peak A), neutral protease (peak B), and lysozyme (peak C) activities. Also observed in peak C were cationic protein species whose cathodal electrophoretic migration was faster than that for lysozyme. Only peak C exhibited a bactericidal activity against the rough mutants of S. typhimurium LT-2 similar to that obtained for the unfractionated granule extract, with susceptibility of the bacterial mutants increasing with a progressive loss of carbohydrate residues in the lipopolysaccharide of the cell wall. The bactericidal activity of the peak C protein fraction was dose dependent. Boiling the unfractionated granule extract or peak C for 30 min had little affect on their antimicrobial activity when reacted against a deep-rough lipopolysaccharide mutant. However, trypsin pretreatment of these fractions significantly reduced their antimicrobial activity for the same mutant chemotype.

We have transferred a gene coding for rabbit beta-globin into the male pronucleus of mouse zygotes by direct microinjection. Some of these zygotes developed into mature mice which contained this gene and appeared to be producing a rabbit globin. Evidence for the presence of the gene in these animals was provided by Southern blot hybridization analysis. Evidence for the expression of the rabbit gene in these transformed mice and their offspring was provided by hemoglobin isoelectric focusing analysis and specific serological reactivity between mouse anti-rabbit hemoglobin antiserum and a hemolysate from the mice that developed from the microinjected zygotes. The use of this zygote transformation may allow the introduction and expression of a broad range of genetic elements in mammals.

The in vitro activity of LY146032, a new cyclic lipopeptide antibiotic, was compared with those of vancomycin, teicoplanin, and either oxacillin or ampicillin by determining agar dilution MIC values for 304 clinical gram-positive isolates. LY146032 had superior in vitro activity against oxacillin-resistant staphylococci when compared to vancomycin or teicoplanin. Against oxacillin-sensitive staphylococci, group JK-diphtheroids, streptococci, Listeria monocytogenes and Clostridium difficile, LY146032 was equally or less active than vancomycin, teicoplanin, or the penicillins tested. When tested by macrobroth dilution MIC/MBC, LY146032 showed good bactericidal activity against all organisms with the exception of Clostridium difficile.

The entry of Chlamydia trachomatis into McCoy cells (fibroblasts) was studied by transmission electron microscopy. On adsorption of elementary bodies (EBs) to host cells at 37 degrees C, the EBs were bound primarily to preexisting cell-surface microvilli. They were also observed in coated pits located at the bases of the microvilli and along smooth surfaces of the host cells and were internalized within coated vesicles at this temperature. Postembedding immunogold labeling on Lowicryl thin sections with anti-clathrin antibody as the primary reagent revealed the gold marker localized in pits and vesicles containing chlamydiae. Some EBs were present in smooth-surfaced invaginations at or near the bases of microvilli and in vesicles devoid of distinguishable coat material. A similar entry process was observed with centrifugation-assisted inoculation of EBs onto the McCoy cells. Individual EBs were initially internalized into tightly bound endocytic vesicles. However, within 1 to 3 h postinfection, multiple C. trachomatis EBs were observed in large, loosely bound vesicles. Evidence suggests that vesicles containing C. trachomatis may have fused with one another early in the infectious process. These results indicate that chlamydiae can exploit the specific process of adsorptive endocytosis for entry into host cells and for translocation to a given intracellular destination, which may be different for each species.

The utility of a rapid latex slide agglutination test (Campyslide; BBL Microbiology Systems, Cockeysville, Md.) in detecting selected Campylobacter spp. was evaluated and compared with that of conventional identification methods. Isolated colonies suggestive of Campylobacter spp. were tested directly from primary selective media after incubation at 42 degrees C under microaerophilic conditions. Stock cultures of Campylobacter jejuni (n = 27) and C. coli (n = 3) were correctly confirmed to the genus level by latex agglutination when tested in pure cultures or isolated from seeded human feces. A total of 50 fresh clinical isolates of Campylobacter spp. (45 C. jejuni and 5 C. coli) were examined, with complete agreement observed between the latex test and conventional methods. Of 173 non-Campylobacter isolates tested from primary plates, only 1 rough strain of Pseudomonas aeruginosa produced a false-positive result. Although the manufacturer recommends a 30-min antigen extraction, 1 or 5 min was found to be sufficient. Also, confirmation could be achieved within 24 h of inoculation of clinical specimens, 2 days earlier than with conventional methods.

Nonoxynol-9, a nonionic detergent and active ingredient in spermicidal contraceptives, has been reported to have anti-chlamydial properties. However, in this study exposure of elementary bodies of Chlamydia trachomatis serovar E to nonoxynol-9 (12.5-10,000 micrograms/ml) had no effect on chlamydial infectivity. In contrast, uninfected McCoy cells incubated with increasing concentrations of nonoxynol-9 over 72 h displayed dose-related cytotoxicity. When infected McCoy cells were exposed to nonoxynol-9, the developing chlamydial inclusions did not stain with iodine even though they were similar in number and appearance to the inclusions in unexposed, infected monolayers. Transmission electron microscopy of nonoxynol-treated, infected cells revealed apparent damage to the inclusion membrane and reticulate bodies within. The infectivity of the chlamydiae in the iodine-negative inclusions on subpassage was only 0.3%. We conclude that the primary action of nonoxynol-9 is on the McCoy cell and that there may be secondary effects on the intracellular parasite.

Following the attachment of gonococci to human fallopian tube mucosa in organ culture, the gonococci are endocytosed by specialized low columnar epithelial cells, are transported to the base of the epithelial cells, and are subsequently exocytosed into the subepithelial tissues. This transepithelial transport process by which "invasion" of the host occurs appears to be dependent on microbial factors is designated parasite-directed endocytosis to distinguish it from host-directed endocytosis by cells such as macrophages that eventually degrade the parasites. "Invasion" of the host by a number of human pathogens--bacteria (e.g., Neisseria meningitidis, Haemophilus influenzae, and Listeria monocytogenes), viruses, or protozoa--appears to be accomplished by parasite-directed endocytosis.

The nonoxidative antibacterial properties of isolated rat polymorphonuclear leukocyte granule contents were examined using Salmonella typhimurium LT-2 and a series of progressively rough lipopolysaccharide mutants of this strain as target bacteria. The granule extract was most active at 37 °C, with a substantial decrease in activity observed at lower temperatures. Deep rough bacterial mutants were killed best within a pH range of 6–8, while killing of mutants with increased lipopolysaccharide content was most efficient at an acid pH of 5. The activity of the extract was dependent on incubation time but was independent of the number of bacterial cells present in the assay mixture. The killing action of the granule extract was inhibited by the cations Na+, K+, Mg2+, Ca, and Fe2+. The degree of inhibition was dependent on the type and concentration of ion used. Rough mutants grown with aeration to log phase were killed more efficiently than the same mutants grown to stationary phase under static conditions. Also, gram-positive bacteria were more susceptible to the extract than were gram-negative organisms.