Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns.

Ureaplasma urealyticum, a common commensal of the urogenital tract of sexually mature humans, is gaining recognition as an important opportunistic pathogen during pregnancy. While its etiologic significance in many aspects of adverse pregnancy remains controversial, recent evidence indicates that U. urealyticum in the absence of other organisms is a cause of chorioamnionitis. Furthermore, ureaplasmal infection of the chorioamnion is significantly associated with premature spontaneous labor and delivery. In at least some cases, it appears to be causal. Present evidence indicates that U. urealyticum is a cause of septicemia, meningitis, and pneumonia in newborn infants, particularly those born prematurely. There is strong but not definitive evidence that ureaplasmal infection of the lower respiratory tract can lead to development of chronic lung disease in very low-birth-weight infants. Although risk factors for colonization of the lower genitourinary tract have been identified, little information is available concerning risk factors for intrauterine infection and host immune responses to invasive infection. Recent establishment of animal models of respiratory and central nervous system diseases should provide an opportunity to evaluate risk factors, pathogenic mechanisms, and operative immune mechanisms. However, the most critical need is additional information concerning indications for diagnosis and treatment as well as efficacy of treatment.     

Subunit structure of the variable V-1 antigen of Mycoplasma pulmonis.

It was previously shown that multiple structural variants of the V-1 antigen (variable antigen 1) of Mycoplasma pulmonis could be found within a single strain. This antigen is unusual in that it produces a ladder pattern after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The present study showed that some variants of V-1 could be extracted into the aqueous phase of a phenol-H2O system. Analysis with anti-V-1 monoclonal antibodies showed that the phenol-H2O-extracted V-1 had a regular spacing of 3.1 kilodaltons (kDa) between bands and trypsinization of this extracted V-1 resulted in the gradual symmetrical collapse (2.9-kDa increments) of the ladder into a single band, suggesting the presence of multiple identical subunits within the V-1 structure. The upper band from the phenol-H2O-extracted V-1 was isolated and analyzed by SDS-polyacrylamide gel electrophoresis immunoblotting, resulting in the regeneration of the original ladder pattern with 3.1-kDa spacing between bands. When V-1 was boiled for increasing times in the presence of SDS, the staining intensity of the upper band decreased with the concurrent appearance of additional lower-molecular-weight bands. Finally, by using whole cells, it was found that the lower-molecular-weight species of the ladder pattern selectively partitioned into the hydrophobic phase of a Triton X-114 phase partitioning system, and the higher-molecular-weight bands were found in the aqueous phase. These data indicate that the V-1 bands are composed of subunits which may aggregate via hydrophobic interactions and that these aggregates at least partially dissociate when exposed to harsh denaturing conditions, resulting in the characteristic ladder pattern of V-1.     

Routine analysis of amphetamine class drugs as their naphthaquinone derivatives in human urine by high-performance liquid chromatography

We describe a simple HPLC method which is suitable for the routine confirmation of immunoassay positive amphetamine urine samples. The precolumn derivisation method employing sodium naphthaquinone-4-sulphonate was found to have adequate sensitivity, selectivity and precision for the measurement of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) at 500 microg/l cutoff level for confirmatory analysis of amphetamines in urine. The specificity of the method is enhanced by detecting the peaks at two different wavelengths. The ratios of the peak heights measured at the two wavelengths were different for each of the 5 amphetamines analysed. There was no interference from other phenylethylamine analogues that are commonly found in "over the counter" preparations. The HPLC method is compared to a commercial TLC system for detecting amphetamines in urine of drug abusers attending drug rehabilitation programmes. The HPLC confirmatory method described is a viable alternative to GC or to the more complex and costly GC-MS techniques for confirming amphetamine, methamphetamine, MDMA, MDA and MDEA in urine of drug abusers especially when used in a clinical care setting.     

Blood Cultures Positive for Coagulase-Negative Staphylococci: Antisepsis,Pseudobacteremia, and Therapy of Patients

A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (P = 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (P = 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians’ intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.Coagulase-negative staphylococci (CoNS), the most frequent blood culture isolates, are predominantly blood culture contaminants, but they are also a significant cause of bacteremia (2–5, 7, 9, 13). Institution-specific contamination rates vary from 2 to more than 6% (3, 5, 23, 26, 27). In the past 5 years, estimated contamination rates at our hospitals ranged from 2.5 to 3.5%. During this period, CoNS accounted for 45 to 60% of total blood isolates, and we estimated, using laboratory criteria, that 10 to 12% of CoNS isolates from blood were implicated in significant bloodstream infections. A relatively large proportion of the patient population with presumed false-positive blood cultures due to contaminants (pseudobacteremia) were treated with antimicrobial agents, in particular, vancomycin.Clinical and microbiologic guidelines for the differentiation of true bacteremia from pseudobacteremia or contamination have been published (5, 13, 15). Suggested laboratory criteria for true bacteremia include growth within 48 h and multiple blood cultures positive for the same organism. In contrast, increased duration of time before positivity, polymicrobial growth of skin organisms, or growth during antibiotic treatment suggest contamination. Others recommended that the addition of clinical guidelines is essential for the appropriate classification of bacteremia (4, 8, 9, 15, 18).We conducted a cohort study to evaluate clinical and laboratory data for adult patients with blood cultures positive for CoNS. The study was done at two tertiary-care teaching centers, Deaconess Medical Center (DMC) and Sacred Heart Medical Center (SHMC), with a combined capacity of 900 beds. We examined problems associated with false-positive bacteremia and determined the incidence of significant bacteremia. Our goal was to make recommendations to improve clinicians’ ability to recognize the significance of potentially contaminating organisms and to evaluate treatment given to patients with CoNS-positive blood cultures. To attempt to minimize contamination, we evaluated the nontoxic, antiseptic and disinfectant chlorine peroxide in comparison to a standard disinfectant.(This work was previously presented in abstract form at the 96th General Meeting of the American Society for Microbiology, New Orleans, La., 19 to 23 May 1996 [24a].)     

Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies

Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.     

Colchicine myopathy and neuropathy

Although colchicine has been used for centuries, its neuromuscular toxicity in humans is largely unrecognized. In this report we describe a characteristic syndrome of myopathy and neuropathy and present 12 new cases of the condition. Colchicine myopathy may occur in patients with gout who take customary doses of the drug but who have elevated plasma drug levels because of altered renal function. It usually presents with proximal weakness and always presents with elevation of serum creatine kinase; both features remit within three to four weeks after the drug is discontinued. The accompanying axonal polyneuropathy is mild and resolves slowly. Electromyography of proximal muscles shows a myopathy that is marked by abnormal spontaneous activity. Because of these features, colchicine myoneuropathy is usually misdiagnosed initially, either as probable polymyositis or as uremic neuropathy. The myopathy is vacuolar, marked by accumulation of lysosomes and autophagic vacuoles unrelated to necrosis or to the mild denervation in distal muscles. The morphologic changes in muscle suggest that the pathogenesis involves disruption of a microtubule-dependent cytoskeletal network that interacts with lysosomes. Correct diagnosis may save patients with this disorder from inappropriate therapy.     

Reinterpretation of the Dick test: role of group A streptococcal pyrogenic exotoxin.

Because of the association of the group A streptococcal pyrogenic exotoxins (SPEs) with erythrogenic toxin used in the classical Dick test, the involvement of the SPEs in production of erythematous skin reactions was assessed. Unless they had been presensitized, young adult rabbits failed to show skin reactions after intracutaneous challenged with SPEs. Rabbits presensitized to purified protein derivative exhibited enhanced skin reactivity when given purified protein derivative plus SPE C; the enhancement was neutralized by antiserum to SPE C. Rabbits sensitized to bovine serum albumin showed extensive red rash development resembling scarlet fever rashes when given bovine serum albumin containing SPE C. Desquamation occurred 5 to 10 days after injection. Animals sensitized to one SPE type showed enhanced skin reactivity to challenge with homologous or heterologous SPE types, indicating the presence of a cross-reactive determinant within the SPE molecules. Repeated challenge of SPE-sensitized animals with homologous toxin resulted in concomitant antitoxin production with reduction of the enhanced skin reactivities, until typical delayed-hypersensitivity skin reactions remained. The data indicate that, in addition to the toxic reaction previously described, SPEs enhance Arthus and delayed-hypersensitivity skin reactions. It follows that erythrogenic toxin represents the enhancement of acquired skin reactivity to streptococcal antigens by one or more SPE types. Therefore, the Dick test measures SPE-enhanced hypersensitivity to streptococcal products.