No evidence of ongoing HIV replication or compartmentalization in tissues during combination antiretroviral therapy: Implications for HIV eradication.

HIV persistence during combination antiretroviral therapy (cART) is the principal obstacle to cure. Mechanisms responsible for persistence remain uncertain; infections may be maintained by persistence and clonal expansion of infected cells or by ongoing replication in anatomic locations with poor antiretroviral penetration. These mechanisms require different strategies for eradication, and determining their contributions to HIV persistence is essential. We used phylogenetic approaches to investigate, at the DNA level, HIV populations in blood, lymphoid, and other infected tissues obtained at colonoscopy or autopsy in individuals who were on cART for 8 to 16 years. We found no evidence of ongoing replication or compartmentalization of HIV; we did detect clonal expansion of infected cells that were present before cART. Long-term persistence, and not ongoing replication, is primarily responsible for maintaining HIV. HIV-infected cells present when cART is initiated represent the only identifiable source of persistence and is the appropriate focus for eradication.

Evaluation of growth characteristics on blood agar and eosin methylene blue agar for the identification of Candida (Torulopsis) glabrata.

Candida albicans and Candida (Torulopsis) glabrata are the most common species of yeast encountered in the clinical laboratory. In this study, we sought to evaluate simple means of screening cultures for the presence or absence of C. glabrata. Twelve thousand five hundred (12,500) consecutive cultures were evaluated for sufficient yeast growth to warrant identification. When detected (369 isolates), the amount of growth on eosin methylene blue agar (EMB) versus sheep blood agar (BAP) (both incubated in 5% CO2), wet mount morphology, and germ tube production were evaluated. All germ tube-negative yeasts were definitively identified using the Vitek YBC card. Of the 369 yeast isolates included in this study, 225 were C. albicans, 102 C. glabrata, and 42 other Candida species. Growth on EMB was greater than BAP for 92 isolates; all identified as C. glabrata. When EMB growth was equal to or less than BAP, 10 isolates were C. glabrata and 267 were other Candida ssp. An accurate presumptive identification of C. glabrata may be made using the observation of greater growth on EMB versus BAP. When coupled with the germ tube test, the majority of yeast isolates could be identified by these simple methods in our laboratory.

Vitek GPS card susceptibility testing accuracy using direct inoculation from BACTEC 9240 blood culture bottles.

The emergent need for antimicrobial susceptibility testing (AST) data for the therapy of bacteremic patients has led to the development of rapid methods and local procedure modification of some commercial AST products such as the direct inoculation from blood culture systems. We compared the Vitek GPS card results using direct and standardized inoculation with a reference broth microdilution method for 112 consecutive staphylococcal bloodstream infections (seven drugs). Among the 28 Staphylococcus aureus strains, 0%-3.6% total error/drug was observed with both Vitek inoculation procedures. However, the only oxacillin-resistant strain was not detected (100% true very-major error). For 84 coagulase-negative staphylococci (CNS), the direct inoculation procedure had an 11.9% very-major error rate for oxacillin, ampicillin-sulbactam, and cephalothin, plus 4.8% very-major error rate for ciprofloxacin and trimethoprim-sulfamethoxazole (total error rate 4.8%-16.7% for five of seven drugs compared). The Vitek direct inoculation procedure routinely missed 20.4% of oxacillin-resistant CNS strains. The use of Vitek direct inoculation procedures for staphylococcal bloodstream infection isolates (from BACTEC 9240 cultures) produced serious false-susceptible results; this procedure should be avoided in favor of routine package insert-recommended Vitek procedures or other reference-quality overnight incubation susceptibility tests.

Efficient detection and long-term persistence of the carriage of methicillin-resistant Staphylococcus aureus.

The natural history of the carriage of methicillin-resistant Staphylococcus aureus (MRSA) was examined in a 9-year retrospective cohort study of 102 known carriers. The populations studied consisted of patients admitted to a university hospital from 1989 through 1991; a review extending back to January 1983 was conducted. The focuses of the study included the duration of carriage among patients who were known to have carried MRSA previously and who were readmitted to the hospital (36 patients) and the optimal anatomic site for screening (66 patients). Cultures of the nares (sensitivity, 93%; negative predictive value, 95%) were considerably more valuable for the detection of MRSA colonization than were cultures of cutaneous sites of the axilla, groin, and perineum (sensitivity, < or = 39%; negative predictive value, < or = 69%). The estimated half-life of MRSA colonization in this special population of patients was approximately 40 months. Restriction enzyme analysis of plasmid types of paired isolates from the 12 patients with MRSA carriage persisting for > 12 months revealed five instances (42%) in which both isolates were of the same type. In summary, our results indicate that the majority of readmitted carriers harbor MRSA for > 3 years and that, in this population, culture of the anterior nares alone (with culture of wound or sputum, when present) is a valid and efficient method for the detection of persistent MRSA carriage.

Application of the Etest to antimicrobial susceptibility testing of Legionella spp.

Development of susceptibility tests for Legionella spp. has been difficult because of the specific growth requirements of this organism. The most commonly used media, buffered charcoal-yeast extract (BCYE) agar contains charcoal, which is known to inactivate some antimicrobial agents. This study compared five antimicrobial (erythromycin, clindamycin, ofloxacin, doxycycline, and rifampin) minimum inhibitory concentration (MIC) values as determined by agar dilution using BCYE, agar dilution using this same media without the charcoal [buffered yeast extract (BYE) agar], and the Etest on BCYE media. The MIC50 and MIC90 for this group of Legionella spp. were greater with BCYE than with BYE. Erythromycin and ofloxacin (fourfold change) were the most affected by the charcoal in the medium. The Etest MIC and agar dilution MIC with BYE were comparable. The Etest rifampin results demonstrated that the Legionella spp. strains were very susceptible (< 0.016 micrograms/ml, producing very large zones) requiring use of one half of the Etest strip, a whole test strip on an individual 100-mm plate of BCYE, or use of a new low-MIC-range Etest strip. The Etest on BCYE provides a simple, readily available, and accurate method unaffected by medium components for susceptibility testing of Legionella spp.

An experimental model for study of Candida survival and transmission in human volunteers.

In order to determine the potential for cross-transmission of Candida spp. between health-care workers and patients, the survival of clinical isolates of five species of Candida on the palms of human volunteers was tested. One hundred microliters of a McFarland 1.0 density suspension (5 x 10(5) cfu) from an overnight culture of Candida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida glabrata was used as inoculum. The degree of hydrophobicity of the different Candida species was also tested and did not influence the survival. The half-lives were brief, being 9.5, 12.4, 7.4, 12.8, 9.6 min for Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida tropicalis, respectively, but at 45 min 2.6 x 10(3) to 3 x 10(4) organisms remained on the hands. Survival of Candida albicans for as long as 24 h on inanimate surfaces was observed. Transmission from one hand to a second hand occurred in 69% of the experiments and from the first to a third hand in 38%. Transmission to and from inanimate surfaces was successful in most of the experiments (90%). This experimental model aids in the biological study of Candida spp. and suggests some of the potential mechanisms of transmission.

Antifungal activity of a new triazole, D0870, compared with four other antifungal agents tested against clinical isolates of Candida and Torulopsis glabrata.

D0870 is a new triazole agent with potent, broad-spectrum antifungal activity. We investigated the in vitro activity of D0870, fluconazole, itraconazole, amphotericin B, and 5-fluorocytosine (5FC) against 319 clinical isolates of Candida spp. and Torulopsis glabrata. In vitro susceptibility testing was performed using a microdilution broth method performed according to NCCLS guidelines. D0870 was very active (MIC90 of 0.12 microgram/ml and 0.5 microgram/ml at 24 and 48 h incubation, respectively) against all of the yeast isolates. D0870 was 2- to 32-fold more active than amphotericin B and 2- to 8500-fold more active than 5FC. By comparison with the other triazoles, D0870 was generally 2- to 16-fold more active than itraconazole and > or = 16-fold more active than fluconazole. More than half (53%) of C. albicans isolates with elevated fluconazole and itraconazole MICs (> or = 128 micrograms/ml and > 8.0 micrograms/ml, respectively) were inhibited by < or = 1.0 microgram/ml of D0870. Based on these studies, D0870 has promising antifungal activity and warrants further in vitro and in vivo investigation.

Minimum inhibitory concentration quality-control guidelines for biapenem, DU-6859a, FK-037, levofloxacin, grepafloxacin, and ceftizoxime when using various National Committee for Clinical Laboratory Standards susceptibility test methods. Quality Control Study Group.

Several multilaboratory collaborative studies using broth microdilution and agar dilution (gonococcal tests only) methods of susceptibility testing were performed to establish quality-control (QC) giudelines. Replicate dilution tests with multiple lots of media were performed with National Committee for Clinical Laboratory Standards recommended QC strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213) by using the following antimicrobials: biapenem, DU-6859a, FK-037, levofloxacin, and grepafloxacin. In addition QC MIC ranges, using appropriate medium modification for Haemophilus influenzae ATCC 49247 and Neisseria gonorrhoeae ATCC 49226 were reported on four (DU-6859a, FK-037, levofloxacin, and grepafloxacin) and two (ceftizoxime and FK-037) antimicrobials, respectively.

Multicenter comparison of a colorimetric microdilution broth method with the reference macrodilution method for in vitro susceptibility testing of yeast isolates.

This multicenter study was performed to compare a colorimetric microdilution method with the NCCLS M27-P reference macrodilution method for the testing of yeast isolates against amphotericin B, fluconazole, and 5-fluorocytosine (5FC). Testing was performed on ten yeast isolates in five independent laboratories. All sites tested each isolate a total of 20 times with both methods. MICs were read after 48 h incubation. The macrodilution MIC reference range was defined as the modal MIC +/- 1-log2 dilution for each organism-antifungal agent combination. Agreement between the M27-P reference range results and the microdilution MICs was 86% with amphotericin B, 90% with fluconazole, and 93% with 5FC. Based on these data, it is apparent that new approaches, such as the colorimetric microdilution method, will provide MIC values comparable to the M27-P macrodilution method in a format that is more practical for use in a busy clinical laboratory.

Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species.

Three commercially available systems (API Staph-Trac, API 20GP, and Vitek GPI), used to identify coagulase-negative staphylococci, were evaluated against 277 bloodstream isolates, including 94 isolates of Staphylococcus epidermidis and 183 isolates of other coagulase-negative Staphylococcus species. The conventional method of Kloos and Schleifer served as the reference method. Controls included 14 ATCC type culture strains of coagulase-negative staphylococci. The API Staph-Trac system showed the highest rate of agreement with reference method, correctly identifying 73% of the isolates. The Vitek GPI System had an overall rate of agreement of 67% and the API 20GP system correctly identified 61%. The API Staph-Trac system correctly identified 94% of the isolates of S. epidermidis compared with 64% by both Vitek GPI and API 20GP. The most common error for both Vitek GPI and API 20GP systems was the failure to identify organisms contained within the database of the systems. Because none of the tested commercial identification systems identified "non-epidermidis" coagulase-negative Staphylococcus species with a high degree of accuracy, the systems need to be markedly improved or new systems developed.

Effects of blood medium supplements on activities of newer cephalosporins tested against enterococci.

This comparative study determined the effect of blood on the antienterococcal activities of the newer cephalosporins. Standardized disk diffusion susceptibility tests were performed with 57 strains of enterococci (30 Enterococcus faecalis strains) on Mueller-Hinton agar with and without 5% sheep blood supplementation. Twelve cephalosporins representing five different structural groups (based on the 7-alpha position substitution) were tested. The greatest frequency of activity enhancement by blood was observed with cefdaloxime and cefdinir (7-alpha hydroxyimino group) against E. faecalis. Cephalosporins with a 7-alpha methoxyimino group (cefpodoxime, cefepime, and cefpirome) had marked increases in zone diameters (3 to > 9 mm) when tested with the blood supplement. Cephems with 7-alpha amino or carboxy substitutions did not demonstrate any enhanced activity. Awareness of this phenomenon is important for the interpretation and accuracy of cephalosporin susceptibility testing.

The survival of bacteria exposed to desiccation on surfaces associated with farm buildings.

The survival of 11 species of Gram-negative and Gram-positive bacteria was examined on different surfaces exposed to desiccation. There were large variations between species; Pseudomonas spp. and Rhizobium leguminosarum biovars survived for less than 2 d, whilst Enterococcus spp. survived for more than 11 weeks. The type of surface on to which the bacteria were deposited affected survival, but with different effects between species. In addition the survival of spontaneous nalidixic acid-resistant (Nal-r) mutants of a natural Escherichia coli isolate were compared. Overall the differences were slight, but of seven resistant mutants, five survived better than the parent whilst one survived less well. Nine transposon insertion derivatives of one of the Nal-r mutants (ECO80) which survived better than the parent were compared; all survived similarly to the parent except ECO883 which survived less well. The growth characteristics of ECO883 and ECO80 were compared; at high osmotic pressures (> 0.4 mol 1-1 NaCl) ECO883 grew more slowly and showed a longer lag time than the parent. Of the osmoregulatory functions studied, ECO883 appeared to be altered with respect to K+ transport or accumulation, although the transposon insertion had occurred in a gene distant from known K+ transport genes.

Quality control guidelines for cefdinir, cefepime, cefetamet, cefmetazole, cefpodoxime, cefprozil, and clinafloxacin (CI-960) for various National Committee for Clinical Laboratory Standards susceptibility testing methods. Quality Control Study Group.

Several multilaboratory studies to determine quality control (QC) ranges for a variety of National Committee for Clinical Laboratory Standards (NCCLS) susceptibility tests are summarized. Replicate testing used multiple lots of media and antimicrobial disks in accordance with NCCLS recommendations, including the appropriate medium modifications for tests with Haemophilus spp. and Neisseria gonorrhoeae. QC ranges for MIC and disk diffusion testing of N. gonorrhoeae ATCC 49226 were proposed for cefepime, cefetamet, cefmetazole, and cefpodoxime. Disk diffusion QC ranges for Haemophilus influenzae ATCC 49247 or ATCC 49766 were recommended with cefepime, cefetamet (10- and 30-microgram disks), cefmetazole, cefpodoxime, and cefprozil. Disk diffusion QC ranges for Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 with cefdinir and clinafloxacin and those for Pseudomonas aeruginosa ATCC 27853 with clinafloxacin were also proposed.

In vitro antimicrobial activity of tioconazole and its concentrations in vaginal fluids following topical (vagistat-1 6.5%) application.

In vitro assays demonstrated that clinical yeasts were significantly more inhibited by tioconazole (MIC50, < or = 0.5 microgram/ml) than by fluconazole (MIC50, 8 micrograms/ml). Tioconazole also exhibited high potency against most molds (Alterneria spp. and Acremonium spp.). All Candida tropicalis isolates had MICs of 8 micrograms/ml, four-fold greater than any other Candida spp. Generally Gram-negative bacteria were less susceptible to tioconazole. Moraxella catarrhalis (MIC90, 2 micrograms/ml) was the most susceptible Gram-negative species. Staphylococci and enterococci were the most susceptible to tioconazole Gram-positive species (MIC50s, 1-8 micrograms/ml). Bacterial species associated with vaginosis. [Gardnerella vaginalis (MIC90, 16 micrograms/ml), Mobiluncus spp. (MIC90, 16 micrograms/ml) and Prevotella biviadisiens (MIC90, 64 micrograms/ml)] were inhibited by tioconazole. Isolates of Lactobacillus spp. were most resistant (MIC90, > or = 256 micrograms/ml) to tioconazole. Vaginal fluid levels of tioconazole (mean, 91.4 micrograms/ml) persisted above the MIC90 levels (1-64 micrograms/ml) for most fungal and bacterial pathogens for 72 h in 19 evaluable female human subjects receiving 300 mg tioconazole in an intravaginal ointment.

Outbreak of Pseudomonas aeruginosa infections in a surgical intensive care unit: probable transmission via hands of a health care worker.

Pseudomonas aeruginosa was isolated from nine patients (16.2 isolations/1,000 patient-days) in a surgical intensive care unit during an outbreak in November 1990; this rate of isolation was three times higher than that noted previously on this unit. Three patients were infected with the same strain, as defined by identical serotypes, pyocin types, and contour-clamped homogeneous electric field (CHEF) electrophoresis patterns of digested genomic DNA. The hands of 80 health care workers were cultured, and a strain of P. aeruginosa identical to that infecting the three patients was isolated from the hands of a nurse providing care to all three. Environmental surfaces, medical devices, and ward stock supplies were cultured; none of these cultures yielded this strain. No clusters of infection with this strain or other strains of P. aeruginosa were observed after compliance with hand-washing and universal precautions was reemphasized. Thus this outbreak was linked to the carriage of P. aeruginosa on the hands of a health care worker. It could not be determined definitively whether this carriage was the source of the cluster or a consequence of it. However, the geographic and temporal clustering of carriage with an outbreak due to a strain of an apparently identical molecular type underlines the importance of routine hand washing between contacts with different patients.

Species identification and determination of high-level aminoglycoside resistance among enterococci. Comparison study of sterile body fluid isolates, 1985-1991.

Enterococcus spp. have become the third most common cause of nosocomial infections. High-level aminoglycoside resistance (HLR), an important clinical concern, has been associated with some species of the enterococci. We evaluated the Vitek and API 20S systems for species identification and the Vitek for the detection of HLR. Enterococci from nosocomial infections (208 strains) at the University of Iowa Hospital (1985-1991) were tested by Vitek, API 20S, and reference methods. The error rate for species identification was 6.7% for the API 20S and 5.8% for the Vitek Gram-positive identification (GPI) cards. Both systems tended to incorrectly identify other enterococcal species as Enterococcus faecium. HLR was found in Enterococcus faecalis and E. faecium isolates only. The highest rates of HLR to streptomycin alone (17.9%) and with gentamicin (13.5%) was observed among E. faecalis strains, and to gentamicin alone (7.3%) was found among E. faecium isolates. No apparent differences in HLR rates were found from year-to-year over the 7-year enterococcus sample interval. Susceptibility errors for Vitek were among the streptomycin tests only. Our results demonstrated acceptable performance by the Vitek cards for enterococcal species identification and the detection of HLR. API 20S also provided an acceptable ability to speciate the enterococci within its data base, however, both systems must be improved by adding other clinical important Enterococcus species.

Disk diffusion quality control guidelines for Haemophilus susceptibility tests using cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.

A multilaboratory study to determine disk diffusion quality control ranges for Haemophilus influenzae ATCC 49247 and five investigational drugs was performed. Multiple lots of Haemophilus Test Medium and antibiotic disks were used for replicate testing in conformance with the recommendations of the National Committee for Clinical Laboratory Standards. Quality control disk zone diameter ranges were proposed for cefdinir, CI-960, fleroxacin, temafloxacin, and trospectomycin.

MIC quality control guidelines for Haemophilus susceptibility tests using cefdinir (FK482), cefepime, cefetamet, cefpirome, ceftibuten, fleroxacin, temafloxacin, clarithromycin, RP59500, and trospectomycin.

A multilaboratory study was performed to establish broth microdilution MIC quality control (QC) guidelines for 10 investigational drugs which previously demonstrated significant activity against Haemophilus influenzae. MIC QC ranges for H. influenzae ATCC 49247 with Haemophilus test medium were determined by using multiple contemporary lots of Haemophilus test medium and the National Committee for Clinical Laboratory Standards' recommended numbers of replicate tests. On the basis of these results, QC ranges (generally modal MIC +/- one log2 dilution) are proposed for cefdinir, cefepime, cefetamet, cefpirome, ceftibuten, fleroxacin, temafloxacin, clarithromycin, RP59500, and trospectomycin. The proposed QC guidelines for clarithromycin and temafloxacin were recently accepted by the National Committee for Clinical Laboratory Standards.

Comparative in vitro activity of the new quinolone sparfloxacin (CI-978, AT-4140) against nosocomial gram-negative bloodstream isolates.

The in vitro activity of sparfloxacin (CI-978, AT-4140), a new quinolone which is active against gram-negative and gram-positive bacteria, and nine other broad-spectrum antibiotics was tested against 128 gram-negative nosocomial bloodstream isolates from separate patients. Sparfloxacin and ciprofloxacin were among the most potent antibiotics against Escherichia coli (n = 40), Enterobacter cloacae (n = 18), Klebsiella oxytoca (n = 13), and Klebsiella pneumoniae (n = 19), with MIC90 values of less than or equal to 0.25 microgram/ml. Ciprofloxacin was slightly more potent than sparfloxacin against Serratia marcescens (n = 14), the MIC90 values being 0.25 and 1.0 micrograms/ml respectively, although all strains were susceptible to both agents. Sparfloxacin was slightly less potent than ciprofloxacin against Pseudomonas aeruginosa (n = 24), the MIC90 values being 4.0 and 0.5 micrograms/ml respectively. Overall, the in vitro activity of sparfloxacin compared favorably with that of ciprofloxacin and the other broad spectrum agents tested against nosocomial gram-negative bloodstream isolates.