A comparison of the Sensititre® MYCOTB panel and the agar proportion method for the susceptibility testing of Mycobacterium tuberculosis.

The agar proportion method (APM) for determining Mycobacterium tuberculosis susceptibilities is a qualitative method that requires 21 days in order to produce the results. The Sensititre method allows for a quantitative assessment. Our objective was to compare the accuracy, time to results, and ease of use of the Sensititre method to the APM. 7H10 plates in the APM and 96-well microtiter dry MYCOTB panels containing 12 antibiotics at full dilution ranges in the Sensititre method were inoculated with M. tuberculosis and read for colony growth. Thirty-seven clinical isolates were tested using both methods and 26 challenge strains of blinded susceptibilities were tested using the Sensititre method only. The Sensititre method displayed 99.3% concordance with the APM. The APM provided reliable results on day 21, whereas the Sensititre method displayed consistent results by day 10. The Sensititre method provides a more rapid, quantitative, and efficient method of testing both first- and second-line drugs when compared to the gold standard. It will give clinicians a sense of the degree of susceptibility, thus, guiding the therapeutic decision-making process. Furthermore, the microwell plate format without the need for instrumentation will allow its use in resource-poor settings.

The identification of mycobacteria from solid media and directly from VersaTREK Myco bottles using the Sherlock Mycobacteria Identification HPLC system.

The Sherlock Mycobacteria Identification HPLC system correctly identified to the species level 61 (67.8%) of 90 isolates growing on solid media, and 73 (45.3%) of 161 isolates directly from positive VersaTREK Myco bottles. When these data were re-analysed with a revised database, correct identifications increased to 91.1% and 83.2%, respectively. All Mycobacterium tuberculosis isolates were identified correctly, regardless of the inoculum source or database used. The use of the revised database with isolates obtained directly from positive VersaTREK Myco bottles allows the identification of most isolates within clinically relevant time-frames.

Osteomyelitis caused by Mycobacterium haemophilum: successful therapy in two patients with AIDS.

OBJECTIVE: Difficulties involved in diagnosis and response to antimicrobial therapy are described in detail for two cases of biopsy-proven osteomyelitis caused by Mycobacterium haemophilum in AIDS patients. SETTING: Two large, private teaching hospitals in New York City, New York, USA. PATIENTS, PARTICIPANTS: A 31-year-old woman with previous diagnoses of candida esophagitis and peripheral neuropathy (patient 1), and a 37-year-old man with Kaposi's sarcoma (patient 2). INTERVENTIONS: One patient was treated with a combination of rifampin, ethambutol, clofazimine, and ciprofloxacin, while the other received rifampin, ciprofloxacin and doxycycline. Both patients also received a short course of intravenous amikacin. MAIN OUTCOME MEASURES: Disease activity was monitored clinically by observing resolution of skin ulcers, lymphadenopathy, and pain and swelling in areas affected by osteomyelitis. RESULTS: Both patients experienced complete resolution of signs and symptoms of M. haemophilum infection. Patient 1 was treated for 17 months and remains well after 10 months without therapy. Patient 2 shows no evidence of infection after 14 months of therapy. CONCLUSIONS: M. haemophilum infection must be considered in the differential diagnosis of osteomyelitis in AIDS patients, although specialized culture techniques are required to isolate and identify this pathogen. Excellent clinical response can be achieved with oral antimicrobial therapy.

The emergence of resistant strains of Acinetobacter baumannii: clinical and infection control implications.

A prospective study was undertaken to determine colonization rates, susceptibility profiles, and outcomes in patients with clinical isolates of Acinetobacter baumannii. Fifty percent of patients became colonized with A. baumannii, and 29% of these patients had clinical and colonizing isolates with discordant susceptibility profiles, without apparent relation to antibiotic use. Barrier infection control measures are necessary to prevent nosocomial transmission.

The ESP culture system for drug susceptibilities of Mycobacterium avium complex.

OBJECTIVE: To validate the non-radiometric, broth-based ESP system for determining Mycobacterium avium complex (MAC) susceptibilities. METHODS: MAC isolates from sterile body sites of 20 adult HIV-infected patients who were failing their present MAC regimen were identified. Susceptibilities were determined and comparisons made between the agar proportion method and the ESP system for clarithromycin, ethambutol, sparfloxacin and cycloserine. RESULTS: Ninety-nine percent of the MICS generated by the ESP system user identical to or lower than the MICs determined by the agar proportion METHOD: In vitro resistance was documented by the ESP system for 86% of the drugs that patients were taking at the time of breakthrough, and no resistance was seen to cycloserine, a drug that no patient was taking. CONCLUSIONS: The ESP system, a fast and reliable method for determining MAC susceptibilities, could be used to optimize MAC regimens in a timely fashion, avoid the use of ineffective drugs, minimize emerging resistance and ultimately improve outcome.

Chloramphenicol treatment for vancomycin-resistant Enterococcus faecium bacteremia.

OBJECTIVE: To evaluate the results of treating vancomycin-resistant Enterococcus faecium (VREF) bacteremia with chloramphenicol. METHODS: We retrospectively reviewed the charts of all adult patients with VREF bacteremia treated with chloramphenicol during the calendar year 1998 at a 522-bed tertiary referral center in New York City. Patients were identified by reviewing microbiology laboratory records. Patients with clinically significant VREF bacteremia who received chloramphenicol for at least 48 h were included in the study. Clinical and microbiological outcomes were determined. Microbiological and molecular tests were performed on a small representative sample of isolates to identify the presence of resistance mechanisms and to look for similarity among the isolates. RESULTS: Seven episodes of significant VREF bacteremia occurred in six patients. Mean age was 54 years. All patients had cancer and three had severe neutropenia. Five of seven episodes were associated with chronic indwelling devices, but in only one of these cases was the device removed. All isolates were susceptible to chloramphenicol in vitro. All six microbiologically evaluable episodes had a favorable response to chloramphenicol treatment, and four of seven (57%) clinically evaluable episodes had favorable outcomes. Only one death may have been due to VREF bacteremia, so the maximal attributable mortality was 14%. The three representative samples that were tested further were indistinguishable from one another and they displayed no evidence of resistance mechanisms. CONCLUSIONS: In a cohort of severely ill cancer patients, chloramphenicol was effective in treating VREF bacteremia. The use of chloramphenicol should be considered in treating infections with this highly resistant organism, where therapeutic options are limited.

In vivo scanning slit confocal microscopy of Acanthamoeba keratitis. A case report.

A 29-year-old woman presented with clinical signs and symptoms of Acanthamoeba keratitis. Scanning slit confocal microscopy revealed a 26-mu-diameter object, resembling an Acanthamoeba cyst, in the anterior stroma. Numerous ovoid objects (possibly inflammatory cells, trophozoites, or altered keratocytes) were present. Normal keratocyte nuclei and the anterior corneal mosaic, readily imaged by scanning slit confocal microscopy of the normal cornea, were noticeably absent. Subsequent corneal biopsy confirmed the diagnosis of Acanthamoeba keratitis.

Obstacles to penicillin use in treating pneumococcal infection.

OBJECTIVES: To determine the pattern of penicillin use in the treatment of pneumococcal pneumonia, and factors contributing to the use of alternative antibiotics. METHODS: This study included all adult inpatients of St. Vincent's Hospital and Medical Center who had documented pneumococcal pneumonia between December 1998 and October 1999. St. Vincent's is a 600 bed tertiary teaching hospital in New York City. Patients who had Streptococcus pneumoniae isolated from a respiratory tract specimen were identified through microbiology laboratory records. A retrospective chart review of these patients was conducted, and those identified with clinical pneumonia were included in this study. Antibiotic use, patient demographics, resistance data, and clinician awareness of the antibiotic susceptibility results were noted. RESULTS: Sixty adult patients hospitalized with documented pneumococcal pneumonia were identified. Thirteen (21.6%) of the 60 patients received penicillin or ampicillin. Susceptibility results were not noted in the medical record in 21 (35.0%) of the 60 patients, and none received penicillin. High rates of reported penicillin allergy in 8 (13.3%) of the 60 patients, and reluctance to use penicillin when isolates demonstrated intermediate susceptibility in 8 (13.3%) of the 60 patients were observed. CONCLUSIONS: Several remediable obstacles to penicillin use were identified in this study. An increased awareness of susceptibility results by physicians and education of practitioners could have increased the use of penicillin as therapy to two-thirds of these patients.

Ciprofloxacin susceptibility testing by MIC and disk elution of drug-resistant Mycobacterium tuberculosis and Mycobacterium avium complex.

The ability to provide susceptibility data for certain species of mycobacteria can be clinically useful. In this study, the disk elution method for susceptibility testing was adapted for testing ciprofloxacin against mycobacterial isolates. Of the 75 Mycobacterium tuberculosis isolates tested, including 23 multiply drug-resistant isolates, 96% were susceptible to ciprofloxacin at a breakpoint concentration of 2 micrograms/ml.

Use of nucleic acid probes to identify mycobacteria directly from Difco ESP-Myco bottles.

Mycobacterial isolates were identified directly from positive ESP-Myco bottles by use of nucleic acid probes. Retrospective analysis of 360 cultures which grew either Mycobacterium tuberculosis, M. avium complex, or M. gordonae showed that 87% were identified by direct testing of an aliquot obtained at the time a positive culture was detected. Another 12% of these cultures gave results in the equivocal range, with only 1% of the isolates yielding negative results on initial testing.

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.

Isolation and characterization of a trypsin-like serine proteinase from the membranes of Walker 256 carcino-sarcoma cells.

A serine proteinase was isolated from Walker-256-carcino-sarcoma plasma-membrane-enriched preparations by affinity chromatography employing soya-bean trypsin inhibitor as the ligand. This enzyme was termed 'memsin' owing to its membrane location and trypsin-like substrate specificity. Analysis of this preparation by steric-exclusion high-pressure liquid chromatography (h.p.l.c.) resulted in a single peak of enzyme activity. Calculations of the rates of inactivation of memsin by peptidyl-chloromethanes and comparison with rate constants obtained with other serine proteinases indicated that memsin closely resembled trypsin and acrosin. Digestion of oxidized ribonuclease by memsin and analysis of the resulting peptides by h.p.l.c. yielded a chromatogram that was very similar to one generated by a tryptic digest of oxidized ribonuclease. This enzyme could possibly play a role in tumour-cell invasion.

Improved isolation and differentiation of enterococci in cheese.

Further documentation of an enterococcus selective differential (ESD) medium was obtained in isolations from eight different cheeses. An improved differentiation of tetrazolium salt (2, 3, 5-triphenyl tetrazolium hydrochloride [TTC])-reducing strains of Streptococcus faecalis from TTC-nonreducing or TTC-faintly-reducing Streptococcus faecium was attained. The sensitivity of the medium was evaluated in comparison with that of KF streptococcal, Pfizer selective enterococcus (PSE), the medium of Reinbold, Swern, and Hussong (RSH), and the medium of Saraswat, Clark, and Reinbold (SCR). Selective counts, rate of colony formation, and ease of isolation and differentiation of colonies were examined. The specificity of the medium was also investigated. ESD supported the fastest rate of growth and the maximum size of colonies; counts in this medium were in most cases possible with 17 h of incubation, whereas the other media required 24 to 48 h. A presumptive identification of 1,077 isolates by four biochemical tests disclosed that SCR, RSH, and ESD selected high, comparable percentages of strains that approximated most closely the typical description of enterococci (66, 60.1, and 58%, respectively). Low percentages (21.1 and 30.7%) were yielded by KF and PSE. The utility of ESD for a rapid, presumptive identification of enterococci was confirmed by serological and biochemical testing of color TTC-differentiated colonies isolated from 18 cheeses.