Disseminated Mycobacterium haemophilum infection in two patients with the acquired immunodeficiency syndrome.

In two patients with the acquired immunodeficiency syndrome (AIDS), multiple erythematous cutaneous lesions developed, revealing acid-fast organisms on Fite's stain. Initial culture results on the first patient were negative; however, repeat cultures on hemin-enriched media were positive for Mycobacterium haemophilum. Despite antimycobacterial therapy, lesions persisted until the patients' death. M. haemophilum should be a diagnostic consideration when smears of peripheral (especially skin) lesions from AIDS patients show acid-fast organisms. Cultures in such patients should be done with iron hemin- or ferric ammonium citrate-enriched media.

Invasive infection with Sarcinosporon inkin in a patient with chronic granulomatous disease.

Sarcinosporon inkin, a rare skin fungus, was found to have caused progressive pneumonia in a young male with chronic granulomatous disease. Histologic sections of right upper lobe lung tissue showed clusters of globose to oblong hyalin-walled, septate, sporangia throughout the necrotic areas within the pyogranulomas. Pure cultures of S. inkin were recovered from the surgical specimen of the lung. Current status of the taxonomy of S. inkin is reviewed and clarified. Treatment of the patient with Amphotericin B and white blood cell transfusions led to clinical and radiographic response. This is the first documented case of systemic infection caused by S. inkin.

The laboratory diagnosis of mycobacterial diseases.

Recent developments in diagnostic mycobacteriology are surveyed with emphasis on laboratory capabilities relevant to the more rapid and accurate detection and identification of mycobacterial pathogens. Some terminologic problems are reviewed, and some newly recognized mycobacterial pathogens are discussed. Newer methodologies presented in some detail include the radiometric technique for detection, identification, and susceptibility testing of mycobacteria, and the use of DNA probes for identification of mycobacterial species and species complexes. The utility of other developing methodologies, such as polymerase chain reaction technology, analysis of body fluids for tuberculostearic acid, and the use of ELISA are also assessed.

Malignant lymphoma of the jejunum in a patient with Wiskott-Aldrich syndrome. Surgical treatment.

An 8-year-old boy with Wiskott-Aldrich syndrome underwent laparotomy and resection of a stage 1E malignant lymphoma of the jejunum. Although preoperative platelet counts were less than 10,000/cu mm, intraoperative bleeding was minimal, and postoperative bleeding from the wound was easily controlled with platelet infusions and local application of epinephrine. Six weeks later, he underwent reoperation for small intestinal obstruction and no residual tumor could be identified. The patient died 4 1/2 months following the initial surgery from an intracerebral hemorrhage. Postmortem examiniation did not reveal residual lymphoma.

Inter-bottle transfer of mycobacteria by the BACTEC 460.

As a result of several episodes of inter-bottle transfer of molds and mycobacteria in our BACTEC 460 TB System (Johnston Laboratories, Towson, MD), we designed some experiments to attempt to reproduce the transfer process. We demonstrated that organism transfer could occur with our instrument during routine use without any indication of instrument malfunction. Utilizing a redesigned A1B4 circuit board that extends the time of the needle heating cycle to 85 sec and increases the needle heater maximum temperature, we were not able to effect organism transfer even from bottles with very high growth indices. We also demonstrated that even with the redesigned A1B4 circuit board, the needles were not heated sufficiently to sterilize them after direct insertion into a mycobacterial suspension. Organism transfer also occurred while using a second redesigned A1B4 circuit board which extends the needle heating cycle to 75 sec. Users should be aware that organism transfer can occur with the BACTEC 460 under certain circumstances. Maintenance and testing recommendations from Johnston Laboratories should be scrupulously followed, and we suggest some additional procedures that might further reduce the possibility of organism transfer.

Comparison of three techniques for concentrating positive BACTEC 13A bottles for mycobacterial DNA probe analysis.

Three methods of concentrating 1 ml aliquots from BACTEC 13A bottles containing patient blood samples were evaluated for testing with the Gen-Probe Rapid Diagnostic System for Mycobacteria avium complex: 1. using no reagents, 2. using both lysing and wash reagents; and 3. using lysing reagent only. Aliquots from 13As containing human blood and seeded with eight mycobacterial species were also concentrated directly and using both reagents. Results for samples containing M. avium were as follows: 1. using the direct concentration technique 34 of 47 samples (72%) gave unequivocally positive results; 2. 43 of 47 samples (92%) concentrated using both reagents gave positive results; 3. the technique using lysing reagent only was not found useful. There were no false positives with any of the seeded specimens. We were also able to define the minimum Growth Index necessary to ensure un-equivocally positive results for each concentration technique. For those samples containing M. avium these values were 42 for the technique using both reagents and 86 for the direct technique. Direct or reagent concentration of 13A aliquots for testing with Gen-Probe DNA probes provides a rapid, sensitive, and specific means for the identification of M. avium complex bacteremia.

Mycobacteremia caused by simultaneous infection with Mycobacterium avium and Mycobacterium intracellulare detected by analysis of a BACTEC 13A bottle with the Gen-Probe kit.

Simultaneous infection with Mycobacterium avium and Mycobacterium intracellulare in an AIDS patient was suspected after direct analysis of two BACTEC 13A blood cultures with the Gen-Probe kit for M. avium complex. A mixed infection was confirmed by evaluating isolated colonies. The Gen-Probe kit may provide a simple technique for detecting mixed M. avium-M. intracellulare infections.

Lysis-centrifugation blood cultures in the detection of tissue-proven invasive candidiasis. Disseminated versus single-organ infection.

Several studies have demonstrated significantly higher frequency and more rapid detection of candidemia with blood culture methods performed by lysis-centrifugation (LC) in comparison with other techniques. Little is known, however, about the ability of LC blood culture methods to detect tissue-proven invasive candidiasis. We therefore investigated the sensitivity of LC blood cultures in the detection of tissue-proven invasive candidiasis. Between 1985 and 1991, invasive candidiasis was detected in 41 (5.1%) of 803 autopsies at the Clinical Center of the National Institutes of Health (Bethesda, MD, USA). Cases were classified as single-organ (SO) candidiasis (n = 20) and as disseminated candidiasis (DI) (n = 21). Patients with DI were more likely than those with SO to have a hematologic malignancy (71% vs 15%, P < 0.001) and to have gastrointestinal mucosal candidiasis (76% vs 25%, P = 0.003). LC detected fungemia in 16 (43%) of all 37 cases with blood cultures. When analyzed by classification, Candida spp. were isolated from blood in 11 (58%) of 19 patients with DI and in five (28%) of 18 patients with SO (P = 0.13). When analyzed by number of organs infected, blood cultures were positive in seven (78%) of nine patients with > 3 organs infected by Candida in comparison to five (28%) of 18 patients with one organ infected (P = 0.024). The mean recovery time for Candida in blood cultures was 2.6 days in DI and 3.2 days in SO (P = 0.017). There was no difference in colonies of organisms per LC tube between patients with DI and those with SO.(ABSTRACT TRUNCATED AT 250 WORDS)

Controversies in antimicrobial susceptibility testing.

This article examines a number of areas of disagreement surrounding antimicrobial susceptibility testing and discusses some of the useful susceptibility testing techniques for which no standardized procedures have been established.

Identification of mycobacteria by conventional methods.

The methodology and use of the conventional tests employed in the identification of the currently recognized human mycobacterial pathogens are reviewed. The common disease presentations of each species are briefly noted. Tabular summaries of the phenotypic characteristics of these organisms have also been provided. It should be re-emphasized that the use of conventional methods, unlike the rapid methods now available, is not recommended for the initial identification of the M. tuberculosis complex. We also urge caution in the identification of unfamiliar or atypical isolates. It is to be expected that additional species of human mycobacterial pathogens will be characterized in the future; many of these may be represented by isolates that differ phenotypically little, if at all, from species currently recognized.

Multicategory interpretive reporting of susceptibility testing with selected antimicrobial concentrations. Ten years of laboratory and clinical experience.

For the last 10 years, the NIH Microbiology Laboratory has been using a broth microdilution method to perform antibiotic susceptibility tests. Instead of using a continuous twofold dilution series as we had done for the previous 10 years, in 1978 we introduced a susceptibility panel that utilized a minimal number of selected (discontinuous) antimicrobic concentrations. The concentrations selected were those thought to be the most clinically relevant, based on known pharmacologic properties of each antimicrobic agent, as well as known MIC population distributions that we had acquired on 50,000 organisms in the preceding years. In addition to using selected concentrations, we also added interpretive codes to aid the physician in selecting the best antimicrobic agent to use. We had previously only reported quantitative MIC results without qualitative interpretations. The present interpretive criteria inform the physician not only if the organism is susceptible or resistant but also if intramuscular or intravenous doses are needed, if the organism is susceptible to an antimicrobic agent but only for lower urinary tract infections, if the organism is resistant to penicillin by virtue of penicillinase production, and in the case of streptococci, if streptomycin or gentamicin can be expected to show synergy when combined with a penicillin. The use of clinically relevant selected concentrations combined with clear interpretive criteria has worked well in our hospital setting. Physicians are able to understand and utilize the information effectively and have found almost no need for exact MICs using a twofold dilution series.

College of American Pathologists Mycobacteriology E Proficiency Testing Survey. Summary of participant performance, 1979-1992.

The College of American Pathologists first offered a program of proficiency testing in mycobacteriology in 1969 to laboratories that offered any extent of diagnostic service. This program was intended to provide a mechanism for evaluation of methods of staining, culture, identification, and susceptibility testing. From 1979 to 1992, the period covered by this review, participation in the Mycobacteriology E Survey increased almost sixfold. On graded smears to be stained for detection of acid-fast bacilli, more than 85% of Extent 4 and Extent 3 laboratories and more than 80% of Extent 2 laboratories responded correctly to all specimens except one. Performance on specimens that contained Mycobacterium tuberculosis was similar for Extent 4 and Extent 3 laboratories. For all specimens containing M tuberculosis, a mean of more than 90% of Extent 4 and Extent 3 laboratories provided a correct identification each year except 1979, when a mean of 83% of Extent 3 laboratories responded correctly. Only Extent 4 laboratories were required to identify isolates other than M tuberculosis to the species level. For specimens that contained nontuberculous mycobacteria, the means of the yearly averages of correct responses for Extent 4 laboratories were 90% or greater for M kansasii, M marinum, M avium complex, and M fortuitum-chelonae complex and less than 85% for M bovis, M simiae, M scrofulaceum, M szulgai, M flavescens, M xenopi, M terrae complex, and M gastri. In general, on these same specimens, a slightly higher percentage of Extent 3 laboratories (which were required to identify only M tuberculosis to the species or complex level) gave correct or acceptable responses, and the performance of Extent 2 laboratories (which were only required to report whether or not a mycobacterium was present) was the best of all extents. The data suggest that laboratory performance improved somewhat after initial experience with uncommonly encountered organisms. For the most part, however, performance with a given species changed minimally from year to year.

Current status of mycobacterial testing in clinical laboratories. Results of a questionnaire completed by participants in the College of American Pathologists Mycobacteriology E survey.

To learn what methods are used in clinical microbiology laboratories for detection, identification, and susceptibility testing of mycobacteria, questions addressing these issues were added to the College of American Pathologists' Mycobacteriology E proficiency testing survey, and participants in the survey were asked to complete the questionnaire. Other questions related to numbers of mycobacteriology tests performed and the frequency with which drug-resistant Mycobacterium tuberculosis is isolated. Just over half of the respondents stained smears for acid-fast bacilli with the fluorochrome stain. Only 26% of respondents processed respiratory specimens daily, potentially providing results within 24 hours of receiving the specimen. The most rapid methods currently available for detection of mycobacteria and for identification of M tuberculosis, respectively, were used by 30% and 35% of the respondents. Half of the respondents who performed susceptibility testing on isolates of M tuberculosis used rapid methods. Approximately 26% of the respondents indicated that they usually provided a final identification of M tuberculosis within 21 days of receiving a specimen, and 11% indicated that identification and susceptibility test results were reported within 28 days. Participants' responses indicated that the mean number of specimens received for mycobacterial culture per month was higher during the first 5 months of 1992 than it had been in 1991. Moreover, the number of drug-resistant M tuberculosis isolated per month from January through May 1992 was about double the number isolated per month in 1991.(ABSTRACT TRUNCATED AT 250 WORDS)

Susceptibility testing of Mycobacterium avium complex in clinical laboratories. Results of a questionnaire and proficiency test performance by participants in the College of American Pathologists Mycobacteriology E Survey.

OBJECTIVES: To obtain information regarding the frequency and methodology of susceptibility testing of Mycobacterium avium complex (MAC) in clinical microbiology laboratories, and to assess interlaboratory reproducibility of MAC susceptibility testing. DESIGN: Questions addressing MAC susceptibility testing were added to the College of American Pathologists' 1994 Mycobacteriology E Proficiency Testing Survey, and participants were asked to complete the questionnaire. In addition, participants in the 1994 E Survey were asked to test susceptibility of a MAC isolate recovered from a proficiency testing specimen as an ungraded exercise if they offered such testing for patients. RESULTS: Of the 1003 participants enrolled in the 1994 Mycobacteriology E-A Survey, 806 responded to one or more supplemental questions. In regard to the demand for MAC susceptibility testing, 606 participants indicated that the test is requested by physicians in their institutions, and 188 said that they do the test routinely on at least one MAC isolate per patient. Eighty-two percent (630/765) of participants refer the test to an outside laboratory, most commonly a commercial reference laboratory or state health laboratory. Of the 70 participants who perform MAC susceptibility testing in-house and indicated the method on the questionnaire, 54 (77%) used a solid medium, whereas only 14 (20%) used BACTEC TB, which currently is the recommended method. The most frequently tested drugs were ethambutol, rifampin, isoniazid, and streptomycin; other commonly evaluated agents were ciprofloxacin, amikacin, and clarithromycin. Only eight participants modify the pH of the medium when testing a macrolide. In regard to reporting test results, 56% (45/80) report a qualitative result only, 35% (28/80) report a quantitative result with a qualitative interpretation, and 9% (7/80) report only a quantitative result. Participant performance on the MAC proficiency testing specimen showed lack of interlaboratory reproducibility; 80% or fewer participants reported the correct result for all drugs except amikacin, for which 92% (11/17) of laboratories responded correctly. CONCLUSIONS: Given the obvious interest in MAC susceptibility testing, standardized methodology that demonstrates interlaboratory reproducibility and, optimally, shows some correlation with clinical outcome is needed. Moreover, recommendations concerning indications for performing the test would be useful.

Mycobacterial testing in clinical laboratories that participate in the College of American Pathologists Mycobacteriology Surveys. Changes in practices based on responses to 1992, 1993, and 1995 questionnaires.

OBJECTIVE: To determine whether the trend of increasing use of rapid methods of mycobacterial testing among participants in the College of American Pathologists (CAP) Mycobacteriology E Proficiency Testing Survey noted between 1992 and 1993 continued through 1995, and to collect information concerning mycobacterial staining and culture protocols from laboratories that do limited mycobacterial testing. METHODS: The 1993 CAP E Survey questionnaire addressing mycobacterial laboratory practices, test volumes, and rate of recovery of drug-resistant Mycobacterium tuberculosis was included with the CAP 1995 E-A Survey. A shortened list of these same questions, excluding those addressing mycobacterial identification and susceptibility test methods, was added to the CAP 1995 E1-A Survey, to which laboratories that do limited mycobacterial testing subscribe. RESULTS: A total of 802 and 1490 participants in the E and E1 surveys, respectively, returned responses to the CAP by the cutoff date for data analysis. For E Survey participants who answered questions concerning test methods in the years being compared, the percentage who used rapid techniques increased significantly over the study period. More participants used the fluorochrome stain (58% in 1992, 62% in 1993, and 72% in 1995), BACTEC TB plus a solid medium for culture (36% in 1992, 42% in 1993, and 50% in 1995), DNA probes for identification of M tuberculosis (68% in 1993, 79% in 1995), and BACTEC TB for susceptibility testing (65% in 1993, 71% in 1995). The percentages of E1 Survey participants who used a fluorochrome stain for detection of acid-fast bacilli and both a liquid and a solid medium for mycobacterial culture were lower than the percentages of E Survey participants who used these methods. Among participants who responded in all years being compared, the percentage processing respiratory specimens at least 7 times per week increased from 26% in 1992 to 30% in 1993 and 43% in 1995 (P < .001), and the percentages reporting an identification of M tuberculosis within 21 days and susceptibility test results within 28 days increased significantly over the study period (29% in 1992, 40% in 1993, and 56% in 1995 for identification; 13% in 1992, 19% in 1993, and 30% in 1995 for susceptibility testing). Turnaround times for E Survey participants were significantly shorter than those for E1 Survey participants. The number of specimens tested per month appeared to remain relatively stable between 1993 and 1995; however, the number of new patients with tuberculosis and the number of known tuberculosis patients with positive cultures declined significantly. CONCLUSIONS: The recent emphasis placed on utilization of rapid methods of mycobacterial testing appears to have influenced laboratories that subscribe to the CAP E Survey. Significantly more of these laboratories were following the Centers for Disease Control and Prevention's recommendations in 1995 than in 1993 and 1992. However, many laboratories that provide only limited mycobacterial testing still have not adopted the more rapid techniques. Because tuberculosis remains a public health problem, the efforts directed at its control must not wane if the recent downward trend in incidence is to be maintained.

The denervated Meissner corpuscle. A sequential histological study after nerve division in the Rhesus monkey.

The effects of denervation upon the Meissner corpuscle were evaluated by sequential fingertip biopsies in 3 rhesus monkeys, following transection of all the sensory innervation of the hand. Histological techniques were used to identify changes in the neural, connective, and enzymatic components of the Meissner corpuscles. Denervation of the Meissner corpuscle resulted in rapid and complete degeneration of the axon terminal and a slowly progressive degeneration of the connective tissue component of the corpuscle, characterized by loss of lobulation, lamellar collapse, and a steadily diminishing corpuscular size. The physiological basis and the clinical implications of these findings are discussed. The literature is reviewed.