Increased sensitivity of the BACTEC 460 mycobacterial radiometric broth culture system does not decrease the number of respiratory specimens required for a definitive diagnosis of pulmonary tuberculosis.

The BACTEC 460 radiometric mycobacterial broth culture system has consistently demonstrated faster and increased recovery of Mycobacterium tuberculosis from respiratory specimens of patients with pulmonary tuberculosis than conventional culture methods. We thus questioned whether three sputa were still necessary to definitively diagnose pulmonary tuberculosis if the BACTEC radiometric culture system were in use. We performed a retrospective analysis of 430 sequential respiratory specimens submitted from 143 patients and from which M. tuberculosis had been recovered by in vitro culture and simultaneously assessed the diagnostic yield of acid-fast smear in this same cohort. M. tuberculosis was recovered from the first specimen for 117 (82%) of the 143 patients, from the second for 14 patients (10%; cumulative rate, 92%), and from the third for 12 patients (8%; cumulative rate, 100%). With the exception of those for bronchial brushings, recovery rates of M. tuberculosis were comparable for all respiratory specimen types (expectorated sputum, induced sputum, tracheal aspirates, bronchoalveolar lavage fluids). Only 46 (32%) of these 143 patients had acid-fast bacilli detected in smears; acid-fast bacilli were detected in the first submitted specimen for 44 patients (96%) and in the second for the remaining 2 patients (4%; cumulative rate, 100%). Culture- or smear-positive rates for sequential specimens obtained from AIDS patients were comparable to those for non-AIDS patients. Overall, the diagnostic culture yield of sequentially submitted specimens was not different from previously published studies in which the BACTEC radiometric culture system had not been used. Despite the documented enhanced ability of the BACTEC 460 radiometric mycobacterial culture system to recover M. tuberculosis more often and faster than conventional methods, three sequential respiratory specimens (regardless of type) were still necessary to definitively diagnose pulmonary tuberculosis.

Emergence of trimethoprim-sulfamethoxazole resistance in the AIDS era.

Trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for Pneumocystis carinii pneumonia prophylaxis in human immunodeficiency virus (HIV)-infected patients, but little is known about the effects of this practice on the emergence of TMP-SMX-resistant bacteria. A serial cross-sectional study of resistance to TMP-SMX among all clinical isolates of Staphylococcus aureus and 7 genera of Enterobacteriaceae was performed at San Francisco General Hospital. Resistance among all isolates was <5.5% from 1979 to 1986 but then markedly increased, reaching 20.4% in 1995. This was most prominent in HIV-infected patients: resistance increased from 6.3% in 1988 to 53% in 1995. The largest increases in resistance were in Escherichia coli (24% in 1988 to 74% in 1995) and S. aureus (0% to 48%) obtained from HIV-infected patients. A rapid increase in the use of prophylactic TMP-SMX in HIV disease was also observed during this time in San Francisco and is likely responsible for the increase in TMP-SMX resistance.

Extrapulmonary pneumocystosis.

Extrapulmonary pneumocystosis is an exceedingly rare complication of Pneumocystis carinii pneumonia (PCP). Prior to the advent of the human immunodeficiency virus type 1 (HIV-1) epidemic, only 16 cases of extrapulmonary pneumocystosis in individuals who were immunocompromised by a variety of underlying diseases had been reported. Since the beginning of the HIV-1 and related PCP epidemic, at least 90 cases of extrapulmonary pneumocystosis have been reported. This review briefly presents a history of the discovery of P. carinii and its recognition as a human pathogen, the controversy regarding its taxonomy, and the epidemiology of this organism. A more detailed analysis of the incidence of extrapulmonary pneumocystosis in HIV-1-infected individuals and its occurrence despite widespread prophylaxis for PCP with either aerosolized pentamidine or systemic dapsone-trimethoprim is presented. The clinical features of published cases of extrapulmonary pneumocystosis in non-HIV-1-infected individuals are summarized and contrasted with those in HIV-1 infected individuals. The diagnosis of extrapulmonary pneumocystosis is discussed, and because clinical microbiologists and pathologists are the key individuals in establishing the diagnosis, the characteristic microscopic morphology of P. carinii as its appears when stained with a variety of stains is presented and reviewed. The review concludes with a brief discussion of treatments for extrapulmonary pneumocystosis.

In vitro activities of rifabutin, azithromycin, ciprofloxacin, clarithromycin, clofazimine, ethambutol, and amikacin in combinations of two, three, and four drugs against Mycobacterium avium.

Multidrug therapy is recommended for treatment of Mycobacterium avium complex (MAC) bacteremia in patients with AIDS. Azithromycin, clarithromycin, rifabutin, ciprofloxacin, ethambutol, clofazimine, and amikacin have all been suggested for use in treating MAC bacteremia, but the most active combinations of these drugs have not been identified, nor has the minimum number of drugs needed for effective therapy been determined. To address the former, the in vitro bactericidal activities of all two-, three-, and four-drug combinations of these seven agents was determined by using 10 blood-derived strains of MAC isolated from patients with AIDS. The activities of the 132 drug combinations were compared by statistical analysis of survival means (analysis of variance) and further evaluated by determining the percentage of strains considered susceptible to each combination. When susceptibility was defined as a decrease in CFU of > or = 2 log10, no two- or three-drug combination and only two four-drug combinations were active against all 10 MAC strains. When a less stringent definition was applied (> or = 1 log10 decrease in CFU), 1 two-drug combinations, 9 three-drug combinations, and 31 four-drug combinations showed activity against all 10 strains. Eighteen selected drug combinations were also tested for intracellular activity in MAC-infected J774 cells. Combinations which contained amikacin as a component were considerably less active against intracellular MAC organisms than against organisms in broth. The opposite result was obtained for the combination of clarithromycin plus clofazimine.

Can penicillins and other beta-lactam antibiotics be used to treat tuberculosis?

An increase in the number of tuberculosis cases caused by multiple-drug-resistant strains of Mycobacterium tuberculosis has stimulated search for new antituberculous agents. Beta-lactam antibiotics, traditionally regarded as ineffective against tuberculosis, merit consideration. Four major penicillin-binding proteins (PBPs) with approximate molecular sizes of 94, 82, 52, and 37 kDa were detected by fluorography of [3H]penicillin-radiolabeled membrane proteins prepared from M. tuberculosis H37Ra. The presence of membrane-associated beta-lactamase precluded the use of membranes for assaying the binding affinities of beta-lactam antibiotics. Therefore, ampicillin affinity chromatography was used to purify these four PBPs from crude membranes in order to assay the binding affinities of beta-lactam antibiotics. Ampicillin, amoxicillin, and imipenem, beta-lactam antibiotics previously reported to be active in vitro against M. tuberculosis, bound to M. tuberculosis PBPs at therapeutically achievable concentrations. Binding of the 94-, 82-, and 52-kDa PBPs, but not the 37-kDa PBP, was associated with antibacterial activity, suggesting that these PBPs are the critical targets. Studies of mycobacterial cell wall permeability, which was assayed with a panel of reference cephalosporins and penicillins with different charge positivities, indicated that the rate of penetration of beta-lactam antibiotics to the target PBPs could not account for resistance. Resistance could be reversed with the beta-lactamase inhibitors clavulanate or sulbactam or could be circumvented by the use of a beta-lactamase-stable drug, imipenem, indicating that mycobacterial beta-lactamase, probably in conjunction with slow penetration, is a major determinant of M. tuberculosis resistance to beta-lactam antibiotics. These findings confirm in vitro data that M. tuberculosis is susceptible to some beta-lactam antibiotics. Further evaluation of these drugs for the treatment of tuberculosis in animal models and in clinical trials is warranted.

Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis.

A colorimetric method for quantitative measurement of the susceptibility of Mycobacterium tuberculosis to antimicrobial agents is described. The method utilizes an oxidation-reduction dye, Alamar blue, as an indicator of growth. By this method, MICs of isoniazid, rifampin, streptomycin, and ethambutol were determined for 50 strains of M. tuberculosis. Colorimetric MIC results were available on the 7th, 10th, or 14th day of incubation for 29 (58%), 14 (28%), and 7 (14%) of the 50 strains, respectively. When MIC susceptibility results were compared with results obtained by the agar proportion method, increased levels of resistance detected by agar proportion were associated with higher MICs obtained by the colorimetric method. Tentative interpretive criteria for colorimetric MIC results which showed good agreement with results obtained by the agar proportion method were established. Interpretive agreement between the two methods was 98% for isoniazid, rifampin, and ethambutol and 94% for streptomycin. Overall, there was agreement between the two methods for 194 of 200 test results (97%). The colorimetric method is a rapid, quantitative, nonradiometric method for determining the antimicrobial susceptibility of M. tuberculosis.

Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by the Amplicor PCR assay.

The minimum number of Mycobacterium tuberculosis CFU detectable in clinical sputum specimens by the Amplicor PCR test was estimated by performing the test on duplicate samples of quantitatively cultured serial dilutions of sputum. Positive PCR test results were obtained for all samples that contained 42 CFU of M. tuberculosis. The detection limits of the PCR assay for decontaminated (N-acetyl-L-cysteine [NALC]-NaOH) and nondecontaminated (NALC only) specimens were equivalent, even though the number of CFU cultured from decontaminated samples was only 11 to 20% of the number cultured from nondecontaminated samples. Thus, the 42 CFU that could be detected in nondecontaminated specimens by the Amplicor PCR test correspond to the approximately 8 CFU (0.20 x 42) that could be recovered in culture after decontamination with NALC-NaOH.

Suspected Pneumocystis carinii pneumonia with a negative induced sputum examination. Is early bronchoscopy useful?

In U.S. patients with the acquired immunodeficiency syndrome (AIDS), Pneumocystis carinii pneumonia is the most frequent AIDS-defining opportunistic infection. Sputum induction and bronchoscopy are effective techniques for obtaining specimens used to identify P. carinii although debate continues over their optimal use, specifically whether to perform bronchoscopy after a negative induced sputum examination for P. carinii. To evaluate the usefulness of bronchoscopy in this situation, we reviewed all cases of suspected P. carinii pneumonia in which sputum induction for P. carinii was performed at San Francisco General Hospital during a 4-yr period. Bronchoscopy, performed after a negative induced sputum examination, yielded a diagnosis in 50.5% of evaluations. The most frequent diagnoses were P. carinii pneumonia (192), tracheobronchial Kaposi's sarcoma (93), tuberculosis (28), and Cryptococcus neoformans pneumonia (9). Bronchoscopy provided either the only or an earlier diagnosis in 64.3% of tuberculosis cases. Bronchoscopy with BAL was free of complications, and, importantly, a negative BAL examination for P. carinii allowed physicians to discontinue empiric P. carinii pneumonia treatment in 95%. In patients with suspected P. carinii pneumonia with a negative induced sputum examination for P. carinii, early bronchoscopy with BAL should be performed.

Clinical utility of a commercial test based on the polymerase chain reaction for detecting Mycobacterium tuberculosis in respiratory specimens.

Several studies have reported using methods based on polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in respiratory tract specimens. However, little is known about the actual clinical utility of PCR-based tests, and it is uncertain if PCR technology can be transferred to the clinical laboratory. To determine its utility, we evaluated a commercially developed PCR test system in a clinical laboratory using consecutive respiratory tract specimens. Microscopic examination of smears stained with acid-fast bacilli (AFB), culture, and a PCR-based test (Amplicor Mycobacterium tuberculosis assay; Roche Molecular Systems) were used to evaluate 535 consecutive sputum and bronchoalveolar lavage specimens from 227 patients. A clinical case definition of tuberculosis was used as the reference-standard to determine the utility of all diagnostic tests. For all specimens from patients with a new or a treatment-failure case of pulmonary tuberculosis, the positivity rate of PCR (58%) was similar to that of culture (56%) (p > 0.90) and substantially greater than microscopic examination of AFB-stained smears (22%) (p < 0.001). PCR and culture detected M. tuberculosis in 46 and 43%, respectively, of the specimens from patients who did not have AFB on microscopic examination of their respiratory tract specimens (p > 0.90). PCR had a false positive rate of 0.8%. In several instances, PCR detected M. tuberculosis when culture did not; and vice versa. The clinical utility of this PCR-based test is similar to that of culture for detecting M. tuberculosis in respiratory tract specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycobacterium avium complex in water, food, and soil samples collected from the environment of HIV-infected individuals.

As part of an epidemiologic study of Mycobacterium avium complex (MAC) infection in San Francisco, water, food and soil samples were collected from the home environment of 290 persons with human immunodeficiency virus (HIV) infection and cultured for mycobacteria. Isolates recovered from the environment were compared with isolates cultured from study patients. Although mycobacteria were recovered from numerous environmental samples, isolates reactive with MAC-specific DNA probes were recovered from only four of 528 (0.76%) water samples and one of 397 (0.25%) food samples. The species M. avium was recovered from one water (0.19%) and one food sample. In contrast, MAC was recovered from 55% and M. avium from 27% of soil samples taken from potted plants in patients' home. Speciation of 76 MAC isolates from study patients showed all isolates belonged to the species M. avium. With use of serotype and multilocus enzyme electrophoresis analysis, some of the soil isolates were found to be similar to isolates recovered from study patients. The results of this study suggest that soil, rather than water, may be a significant reservoir of organisms causing MAC infection in San Francisco.

Predicting Mycobacterium avium complex bacteremia in patients infected with human immunodeficiency virus: a prospectively validated model.

In cases of advanced infection with human immunodeficiency virus, mycobacterial blood cultures are frequently used to diagnose disseminated infection with the Mycobacterium avium complex (MAC). However, no prospectively validated guidelines exist for the use of such cultures. In this study, a two-part model for predicting MAC bacteremia was developed and then validated prospectively. First, a CD4+ cell count of < or = 50/microL was used to predict bacteremia. Then, among patients with < or = 50 CD4+ cells/microL, the documentation of fever on more than 30 days during the preceding 3 months, a hematocrit of < 30%, or a serum albumin concentration of < 3.0 g/dL was used to predict bacteremia. This model had a sensitivity of 89% and positive and negative predictive values of 30% and 98%, respectively, for the identification of patients with bacteremia. Had the model been applied to patients in this study, the number of blood cultures performed would have decreased by 61%, but 11% of the positive cultures would have been missed. In short, this model can predict MAC bacteremia and can potentially guide the use of mycobacterial blood cultures.

The impact of Mycobacterium avium complex bacteremia and its treatment on survival of AIDS patients--a prospective study.

It is currently recommended that patients with AIDS and Mycobacterium avium complex (MAC) bacteremia receive antimycobacterial treatment. However, no study has prospectively evaluated the impact of this infection and its treatment on survival. This study prospectively followed a cohort of 367 AIDS patients with < or = 50 CD4+ cells/microL and found that MAC bacteremia was independently associated with an increased risk of death (relative hazard [RH] = 1.8, 95% confidence interval [CI] = 1.3-2.4, P < .001). Patients with MAC bacteremia who were treated had a longer median survival than those who were not (263 vs. 139 days, P < .001); treatment was independently associated with a lower risk of death (RH = 0.45, 95% CI = 0.23-0.89, P < .001). However, 23% of patients with bacteremia died within 28 days of that diagnosis; few were treated. MAC bacteremia contributes to the death of patients with AIDS, and treatment increases survival. However, many patients will not survive long enough to receive treatment. These results underscore the importance of early diagnosis and chemoprophylaxis for MAC bacteremia.

High predictive value of the acid-fast smear for Mycobacterium tuberculosis despite the high prevalence of Mycobacterium avium complex in respiratory specimens.

The value of the smear for acid-fast bacilli in predicting pulmonary tuberculosis is unclear in a setting where there is a high prevalence of Mycobacterium avium complex in respiratory specimens. To evaluate the impact of a high prevalence of M. avium complex on the predictive value of the acid-fast bacilli smear for tuberculosis, we reviewed findings on smears and results of cultures over a 3-year period at a hospital where M. avium complex is the predominant mycobacterial isolate. In this setting, the predictive value of the acid-fast bacilli smear for Mycobacterium tuberculosis was 92% for expectorated sputum specimens, 71% for induced sputum specimens, and 71% for bronchoalveolar lavage specimens. When multiple specimens collected from the same patient were excluded from the data base, the predictive values were 87%, 70%, and 71%, respectively. Smears of sputum samples were positive at the same rate for patients with tuberculosis who had AIDS and for patients with tuberculosis who did not have AIDS.

Environmental risk factors for acquisition of Mycobacterium avium complex in persons with human immunodeficiency virus infection.

A case-control study was done to determine risk factors for Mycobacterium avium complex (MAC) disease in persons infected with human immunodeficiency virus (HIV) with < 50 CD4+ cells/mm3. In univariate analysis, cases (n = 83) had lower CD4+ cell counts than controls (n = 177) (median, 10 vs. 17/mm3; P < .001) and were more likely to have consumed hard cheese (odds ratio [OR], 5.44; 95% confidence interval [CI], 1.61-18.4) but were less likely to have taken daily showers (OR, 0.55; 95% CI, 0.33-0.94). In multivariate analysis, CD4+ cell count < 25/mm3 (OR, 3.58; 95% CI, 1.71-7.49) and consumption of hard cheese (OR, 5.63; 95% CI, 1.58-20.1) remained associated with disease, while daily showering (OR, 0.58; 95% CI, 0.28-0.88) remained protective. Increased risk for MAC disease in persons with HIV infection and low CD4+ cell counts is not associated with exposure to water or a variety of other environmental sources but may be associated with consumption of hard cheese.

Mycobacterium avium complex in the respiratory or gastrointestinal tract and the risk of M. avium complex bacteremia in patients with human immunodeficiency virus infection.

Mycobacterium avium complex (MAC) is frequently isolated from the respiratory or gastrointestinal tract of patients with advanced human immunodeficiency virus (HIV) infection. Whether they are at increased risk of MAC bacteremia and whether culture of respiratory tract or stool specimens is useful for predicting bacteremia are unclear. HIV-infected patients with < or = 50 CD4+ cells/microL were prospectively studied. The risk of MAC bacteremia was approximately 60% within 1 year for patients with MAC in either the respiratory or gastrointestinal tract and was greater than for those without MAC in these sites (relative hazards for respiratory and gastrointestinal tract, 2.3 and 6.0; 95% confidence intervals, 1.1-4.6 and 2.5-14.6, respectively). Both respiratory tract specimen and stool culture had poor sensitivities (22% and 20%, respectively) but good positive predictive values (approximately 60%) for bacteremia. Symptomatic HIV-infected patients with MAC in the respiratory or gastrointestinal tract are at a substantial risk for developing MAC bacteremia; culture of these sites has limited usefulness as a screening test.

Randomized, placebo-controlled trial of rifampin, ethambutol, and ciprofloxacin for AIDS patients with disseminated Mycobacterium avium complex infection.

Patients with AIDS and disseminated Mycobacterium avium complex (MAC) infection received rifampin (600 mg) plus ethambutol (25 mg/kg) plus ciprofloxacin (750 mg) or matching placebos daily for 8 weeks. Patients were monitored every 2 weeks clinically and by quantitating MAC colony-forming units (cfu) per milliliter of blood. Analysis of baseline characteristics revealed no significant differences between groups. After 8 weeks, MAC cfu had decreased by > or = 1 log/mL in 4 of 9 treated patients versus 0 of 10 placebo recipients while increasing by > or = 1 log/mL in 1 and 7, respectively (P = .006). While the average combined clinical response score declined in both groups, it tended to decrease less in treated patients (P = .36). On the other hand, dose-limiting toxicity (primarily nausea and adverse drug interactions) occurred in 9 of 12 treatment versus 1 of 12 placebo patients (P = .005). Combined rifampin [corrected]-ethambutol-ciprofloxacin therapy for disseminated MAC infection had significant microbiologic efficacy with some evidence of clinical efficacy but was associated with drug intolerance.

Update on laboratory tests for the diagnosis of pulmonary disease in HIV-1-infected individuals.

Various diagnostic tests, both specific and nonspecific, are available in the clinical laboratories for diagnosing human immunodeficiency virus-1 (HIV-1) infection and associated respiratory pathogens. Pneumocystis carinii pneumonia remains the most common pulmonary disease in HIV-1-infected individuals and there have been no significant advances in the laboratory diagnosis of the pathogen beyond the traditional microscopic examination of specimens. In contrast, the greatest revolution in laboratory diagnostic testing has been for mycobacteria, with major advances resulting in significant reduction in the time necessary for isolation and identification to the species level. The application of the polymerase chain reaction for the identification of a variety of pulmonary pathogens observed in HIV-1 infected individuals is discussed.