A nosocomial pseudo-outbreak of Mycobacterium xenopi due to a contaminated potable water supply: lessons in prevention.

OBJECTIVES: To determine risk factors for Mycobacterium xenopi isolation in patients following a pseudo-outbreak of infection with the organism. DESIGN: Retrospective cohort analysis of mycobacteriology laboratory specimen records and frequency-matched case-control study of hospital patients. SETTING: General community hospital. PATIENTS: For the case-control study, 13 case patients and 39 randomly selected controls with mycobacterial cultures negative for M xenopi, frequency matched by specimen source, whose specimens were submitted from June 1990 through June 1991. RESULTS: Between June 1990 and June 1991, M xenopi was isolated from 13 clinical specimens processed at a midwestern hospital, including sputum (n = 6), bronchial washings (2), urine (4), and stool (1). None of the patients with M xenopi-positive specimens had apparent mycobacterial disease, although five received antituberculosis drug therapy for a range of one to six months. Specimens collected in a nonsterile manner were more likely to grow the organism than those collected aseptically (3.1% versus 0, relative risk = infinity, P = 0.003). M xenopi isolation was attributed to exposure of clinical specimens to tap water, including rinsing of bronchoscopes with tap water after disinfection, irrigation with tap water during colonoscopy, gargling with tap water before sputum collection, and collecting urine in recently rinsed bedpans. M xenopi was isolated from tap water in 20 of 24 patient rooms tested, the endoscopy suite, and the central hot water mixing tank, but not from water in the microbiology laboratory. The pseudo-outbreak occurred following a decrease in the hot water temperature from 130 degrees F to 120 degrees F in 1989. CONCLUSIONS: Maintenance of a higher water temperature and improved specimen collection protocols and instrument disinfection procedures probably would have prevented this pseudo-outbreak.

Emergence of Buruli ulcer disease in the Daloa region of Cote d'Ivoire.

Recent reports have suggested increases in Buruli ulcer (BU), an infection caused by Mycobacterium ulcerans in west Africa. In 1991, we conducted surveillance for BU in a rural area of Cote d'Ivoire and identified 312 cases of active or healed ulceration. A case-control study was then performed to investigate risk factors for this infection. The rate of illness did not appear to differ between males and females (5.2% versus 7.5%; P = 0.11). The highest rate of illness was seen in the 10-14-year-old age group (143 cases per 1,000 population). New cases increased more than three-fold between 1987 and 1991, and local prevalence of BU was as high as 16.3%. Twenty-six percent of persons with healed ulcers had chronic functional disability. Participation in farming activities near the main river in the region was identified in the case-control study as a risk factor for infection (odds ratio [OR] for each 10-min decrease in walking distance between the fields and the river = 1.52, 95% confidence interval [CI] 1.01, 2.28, P = 0.046). Wearing long pants was protective (OR 0.20, 95% CI 0.06, 0.62, P < 0.005). We conclude that the incidence of BU is increasing rapidly in Cote d'Ivoire. Specific causes of this increase were not identified, but wearing protective clothing appeared to decrease the risk of disease.

The modern mycobacteriology laboratory. How it can help the clinician.

The mycobacteriology laboratory provides the information necessary to diagnose mycobacteriosis and to suggest proper patient management. When the definite diagnosis of disease due to M. tuberculosis is made on the basis of the laboratory result, contact follow-up studies should continue and the patient may be considered infectious if the bacilli were isolated from the sputum. A report of another Mycobacterium species suggests that a contact follow-up is not necessary, that the patient need not be isolated, and that a therapeutic regimen should be based on the results of drug-susceptibility tests with the isolate. Techniques of molecular biology are being used in the laboratory to provide the necessary information quickly for patient management. BACTEC has been accepted into most clinical laboratories to speed reporting of results. Other methods such as genetic and immunologic probes are under development. New DNA probes have been marketed by GenProbe for the identification of cultures of M. tuberculosis, M. avium, and M. intracellulare. HPLC and GLC have been used for the identification of cultures based on unique mycolic acid patterns of the species. Immunologic probes may eventually be the most specific and sensitive, but additional development is necessary. Mycobacteriophage typing of M. tuberculosis isolates has been developed as a tool to aid in epidemiologic studies. These and other technologies are essential in the support of programs for the elimination of tuberculosis.

Biosafety in the clinical mycobacteriology laboratory.

This article presents an expanded agent summary statement for laboratorians working with Mycobacterium tuberculosis. It focuses on reducing the serious risk of infection in clinical laboratories that process specimens from tuberculosis patients or that work with purified cultures of tubercle bacilli. Administrative and engineering controls, practices and procedures, and personal protective equipment are discussed. Guidelines for packaging specimens for transfer to another laboratory also are presented.

Geographic distribution, frequency, and specimen source of Mycobacterium avium complex serotypes isolated from patients with acquired immunodeficiency syndrome.

Isolates of Mycobacterium avium complex from 727 patients with acquired immunodeficiency syndrome (AIDS) were submitted by medical centers across the United States to the Centers for Disease Control for serotyping. We were able to type 630 (87%) of these isolates by our seroagglutination procedure. Almost all typeable isolates were M. avium (serotypes 1 to 6 and 8 to 11). Blood was the major specimen source for both M. avium and the nontypeable isolates. M. intracellulare serotypes made up only 3% of all isolates from AIDS patients, with sputum being the major specimen source. More than 50% of the isolates originated from either New York or California, with serotype 4 being isolated most frequently in New York and serotype 8 appearing most frequently in California. AIDS patients in Los Angeles had a significantly higher isolation frequency for serotype 8 and a significantly lower one for serotype 4 in comparison with patients in either San Francisco or New York City.

Mycobacterial taxonomy.

The minimal standards for including a species in the genus Mycobacterium are i) acid-alcohol fastness, ii) the presence of mycolic acids containing 60-90 carbon atoms which are cleaved to C22 to C26 fatty acid methyl esters by pyrolysis, and iii) a guanine + cytosine content of the DNA of 61 to 71 mol %. Currently, there are 71 recognized or proposed species of Mycobacterium which can be divided into two main groups based on growth rate. The slowly growing species require > 7 days to form visible colonies on solid media while the rapidly growing species require < 7 days. Slowly growing species are often pathogenic for humans or animals while rapidly growing species are usually considered nonpathogenic for humans, although important exceptions exist. The taxonomic and diagnostic characteristics of medically important species and of newly described species of the Mycobacterium genus are reviewed.

Diagnostic mycobacteriology laboratory practices.

The resurgence of tuberculosis has forced clinical laboratories to improve the methods used for detection of M. tuberculosis. Current recommendations for diagnostic laboratory performance [7] include (1) daily processing of specimens (i.e., handling these specimens in the same way that all other specimens sent to the laboratory are handled); (2) inoculation of liquid media (e.g., BACTEC) for the primary culture; (3) use of nucleic acid probes or the NAP test for identifying isolates as M. tuberculosis as soon as possible; (4) determining drug susceptibility with use of liquid media; and (5) reporting results of each step to physicians in a timely manner. The immediate goals are to report identification of M. tuberculosis within 10-14 days of receipt of the specimen and to report drug susceptibilities within 15-30 days. This can be done if current technologies are fully utilized. The amplification-based systems for the identification of M. tuberculosis and the luciferase-based systems for rapid determination of drug susceptibilities should help further shorten turn-around times. The results to date demonstrate that these systems are feasible, although they must be reduced to formats that can be used routinely in clinical laboratories. The gene-amplification systems may be the most promising, and they are nearing commercial availability. If the assays function as well during routine use as they have during clinical trials, a clinical laboratory may soon be able to report confirmed M. tuberculosis infection to the physician within hours of receiving a specimen, instead of within the typical period of 2-4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory pathogens in monkeys.

Respiratory disease in a dynamic colony of nonhuman primates during a 4-year period was due primarily to infections caused by Klebsiella pneumoniae, Diplococcus pneumoniae, Bordetella bronchiseptica, Pasteurella multocida, and Haemophilus influenzae. The principal secondary invaders were Escherichia coli, Staphylococcus aureus, and streptococci. A high fatality rate was associated with infections caused by each of the primary pathogens, and females appeared to be more susceptible than males. Incidence of respiratory disease was greatest in the fall and early winter; however, at all times newly colonized monkeys had a higher infection rate than conditioned monkeys. Infections were occasionally confined only to the lungs and were sometimes present without grossly observable lung lesions. The information given on susceptibility of 10 species of nonhuman primates to respiratory infections provides a basis for developing disease models.

The usefulness of phage typing Mycobacterium tuberculosis isolates.

Mycobacteriophage typing of Mycobacterium tuberculosis isolates was used as an epidemiologic aid in investigating the transmission of tuberculosis in community, industrial, and institutional outbreaks. The technique was also useful in other situations, e.g., documenting congenital transmission of infection and distinguishing exogenous reinfection from endogenous reactivation. Additional studies are indicated to further explore the value of phage typing for tracking the transmission of tuberculosis in the community.

Evaluation of thermal disinfection procedures for hydrophilic contact lenses.

The kinetics and efficacy of moist heat disinfection for hydrophilic contact lenses were investigated by using representative microorganisms of ophthalmic concern and several heat-resistant species. In replicate challenges, 80 degrees C for 10 min and 75 degrees C for 5 min were proven efficacious for moist-heat disinfection.

Identification of clinically significant Mycobacterium fortuitum complex isolates.

Recent outbreaks of nosocomial infections caused by organisms identified as the Mycobacterium fortuitum complex suggest that species and subspecies identification is epidemiologically important. In a study of 170 strains, M. fortuitum was differentiated from M. chelonei by nitrate reduction and iron uptake. M. fortuitum was further divided into biovariant fortuitum, biovar peregrinum, and an unnamed third biovar by inositol and mannitol utilization. M. chelonei was further divided into subsp. chelonei, subsp. abscessus, and an unnamed subspecies by tolerance to 5% sodium chloride, utilization of mannitol and sodium citrate, and uptake of iron.

Immunoelectrophoresis of Mycobacterium tuberculosis antigens. Comparative analysis of cell extract and culture filtrate antigens.

Immunoelectrophoretic analyses were performed on a cell extract and culture filtrate of Mycobacterium tuberculosis, H37Rv strain using homologous polyvalent goat antisera. General similarity between the cell extract and culture filtrate antigens was found. Significant variation between two anti-culture filtrate antisera was observed. These antigens and antisera also differed significantly from previously described reference reagents. These findings demonstrate the necessity of using reference materials for precise identification of mycobacterial antigens in comparative studies.

Tuberculin-induced lymphocyte transformation and skin reactivity in monkeys vaccinated or not vaccinated with Bacille Calmette-Guérin, then challenged with virulent Mycobacterium tuberculosis.

The in vitro lymphocyte transformation test was compared to the skin test at intervals after aerogenic administration of bacille Calmette-Guerin vaccine to monkeys, and also at monthly intervals after aerogenic challenge of monkeys vaccinated and not vaccinated with virulent strain H37Rv of Mycobacterium tuberculosis. Individual responses varied, but several consistent patterns of sensitivity development could be discerned. The lymphocyte transformation test was more sensitive, and often positive when the skin test was negative, doubtful, or feeble. Conversion to tuberculin reactivity was detected by lymphocyte transformation in vitro earlier in the disease or after vaccination, and persisted longer after sensitivity to the skin test had waned or after the animals had become anergic by the skin test. Monkeys not vaccinated, but challenged, developed larger in vitro skin reactions and responses than animals that were either vaccinated and challenged or only vaccinated; however, the unvaccinated, challenged monkeys developed anergy to tuberculin and progressive disease more rapidly than other groups, and their cells became less responsive to phytohemagglutinin in vitro.

Current practices in mycobacteriology: results of a survey of state public health laboratories.

Fifty-six state and territorial public health laboratories were surveyed to determine whether currently available rapid methods for the identification and drug susceptibility testing of Mycobacterium tuberculosis were being performed. Forty (71%) laboratories use fluorochrome rather than conventional basic fuchsin stains for screening clinical specimens for acid-fast bacilli. Of the 55 laboratories that routinely culture for mycobacteria, 16 (29%) use the more rapid radiometric methods. Species identification of isolates is done by biochemical tests in 13 (23%) laboratories; 40 (72%) use nucleic acid probes, high-performance liquid chromatography, or the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test (rapid tests); 3 laboratories do not perform species identification. Drug susceptibility testing is performed with solid media by 36 of 45 (80%) laboratories, while the more rapid radiometric methods are used by 9 (20%) laboratories. Compared with the laboratories that use conventional methods, laboratories that use rapid methods report results more quickly: for species identification, 43 days (conventional) versus 22 days (rapid); for drug susceptibility testing, 44 days (conventional) versus 31 days (rapid) from specimen processing. Rapid technologies for microscopy and species identification are being used by many, but not all, state and territorial public health laboratories; however, most laboratories do not use the more rapid radiometric methods for routine culture or drug susceptibility testing of mycobacteria. Implementation of such rapid technologies can shorten turnaround times for the laboratory diagnosis of tuberculosis and recognition of drug resistance.

Enteric pathogens in monkeys.

From 1964 to 1967, 6,646 monkeys, representing 10 primate species, were examined for Shigella and Salmonella infections upon arrival at the National Center for Primate Biology. Of these animals, 12% were infected with Shigella, and 75% of the Shigella isolates were S. flexneri 4. The incidence of Salmonella infections decreased from 12 to 3% during the period of study. Epidemiological studies of animals in the colony for 90 days or more indicated no seasonal variation in the occurrence of Shigella and Salmonella. Many of the isolates from incoming monkeys as well as from laboratory-conditioned animals were resistant to chloramphenicol, dihydrostreptomycin, and tetracycline. The possible operation of drug-resistance factors in these infections is discussed.

Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatography.

Conditions are described for the detection of tuberculostearic acid (10-methyloctadecanoate; C18 X CH3) in cerebrospinal fluid and serum of patients with tuberculous meningitis. C18 X CH3 was found in both the cerebrospinal fluid and serum of patients with tuberculous meningitis at concentrations of 25 to 50 fmol (10(-15) mol). The necessary specificity and sensitivity for detection of C18 X CH3 were obtained by extraction under acid conditions with organic solvent, specific functional group esterification with trichloroethanol, cleanup with disposable reverse-phase sorption chromatography columns, analysis on high-resolution polar and nonpolar capillary columns, and detection by a frequency-pulsed electron capture detector. Use of an IBM 9000 computer equipped with CAP software significantly aided comparison between known C18 X CH3 standards and C18 X CH3 in clinical specimens. Scale expansion and attenuation changes were the major contributions obtained by use of the computer. The data indicate that detection of C18 X CH3 by frequency-pulsed electron capture gas-liquid chromatography may be a valuable aid for early detection of tuberculous meningitis.

Changing practices in mycobacteriology: a follow-up survey of state and territorial public health laboratories.

The resurgence of tuberculosis, which includes an increase in the isolation of multidrug-resistant strains of Mycobacterium tuberculosis, emphasizes the need for more rapid laboratory testing for identification of the etiological agent of the disease. In December 1991, state and territorial public health laboratories were surveyed to determine the methods that they were using for testing and reporting of M. tuberculosis. A follow-up survey was conducted in June 1994 to measure changes in the testing and reporting practices that had occurred as a result of efforts focused on the disease and on laboratory improvement. Completed questionnaires were received from 51 of 55 laboratories. Comparative data indicate that the proportion of laboratories reporting testing results within the number of days recommended by the Centers for Disease Control and Prevention has increased. Starting from the time at which the laboratory receives the specimen, the proportion of laboratories reporting the results of microscopic smear examination within the recommended 24 h has increased from 52.1 to 77.6%; the proportion reporting isolation and identification within 21 days has increased from 22.1 to 72.9%; and the proportion reporting results of isolation, identification, and drug susceptibility testing within 28 days has increased from 16.7 to 48.9%. Use of the recommended rapid testing methods has also increased: the proportion of laboratories using fluorescence staining for acid-fast microscopy has increased from 71.4 to 85.7%, the proportion using BACTEC for primary culture has increased from 27.1 to 79.6%, the proportion using rapid methods for M. tuberculosis identification has increased from 74.5 to 100.0%, and the proportion using BACTEC for primary drug susceptibility testing has increased from 26.2 to 73.3%. By implementing the recommended methods for M. tuberculosis testing and reporting, state and territorial public health laboratories are now able to transmit results to physicians more rapidly.