clinical and microbiological assessment of mycobacterium simiae isolates from a single laboratory in...

Clinical and Microbiological Assessment of Mycobacterium simiae Isolates from a SingleLaboratory in Southern ArizonaAuthor(s): Dianna L. Rynkiewicz, Gary D. Cage, W. Ray Butler and Neil M. AmpelSource: Clinical Infectious Diseases, Vol. 26, No. 3 (Mar., 1998), pp. 625-630Published by: Oxford University PressStable URL: http://www.jstor.org/stable/4481430 .

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625

Clinical and Microbiological Assessment of Mycobacterium simiae Isolates from a Single Laboratory in Southern Arizona

Dianna L. Rynkiewicz, Gary D. Cage, W. Ray Butler, and Neil M. Ampel

From the Infectious Diseases Section, University of Arizona College of Medicine, and the Medical Service of the Tucson Veterans

Administration Medical Center, Tucson, and the Clinical and Reference Microbiology Laboratory, Arizona Department of Health Services,

Phoenix, Arizona; and the Division of AIDS, STD, and TB Laboratory Research, National Centers for Infectious Diseases, Centers for Disease

Control and Prevention, Atlanta, Georgia

Mycobacterium simiae was the third most common mycobacterium identified over a 2-year period from a single clinical laboratory in southern Arizona. Thirty-three isolates from 25 patients were identified over 1 year. The isolation of M. simiae was considered clinically significant for only two of 23 evaluable patients. None of five patients with human immunodeficiency virus infection had clinical disease associated with M. simiae. Twenty isolates were available for detailed study. All but one of the 20 isolates were niacin-negative, and 11 were nonphotochromogenic. All 20 isolates had a triple-cluster pattern consistent with M. simiae by high-performance liquid chromatography, and restriction fragment patterns were identical for 16 isolates. Analysis of 16S rDNA confirmed the identity of all the tested isolates as M. simiae. In this study, M. simiae was a frequent clinical isolate but was rarely associated with disease. The organisms isolated were confirmed to be M. simiae but appeared to be phenotypically distinct strains of low virulence.

Mycobacterium simiae was first identified in Macaca rhesus monkeys by Karasseva in 1965 [1]. Since its first isolation from humans in 1971 [2], there have been >400 reported cases of M. simiae isolated from clinical sites. These cases have been associated with a wide range of pathogenicity [1, 3-12]. Several series from the United States have suggested that clinically significant disease occurs in ~20% of patients [6, 10]. In contrast, a large series from Israel revealed no instances of significant disease [5].

We recently noted a high incidence of M. simiae isolated from clinical sites in our mycobacteriology laboratory, which serves two teaching hospitals in southern Arizona. However, we had not seen a corresponding significant prevalence of disease attributable to this organism. In this study, we sought to determine the importance of the isolation of this organism from clinical sites and to analyze in detail the phenotypic and genotypic characteristics of these isolates.

Materials and Methods

We retrospectively reviewed the records from the mycobacterium reference laboratory serving the University Medical Center (Tucson) and the Tucson Veterans Administration Med- ical Center from 1 January 1994 through 31 December 1995.

Received 1 July 1997; revised 27 October 1997. Reprints or correspondence: Dr. Neil M. Ampel, Medical Service (111),

Veterans Administration Medical Center, 3601 South Sixth Avenue, Tucson, Arizona 85723.

Clinical Infectious Diseases 1998;26:625-30 ? 1998 by The University of Chicago. All rights reserved. 1058-4838/98/2603-0012$03.00

The medical records of all patients from whom clinical isolates of M. simiae were recovered from 1 January 1995 through 31 December 1995 were reviewed. All records were reviewed again in April 1996 to assess status on follow-up.

Our case definition was a modification of that employed by Bell and colleagues [6]. A case was defined as follows: defi- nitely clinically significant, isolation of M. simiae was sup- ported by histopathologic evidence of infection, M. simiae was isolated from a sterile tissue specimen with the histopathologic changes, there was no other organism implicated in infection, and there was radiographic evidence of a pulmonary lesion in the case of a respiratory isolate; probably clinically significant, cultures of specimens from a nonsterile source were positive in the absence of a tissue specimen for histopathologic examination or sterile isolation of the organism and the patient had symptoms compatible with a granulomatous infection in the absence of another diagnosis; or not significant, cultures were positive in the absence of evidence of active granulomatous disease after a follow-up of 5 months to 1 year.

In the clinical laboratory, specimens submitted for mycobacterial cultures were placed into a sodium hydroxide/sodium citrate solution before cultivation. This solution was prepared by dissolving sodium hydroxide pellets (Fisher Scientific, Fair Lawn, NJ) and powdered sodium citrate (Curtain Matheson Scientific, Houston) into sterile bottled water (sterile water for irrigation, USP, McGaw, Irvine, CA). Culture of this solution was negative for mycobacteria. All nonpigmented mycobacterial isolates were subjected to DNA probe analysis (Gen-Probe, San Diego) for Mycobacterium tuberculosis and Mycobacte- rium avium complex. DNA probes (Gen-Probe) for Mycobacte- rium kansasii and Mycobacterium gordonae were used when appropriate on the basis of colony pigmentation. Any isolate not positively identified by DNA probe analysis underwent



626 Rynkiewicz et al. CID 1998;26 (March)

mycolic acid profile analysis by HPLC at the Arizona State Health Laboratory (Phoenix) by means of previously described methods [13, 14].

Twenty-two M. simiae isolates were submitted to the Ari- zona State Health Laboratory for examination by biochemical analysis, HPLC, and pulsed-field gel electrophoresis (PFGE) after being subcultured onto Middlebrook 7H11 medium. These isolates also were submitted to the Mycobacterial Laboratory of the Centers for Disease Control and Prevention for genomic analysis. Two isolates from the Maricopa County area of Ari- zona were included in the study for purposes of comparison.

For assessment of growth characteristics and biochemical testing, the isolates were subcultured to L6wenstein-Jensen slants, and the slants were incubated at 37?C. After 3 weeks, colony morphology and pigmentation were examined. Temper- ature studies were performed at 30?C, 37?C, and 42?C. Isolates were tested for niacin accumulation and nitrate reduction with reagents and for urease activity by using the Wayne method with liquid urease. Catalase production, Tween 80 (Remel, Lenexa, KS) hydrolysis, arylsulfatase activity at 3 and 14 days, and 5% sodium chloride tolerance were assessed by means of conventional methods [15]. Photochromogenicity was assessed by exposing Lowenstein-Jensen slants containing mycobacterial colonies to a 60-W bulb for 3 hours and then incubating the slants at 37?C with loose caps for 3 days [15].

For HPLC, colonies were saponified, and mycolic acids were derived according to a recently described standard method [14]. The mycolic acid samples were reconstituted with 100 /pL of dichloromethane, and 2.5 /ug of Ribi high-molecular-weight internal standard (Ribi ImmunoChem Research, Hamilton, MT) was added. A total of 10 uL of the sample and standard mixture was injected into the HPLC system as described previously [16]. Chromatograms were analyzed visually by using Millennium Chromatography software version 2.15 (Waters Corporation, Mil- ford, MA). The Ribi standard was used to compute the relative retention times of the peaks. Criteria for analysis and identification of peaks have been defined previously [14, 16].

Isolates were prepared as a modification of a method previously described for PFGE [17]. DNA was digested with 20 U of Asel in 300 pL of NEB no. 3 buffer (New England Biolabs, Beverly, MA) for 16-20 hours at 37?C. The restriction fragments were separated by means of the GenePath System (Bio-Rad, Richmond, CA) at 14?C for 20 hours, with initial and final switch times of 5.3 seconds and 34.9 seconds, respectively. Gels were stained with 0.5% ethidium bromide, washed with deionized water, and viewed on an ultraviolet transillumi- nator (UVP, San Gabriel, CA). Rare restriction fragment patterns were compared and evaluated by using a stricter interpre- tation of a proposed standard method [18]. Banding patterns of isolates that matched exactly were considered the same strain, while those varying by four or more bands were considered unrelated. Isolates with one to three band differences were considered indeterminate.

For genomic analysis, DNA was extracted from mycobacteria by disruption with siliconized zirconium beads [19]. The

16S gene was amplified by the PCR in 100 [L of reaction mixture with a 10-uL aliquot of the DNA and the oligonucleo- tide primers 5'-GAGAGTTTGATCCTGGCTCAG-3' and 5'-AAGGAGGTGATCCAGCCGCA-3' [20]. Subsequently, a 309-bp region of the 16S rDNA, which included the variable region A (a signature region for slow-growing mycobacteria [21]), was sequenced with use of the primers 5'-TAACAC- ATGCAAGTCGAACG-3' and 5'-CCCACTGCTGCCTCC- CGTAG-3' with the Taq DyeDeoxy terminator cycle sequenc- ing kit (Applied Biosystems Division, Perkin-Elmer, Foster City, CA) following the manufacturer's recommendations. Electrophoretic separation was performed by using an auto- mated ABI model 373A DNA sequencer (Applied Biosystems Division). Alignment of the variable region A was done with the multiple sequence alignment application PILEUP, which is part of the sequence analysis package of the Genetics Computer Group (Madison, WI) [22]. All presently available sequence data on 16S rDNA from mycobacteria that are deposited in the National Institutes of Health's GenBank (Bethesda, MD) were aligned to the sequence data of the test strains.

Results

From 1 January 1994 through 31 December 1995, 54 isolates of M. simiae were recovered from specimens submitted to the Mycobacteriology Laboratory of the Tucson Veterans Admin- istration Medical Center, which additionally serves the Univer- sity Medical Center. M. simiae was the third most common nontuberculous mycobacterium isolated in both years and rep- resented 9.3% and 15.7% of all mycobacterium-positive specimens in 1994 and 1995, respectively.

In 1995, there were 33 M. simiae isolates from 25 patients. Medical records were available for 23 patients. The median age of these patients was 60 years (range, 23-86 years). Nineteen patients were male, and four were female. Nineteen patients were white, and one was black. Race was unavailable for three patients. All patients had at least one underlying condition including tobacco smoking (19 patients), chronic lung disease (14), cancer (6), HIV infection (5), alcoholism (2), and cortico- steroid therapy (1). Further characteristics of the patients are displayed in table 1.

Of the 31 isolates from the evaluable patients, 26 (84%) were of respiratory origin, 2 (6.5%) were from urine, 2 (6.5%) were from blood, and 1 (3%) was from a soft-tissue abscess. In only two (8.7%) of the 23 evaluable cases, isolation of M. simiae was associated with probable clinical disease. The first case occurred in a 64-year-old woman with a history of bronchiectasis who presented with weight loss, night sweats, and productive cough; her chest radiograph revealed pulmonary fibrosis, multiple cavities, and a pulmonary nodule (patient 2, table 1). Acid-fast bacilli were found by examination of multiple sputum smears, and cultures of these specimens yielded M. simiae. Her condition clinically improved while she was treated with a regimen of clarithromycin and ciprofloxacin with aggressive pulmonary toiletting and, at the time of this writing,



Table 1. Characteristics of 23 evaluable patients from whom Mycobacterium simiae was isolated in southern Arizona.

Age Patient (y)/ Sample (no. Underlying Chest radiograph Outcome/final no. sex Source positive/total) Symptom(s) disease finding(s) Treatment diagnosis Significance

1 64/F Outpatient Sputum (3/3) Increased Bronchiectasis Bronchiectasis Doxycycline for Improved/acute No sputum 10 d bronchitis production

2* 64/F Inpatient Sputum (3/3) Cough, sweats, Bronchiectasis Upper lobe fibrosis; Clarithromycin and Improved Yes weight loss multiple cavities ciprofloxacin for

>1 y 3 23/M Inpatient Sputum (2/2) Asymptomatic Cystic fibrosis Increased vascular None Unchanged No

markings 4 55/M Outpatient Urine (1/4) Urethral Transitional Not applicable None Unchanged/recurrent No

stricture cell cancer carcinoma

5 62/M Outpatient Sputum (1/2) Hemoptysis Chronic lung Bullous emphysema Ciprofloxacin for Unchanged/acute No disease 10 d bronchitis

6 81/F Outpatient Sputum (1/1) Chronic cough Chronic lung Apical fibrosis, Clarithromycin for Unchanged/acute No disease bilateral calcified 14 d bronchitis

nodules 7 51/M Inpatient Sputum (1/1) Chronic cough HIV infection Normal None Unchanged No 8 35/F Inpatient Sputum (2/3) Dyspnea HIV infection Transient LUL Ampicillin/ Improved/acute No

density sulbactam for bacterial pneumonia 7d

9 39/M Inpatient Sputum (1/2) Asymptomatic Chronic Left apical thin- None Unchanged/ No bronchitis walled cavity coccidioidomycosis

10 33/M Outpatient Bloodt (2/3) Fevers, HIV infection Normal Macrolide plus Improved over No intermittent ethambutol for following year abdominal <4 w on two pain, anorexia occasions

11 63/M Outpatient Sputum (1/6) Cough, weight Chronic lung Biapical airspace None Unchanged/ No loss disease disease coccidioidomycosis

12 74/M Inpatient Sputum (1/4) Inanition Chronic lung Apical scarring, None Died/metastatic No disease hyperinflation carcinoma

13 62/M Outpatient Sputum (2/4) Cough, night Alcohol abuse Normal Trimethoprim- Improved/acute No sweats sulfamethoxazole bronchitis

for 10 d 14 75/M Outpatient BAL fluidl (1/4) Asymptomatic, Squamous cell LLL nodule, RUL None Unchanged/radiation No

pulmonary carcinoma pneumonitis pneumonitis nodule of lung

15 49/M Outpatient Urine (1/4) Testicular pain History of Normal Amoxicillin/ Improved/bacterial No Hodgkin's clavulanate for epididymitis disease 3 w

16 39/M Inpatient Sputum (1/4) Headache, night Chronic RUL infiltrate Erythromycin for Improved/acute No sweats lymphocytic 7 d bacterial pneumonia

leukemia 17* 86/M Outpatient Sputum? (1/2) Cough, dyspnea, Tobacco LUL nodule, calcified None Chronic cough, death Yes

fatigue smoking hilar lymph node 18 60/M Inpatient Sputum (1/4) Cough, dyspnea Alcohol abuse RUL/RML airspace None Improved/congestive No

disease heart failure 19 75/M Outpatient Sputum (1/3) Asymptomatic Chronic lung Prior surgical scarring None Unchanged No

disease without change 20 56/M Inpatient Open lung Dyspnea, None Diffuse patchy Corticosteroids Improved/fibrosing No

biopsy nonproductive infiltrate alveolitis specimen (1/1) cough

21 69/M Inpatient Sputum (1/3) Dyspnea, Chronic lung Fibrosis, cavitary Cefazolin Unchanged/ No anorexia, disease disease coccidioidomycosis productive cough

22 34/M Inpatient Sputum (1/2) Dyspnea HIV infection Bibasilar alveolar Trimethoprim- Improved/ No disease sulfamethoxazole Pneumocystis

carinii pneumonia 23 39/M Inpatient Soft-tissue Abscess HIV infection Not applicable Cephalexin; Improved before No

(scrotum) isoniazid, receiving specimen (1/1) rifampin, antituberculous

ethambutol, drugs pyrazinamide for 4 mo

NOTE. BAL - bronchoalveolar lavage; LLL = left lower lobe; LUL = left upper lobe; RML = right middle lobe; RUL = right upper lobe. * Considered to have clinically significant disease due to M. simiae. t Blood cultures positive on two separate occasions 5 months apart. t Culture of BAL fluid yielded M. simiae; three other specimens from expectorated sputum were negative. ? Bronchoscopic sample and sputum specimen positive for M. simiae 1 year previously.




has been stable during therapy for 6 months. Unfortunately, no follow-up sputum samples for cultures or chest radiographs were obtained after therapy was initiated.

The second case occurred in an 86-year-old man who presented with a 2-year history of chronic productive cough, progressive dyspnea, and fatigue (patient 17, table 1). One year previously, cultures of bronchoscopic and sputum specimens yielded M simiae. A chest radiograph revealed a left upper lobe parenchymal infiltrate and a calcified left hilar lymph node. Culture of another sputum specimen yielded M. simiae. No other pathogens were found, and cytological examination was negative for malignancy. The patient was not treated, and he died within 6 months. The cause of death was not ascertained, and no follow-up specimens for cultures or a chest radiograph was obtained.

M. simiae was isolated from a sterile source in three patients. However, clinical history and follow-up did not support a diagnosis of significant disease. Two of these patients were infected with HIV. M simiae was isolated from the blood of one HIV- infected patient (no. 10, table 1) on two separate occasions. At the time of the first isolation, the patient complained of low- grade fevers, diarrhea, and intermittent abdominal pain. He was receiving stavudine monotherapy and had a CD4 lymphocyte count of 12/.L. Physical examination revealed moderate abdominal tenderness but no palpable enlargement of the spleen or liver. The chest radiograph was unremarkable.

The patient was treated with clarithromycin, ethambutol, and rifabutin, but treatment was stopped after <4 weeks because of intolerance. The patient's clinical status remained unchanged, and 5 months later, M. simiae was again isolated from the blood. He was treated with azithromycin and ethambutol, but again treatment was discontinued after <4 weeks. Subse- quently, the patient's symptoms resolved, and he remained clinically stable for > 1 year after the isolation of M. simiae despite the lack of specific therapy. A repeated mycobacterial blood culture was negative during this time.

Neither the quantity nor the frequency of isolation of M simiae correlated with the probability of clinical significance. For three evaluable patients, there was 1-2+ growth of M. simiae on cultures of respiratory specimens with no apparent disease. For another patient with underlying bronchiectasis, there were 3+ smear positivity and 3+ growth on a solid culture with no apparent change in clinical status over >5 months of follow-up. Five patients had more than one respiratory specimen positive for M. simiae without evidence of clinical disease.

Twenty of the isolates from 1995 were available for further analysis. All strains were nonpigmented and slow-growing; had a smooth colony morphology; grew at 30?C, 37?C, and 42?C; produced catalase in a semiquantitative test; were negative for Tween 80 hydrolysis; and failed to grow in 5% sodium chloride. Eleven isolates were nonphotochromogenic. Nineteen of 20 strains were negative for niacin accumulation. Further characterization of the strains is shown in table 2.

By means of HPLC, all isolates demonstrated a triple-cluster pattern consistent with M. simiae (figure 1) [14]. The banding

patterns of rare restriction fragments matched exactly in 16 of the isolates by PFGE. One isolate (P 17) differed by more than four bands and was the only one demonstrating a strong urea reaction and a 14-day arylsulfatase reaction. Another isolate (P04) differed by three bands, and two others (P07 and P13) differed by one band. The four isolates that differed in their PGFE profile were all niacin-negative, and all but one (P13) were nonphotochromogenic as shown in table 2. The isolates from Maricopa County (M01 and M02) matched the 16 similar isolates from the study.

On the basis of analysis of the variable region A of 16S rDNA, 19 of the isolates were an exact match of M. simiae (GenBank nucleotide accession number X52931). One isolate (P17) was found to be contaminated and was not sequenced.

Discussion

M. simiae has become an increasingly frequent clinical isolate in certain regions of the world, including Israel, Cuba, and southern Texas [2, 5, 6, 10]. For 2 years (1994 and 1995), M. simiae was a frequent clinical isolate in our Tucson laboratory. Although M. simiae has been isolated from Tucson's municipal water in the past [23], the consistent and frequent isolation of M. simiae over time argues against laboratory contamination as an explanation for this occurrence. We did not culture potable water sources from Tucson as part of this study and cannot comment on the role environmental contamination may have played in the isolation of these strains. Acquisition of organisms from the hospital environment seems unlikely, since about one-half of the patients were seen as outpatients. In addition, sterile water was used for the processing of all mycobacterial specimens, and no mycobacteria were isolated from the processing solution during testing. Moreover, M. simiae was consistently isolated from 1% to 3% of all samples submitted for mycobacterial culture each month for >2 years, thus making laboratory contamination unlikely.

The M. simiae isolates recovered in Tucson appear to be rarely responsible for clinical illness. Only two of the 23 patients in this study were found to have even probable significant disease due to M. simiae. Moreover, one patient with HIV infection had M. simiae isolated from the blood on two separate occasions but remained clinically well without therapy. In contrast, Bell and associates [6] from San Antonio, Texas, found that approximately one-fifth of individuals for whom M. simiae was isolated from respiratory sites had significant disease due to this organism. In addition, a Centers for Disease Control survey in 1987 [24] calculated that 21% of all M. simiae isolates were clinically significant. More recently, Valero and colleagues [10], also from San Antonio, reported that 24% of 63 patients from whom M. simiae was isolated over an 11-year period had definite or probable disease.

Most cases reported as clinically significant have involved chronic pulmonary infection. However, osteomyelitis, peritoni- tis, chronic pyelonephritis, pleuritis, and disseminated disease with bacteremia, primarily in HIV-infected patients, have also



CID 1998;26 (March) Lack of Significance of M. simiae 629

Table 2. Biochemical and morphological characteristics of Mycobacterium simiae isolates recovered in southern Arizona.

Arylsulfatase activity Niacin Nitrate

Isolate accumulation reduction Pigmentation 3 d 14 d Urea reaction

P01 Negative Negative Weakly photochromogenic Negative Weakly positive Weakly positive P02 Negative Positive Nonphotochromogenic Negative Negative Weakly positive P03 Negative Positive Nonphotochromogenic Negative Negative Weakly positive P04 Negative Positive Nonphotochromogenic Negative Negative Weakly positive P05* Negative Negative Nonphotochromogenic Negative Negative Weakly positive P06 Negative Positive Weakly photochromogenic Negative Negative Weakly positive P07 Negative Negative Nonphotochromogenic Negative Negative Weakly positive P08t Negative Positive Nonphotochromogenic Negative Negative Weakly positive P09 Negative Negative Nonphotochromogenic Negative Negative Weakly positive P10 Negative Positive Nonphotochromogenic Negative Negative Weakly positive Pl11 Negative Negative Nonphotochromogenic Negative Weakly positive Positive P12 Negative Positive Weakly photochromogenic Negative Weakly positive Weakly positive P13 Negative Positive Photochromogenic Negative Negative Weakly positive P14 Negative Negative Photochromogenic Negative Negative Weakly positive P15 Weakly positive Negative Weakly photochromogenic Negative Negative Weakly positive P161 Negative Positive Nonphotochromogenic Negative Negative Weakly positive P17 Negative Positive Nonphotochromogenic Negative Negative Weakly positive P18 Negative Positive Weakly photochromogenic Negative Negative Weakly positive P19 Negative Positive Weakly photochromogenic Negative Negative Weakly positive P20 Negative Positive Photochromogenic Negative Negative Weakly positive M01 Negative Positive Weakly photochromogenic Negative Negative Weakly positive M02 Negative Positive Nonphotochromogenic Negative Weakly positive Positive

NOTE. M = Maricopa County isolates; P = Pima County isolates. * Isolate from blood of an HIV-infected person. Isolate from patient 17 who had probably clinically significant disease. Isolate from patient 2 who had probably clinically significant disease.

been described [4, 6-9, 11, 25, 26]. The role of M. simiae as a pathogen in many of these cases was not always clearly defined.

In contrast to most strains isolated in this study, 85% of M. simiae strains accumulate niacin, and >80% are photochromo- gens [15]. Biochemical tests alone may lead to the misidentifica- tion of M simiae as M. avium complex, Mycobacterium flaves- cens, or Mycobacterium scrofulaceum. Since the mycolic acid profile remains constant despite biochemical variability, HPLC is a more accurate method for detecting M simiae [26, 27]. How- ever, the recently described species Mycobacterium genavense, Mycobacterium lentiflavum, and Mycobacterium triplex [22, 28, 29] share similar HPLC patterns with M simiae but show differences in their signature region of 16S rDNA [26]. Our isolates were confirmed to be M. simiae on the basis of their HPLC profiles, PFGE patterns, and analysis of their hypervariable region of 16S rDNA. The PFGE profiles for the M simiae isolates analyzed in this study were remarkably similar. Whether this is a distinctive feature of these clinical isolates or is common to most strains ofM. simiae cannot be answered by this study; future studies should focus on comparing PFGE profiles for clinical isolates from various geographic regions as well as those for strains isolated from environmental sources.

The relative nonpathogenicity of the M. simiae isolates recovered in this study appears to be associated with two factors. First, our definition of a definite or probable case was very

strict and more conservative than those of other investigators [6, 10]. It is possible that several of the cases might be considered clinically significant by means of less stringent criteria. One example is the case of the HIV-infected patient from whom M. simiae was isolated on two separate occasions. However, we could not ascribe any specific illness to the bacteremia that eventually resolved without specific therapy. In addition, the case of the patient with bronchiectasis for whom there was 3 + smear positivity with heavy growth on a solid culture might also be considered clinically significant; this case was not cate- gorized as such in this study because the patient's clinical status remained unchanged over a long period without specific therapy.

Second, the strains of M. simiae isolated in this study, which appear to have distinct phenotypic characteristics, may be less pathogenic than strains reported in other series. On the basis of HPLC, PFGE, and genomic analysis, our strains were all consistent with M. simiae. However, they were unusual in that they were niacin-negative, and many lacked photochromogenicity. Although photochromogenicity can be variable on the basis of the time and intensity of light exposure, we used a standard exposure period to assess this characteristic [15]. In a large series of patients from Israel [5], none of 18 patients who had multiple respiratory specimens positive for M. simiae by HPLC had disease attributable to M. simiae; only two of the isolates were assessed for niacin accumulation, and both




1.0-

0.8 II

0.6

0

STANDARDS

0.0

6 8 10

Retention time (min)

Figure 1. HPLC mycolic acid patterns of 20 strains of Mycobacte- rium simiae isolated from patients in Tucson (P01-P20) as well as those of two strains obtained from Maricopa County, Arizona (M01 and M02), for comparison. STANDARDS = high-molecular-weight internal standards run with each strain; UV = ultraviolet.

were negative. A more recent compilation of isolates from

Europe, which were identified by both HPLC and biochemical analysis, revealed six cases of isolation of niacin-negative M. simiae and a lower frequency of disease [26]. Hence, negative niacin accumulation and lack of photochromogenicity to-

gether may be a marker for a less pathogenic strain of M. simiae.

Acknowledgments

The authors thank Evelyn Pugh for her recultivation of the initial Mycobacterium simiae isolates and Kristina Schaller for her in- valuable help with HPLC. We also thank Marion Wieden, Ph.D., for her careful reading of the manuscript and her thoughtful com- ments.

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