Clinical significance and epidemiology of non-pigmented rapidly growing mycobacteria in a university hospital

Journal of Infection (2007) 54, 135e145

www.elsevierhealth.com/journals/jinf

Clinical significance and epidemiology of non-pigmented rapidly growing mycobacteria in a university hospital ´ndez Roblas, J.I. Garcı´a Cı´a, N. Zamora, A. Ortiz J. Esteban*, R. Ferna Department of Clinical Microbiology, Fundacio´n Jime´nez Dı´az, Av. Reyes Cato´licos 2, 28040-Madrid, Spain Accepted 23 February 2006 Available online 17 April 2006

KEYWORDS Rapidly growing mycobacteria; Molecular epidemiology; RAPD; PCR; Disease

Summary Objetives: To study the clinical significance and epidemiology of Nonpigmented rapidly growing mycobacteria (NPRGM) during a 13-year period. Methods: We performed a retrospective study of patients with isolates of NPRGM to evaluate their clinical significance. We also analyzed the strains using Randomly Amplified Polymorphic DNA (RAPD) analysis to evaluate the relationship between strains. Results: Between 1990 and 2003, 65 patients had an isolate of NPRGM. Twenty of them were considered significant (19 cases) or doubtful (1 case). Many cases were skin and soft tissue infections. Six cases were foreign-body related. All the patients recovered with antibiotic therapy and removal of the foreign body. All the patients were apparently unrelated, despite 56.9% of the isolates were detected between 1995 and 1997. RAPD analysis was performed on 43 strains, and showed only a cluster of two Mycobacterium chelonae isolates. Both of them were related with contamination of a laboratory reactive, and were considered non-significant. Conclusion: In our hospital, almost one-third of the isolates of NPRGM were significant, being this percentage higher for skin and soft tissue isolates. Patients were cured with antibiotic therapy, but the removal of foreign bodies appeared to be necessary for a good outcome. A minor psudo-outbreak was detected. No predominant strain was detected. ª 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ34 915504800x3306; fax: þ34 915442636. E-mail address: [email protected] (J. Esteban). 0163-4453/$30 ª 2006 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2006.02.017

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Introduction Non-pigmented rapidly growing mycobacteria are a relatively heterogeneous group of species that belongs to the genus Mycobacterium that are frequently isolated in the mycobacteriology laboratories and can be the cause of many human infections, many of them nosocomial ones.1,2 From the clinical point of view, the most important members of this group are Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium abscessus, because most of the infections are caused by these species. However, other members of the group, such as Mycobacterium peregrinum, Mycobacterium mucogenicum, Mycobacterium smegmatis or some of the recently described species, such as Mycobacterium goodii, Mycobacterium houstonense or Mycobacterium bonickei, among others, have been described to cause human disease.1 These organisms have an environmental habitat, which includes soil, water and even disinfectant solutions, which can be the source of the clinical isolates.1,3,4 Even in many cases, the infections associated with an environmental source can appear as epidemic outbreaks or pseudo-outbreaks.1,4e8 Some of these outbreaks have been characterized by using molecular epidemiology tools, such as Pulsed-field gel electrophoresis (PFGE)6,7 or randomly amplified polymorphic DNA analysis (RAPD)9,10 or both.5 However, we have encountered only one report where a broad study of the epidemiology of these organisms has been performed using PFGE.11 Here we report our experience with the disease caused by these organisms and a study of the molecular epidemiology of the strains of non-pigmented rapidly growing mycobacteria isolated in our hospital during a 13-year period.

Material and methods Study period and patients selection All the patients with isolates of non-pigmented rapidly growing mycobacteria between 1st January 1990 and 1st January 2003 were selected for the study. Only bacterial isolates from patients from the Fundacio ´n Jime ´nez Dı´az (a 550-bed university hospital which attend a population of 300,000 people) were included. Patients from other centres with samples or strains sent to our laboratory as a reference centre were not included in the analysis of clinical charts, but were included in the RAPD study for comparison purposes because these isolates were found from patients whose addresses

J. Esteban et al. were in the same area than the patients form our hospital.

Evaluation of the clinical significance We retrospectively reviewed the available case records from patients in whom NPRGM had been isolated. We classified them according to their clinical, microbiological and pathological characteristics as clinically significant, of doubtful clinical significance or without clinical significance. We considered pulmonary disease cases of clinical significance only if they fulfilled the American Thoracic Society criteria.12 We regarded skin or soft tissue NPRGM infections as clinically significant if the culture from biopsy or several exudates yielded a positive result or if pathology showed features consistent with mycobacterial infection. When the same organism was isolated from at least 2 different body sites or if blood or bone marrow cultures were positive, cases were grouped as clinically significant disseminated NPRGM disease. For all other body sites, we classified cases as clinically significant when various positive cultures were obtained and/or characteristic pathology was found, in the presence of associated physical signs or symptoms, and improvement after treatment. We considered of doubtful clinical significance those cases with clinical findings previously described for NPRGM in the absence of other possible causes and that improved after treatment with antibiotics active against the mycobacterial isolate. We recorded the followings as possible risk factors: smoking, chronic obstructive airways disease, immunosuppressive drug treatment, malignancy, presence of foreign bodies and HIV infection.

Identification of bacterial isolates During the study period, samples were processed according to the internationally accepted protocols. Briefly, samples were decontaminated with the N-acetyl-cysteineeNaOH technique and respiratory samples were inoculated onto LowensteineJensen and Coletsos slants (Pronadisa, Torrejo ´n de Ardoz, Madrid, Spain), Middlebrook 7H11 agar plates (Beckton Dickinson, Franklin Lakes, NJ, USA). Biopsies and non-respiratory samples were inoculated also in a liquid medium (MBCheck between 1990 and 1997, BACTEC 9000 series in the years 1997e2000 and MGIT 960 from 2000 to 2004, all from Beckton Dickinson, Franklin Lakes, NJ, USA). Isolates were initially identified using common biochemical tests (nitrate reductase,

Infections due to rapidly growing mycobacteria urease, tween hydrolysis, growth on Citrate, Mannitol, Inositol and Sorbitol, growth on 5% NaCl, growth on McConkey agar, and growth on Lowesteine Jensen slants at 45  C, 37  C, 30  C and 24  C), and sent to the Mycobacteria Reference Laboratory (Centro Nacional de Microbiologı´a, Majadahonda, Spain) to confirm the identification. Bacteria were then frozen in sterile skim milk at 20  C until further experiments were performed. Frozen isolates were thawed and inoculated onto Tryptic-soy and Middlebrook 7H10 agar plates and onto LowensteineJensen slants and incubated at 30  C during 2 weeks. Mycobacteria were then checked for purity and reidentified by using recently described biochemical schemes1 and PCReRFLP analysis of the hsp65 gene according to the protocol described previously,13 using for characterization of the isolates the PRASITE web page ().14 No further analysis was performed for those species with only one isolate during the study period.

RAPD protocol Pure mycobacterial isolates were grown onto Middlebrook 7H10 during 4 days at 30  C in a 5% CO2 atmosphere. One loop of the culture was resuspended in 400 ml of Tris 10 mM-EDTA 1 mM, pH 8 buffer, boiled during 30 min and subsequently centrifuged at 13,000  g in a microcentrifuge buffer after extraction with 1 volume of chlorophorm. DNA was then frozen at 20  C. RAPD was performed by using the protocol described by Zhang et al.9 for M. abscessus. We also used the primers OPA-2, OPA-18, IS986-FP and INS-2. The procedure was as follows: 2 ml of the sample were inoculated in PuRe Taq Readyto-go PCR Beads (Amersham Biosciences, UK) together with 23 ml of a mixture of primers, sterile distilled water and MgCl2 to obtain a final concentration of 2.5 mM MgCl2, 100 pM of primer were added to each reaction tube. Samples were amplified in a PTC-100 Peltier Thermal Cycler (MJ Research, USA) with the following protocol: 40 cycles at 94  C for 1 min, 36  C for 1 min, and at 72  C for 2 min. The PCR products were separated by electrophoresis in 3% agarose gels and detected after ethidium bromide staining. All the strains from the same species were analyzed in the same gel to overcome reproducibility problems described with this procedure. Four strains from a well-documented outbreak of postsurgical keratitis due to M. chelonae sent to our laboratory from a different geographical area were used as controls. Comparison of the profiles

137 was performed by using the Dice coefficient, with a 5% homology coefficient (UPGMA), using the BIOPROFIL BioGene Software (Vilber-Lourmat, France). We considered that the strains were identical when the electrophoretic profiles appeared clustered at least with 3 primer sets.

Results Clinical data During the period 1990e2003 NPRGM were detected in 81 samples from 65 patients. The isolated species are shown in Table 1. Thirty-seven (56.9%) of 65 patients were grouped between 1995 and 1997 (Fig. 1). Samples from which the bacteria were isolated include sputum and other respiratory samples (24 patients), wound exudates and skin biopsies (14 patients), bowel biopsies (7 patients), urine (3 patients), blood cultures (3 patients) and several other samples (14 patients). We reviewed the case records of the patients with NPRGM isolates, grouping them as clinically significant in 19 cases, of doubtful clinical significance in 1 and without clinical significance in 45 patients. Clinical manifestations, treatment and outcome of the patients with significant or doubtful disease are shown in Table 2. Some of these data have already been published elsewhere.15 The most frequent clinical syndrome was skin and soft tissue infection (9 cases) followed by disseminated infection (3 cases), osteoarticular infection (3 cases), surgical site infection (2 cases), and diabetic foot infection (2 cases). No case of respiratory infection was found among all cases, although 2 cases of endotracheal stent colonization were found. Risk factors documented in the clinically significant group were diabetes mellitus (3 cases), malignancy (2 cases), and immunosuppressive treatment (2 cases). The 3 cases of surgical infection were secondary to hernia surgery with implantation of polypropylene prosthetic mesh (2 cases) and 1 case of implantation of eye prosthesis.

Table 1 Species of NPRGM isolated between 1990 and 2003 M. M. M. M. M. M. M.

abscessus alvei chelonae fortuitum mucogenicum peregrinum senegalense

2 1 24 23 3 11 1

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J. Esteban et al.

7

of true infection (1 M. abscessus, 6 M. fortuitum, and 7 M. chelonae). Despite lack of purification of the DNA, all strains gave a good readable electrophoretic pattern. Only 1 strain gave no amplification products with primers OPA-2 and OPA-18. Most of the isolates gave more than 4 bands in the amplification product, except OPA-18 primer, which gave a very low number of bands, especially for M. fortuitum. In our experience, primer OPA-2 was the one with a higher discriminative power, as has been documented previously.9,10 After analysis with the Bio Gene software, only 1 cluster of 2 strains of M. chelonae was identified with all the primers (Fig. 2). When the cluster was identified, a careful review of the clinical data was performed. Both isolates were from biopsy samples that were processed the same day, using the same saline serum for grinding them, being the serum discharged after processing both samples. The patients had no clinical data that suggest infection, and the samples were sent to the microbiology laboratory as part of the routine protocol of the hospital. We conclude that both isolates were a contamination probably due to contamination of the saline serum used for grinding in the bacteriology laboratory. Apart from this pseudo-outbreak, 5 clusters with primer IS 986 FP (1 in M. chelonae and 4 in M. fortuitum strains), and 3 with OPA-18 (3 in M fortuitum and 1 in M. peregrinum) were detected, but the strains involved in these clusters do not fulfil the criteria for identity previously stated (Fig. 2).

M.chelonae M.abscessus M.mucogenicum M.fortuitum M.peregrinum

Number of patients

6 5 4 3 2 1 0

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003

Year

Figure 1

Yearly distribution of isolates of NPRGM.

We identified cold wax depilation as an important risk factor in the skin infection group.15 One of the patients with osteomyelitis was a foreign body carrier, and other had a sequestra. All patients with disseminated infection carried a central venous catheter. Ten of the significant and doubtful cases (50%) had a positive acid-fast stain, and 8 of the nonsignificant cases (17.8%) had the same result, including 2 cases clearly identified as laboratory contamination and both cases of tracheal stent colonization. All patients were treated with antimicrobial therapy. In 1 case no systemic therapy was used, and the patient was cured only with topical therapy with antiseptics. The patients with foreign bodies required the removal of the prosthesis after a trial with antibiotics without improvement. All patients were cured. The doubtful case was a patient with arthritis showing granuloma in the pathological study and isolation of M. chelonae. This patient was treated with antituberculous drugs until the identification of mycobacteria, when this therapy was ended. The patient was cured without sequels. No epidemiological link was found among the patients. In 1 case of folliculitis secondary to depilation we tried to isolate the bacteria from cosmetic products without results. The patients with prosthetic mesh infection after hernia surgery appeared with a difference of 6 years.

RAPD analysis Forty-three strains from patients which clinical charts were available for molecular study (1 M. abscessus, 3 M. mucogenicum, 9 M. peregrinum, 13 M. chelonae and 17 M. fortuitum). Fourteen of the strains belonged to the previously described cases

Discussion Non-pigmented, rapidly growing mycobacteria has been described in the literature as the cause of a broad spectrum of human diseases.1,16e18 Our series includes, as expected, different infections, ranging from severe disseminated infections to relatively mild folicullitis syndromes. However, our series showed some original aspects that deserve to be analyzed in detail. One of the most important differences with the previous reports is the absence of respiratory infections. Despite this syndrome has been extensively described in the literature,1,19,20 in our patients all the respiratory isolates were considered as non-significant because the absence of symptoms, the scanty amount of bacteria recovered, and the absence of repeated isolates. The absence in our hospital of patients with underlying diseases that could be infected with these organisms, such

Characteristics of the cases of infection due to NPRGM. Significant (1e19) and probable (20) cases

Case

Year

Sex

Age

Underlying diseases

Syndrome

Positive samples

Acid-fast stain

Therapy

Outcome

Species

1

1990

M

52

Osteomyelitis

AK þ SXT þ E

Cure

M. chelonae

1992

F

68

Bone biopsy, several exudates Skin biopsy

Positive

2

Mediastinal sarcoma, local radiotherapy Haemodialysis

Positive

E þ AK, E

Cure

M. abscessus

3

1993

F

70

Diabetes mellitus

Several exudates

Positive

Cure

M. chelonae

4

1995

F

24

Skin biopsy

Negative

Cure

M. chelonae

5

1995

F

24

Hepatitis C, former drug addiction e

Local therapy with antiseptics CLA

Skin biopsy

Negative

CIP

Cure

M. fortuitum

6

1995

F

31

e

Skin biopsy

Negative

CLA

Cure

M. chelonae

7

1996

F

84

Several exudates

Positive

CLA

Cure

M. chelonae

8

1996

F

70

Catheter exudate, blood cultures

Positive

CIP þ AK þ E/CLA þ CRO/CZ

Cure

M. chelonae

9

1996

F

24

Diabetes mellitus, Ampollous pemphigoid Idiopathic thrombopenic purpura, hypergammaglobulinemia e

Skin biopsy

Negative

CLA

Cure

M. chelonae

10

1997

F

19

e

Skin biopsy

Negative

CLA

Cure

M. chelonae

11

1997

F

51

Hernia surgery

Positive

CIP

Cure

M. fortuitum

12

1997

F

20

e

Several exudates, prosthetic mesh Skin biopsy

Positive

CIP

Cure

M. fortuitum

13

1999

M

79

Diabetes mellitus

Several exudates

Positive

Cure

M. chelonae

14

2000

M

27

Trauma

Negative

Cure

M. abscessus

15

2001

F

39

e

Bone biopsy, several exudates Skin biopsy

A/C, local therapy with antiseptics CLA þ AK þ E

Negative

CLA

Cure

M. chelonae

16

2002

F

50

Positive

CIPþAK

Cure

M. fortuitum

17

2002

M

57

Microcitic lung carcinoma Ocular prosthesis

Not performed

A/C þ TOB (topic)

Cure

M. fortuitum

18

2002

F

23

e

Negative

CIP

Cure

M. peregrinum

19

2003

F

75

Hernia surgery

Negative

CIP þ AK

Cure

M. fortuitum

20

1997

M

61

e

Negative

IþR

Cure

M. chelonae

Disseminated infection Diabetic foot infection Skin and soft tissue infection Skin and soft tissue infection Skin and soft tissue infection Skin and soft tissue infection Disseminated infection Skin and soft tissue infection Skin and soft tissue infection Surgical site infection Skin and soft tissue infection Diabetic foot infection Osteomyelitis Skin and soft tissue infection Disseminated infection Conjunctivitis Skin and soft tissue infection Surgical site infection Arthritis

Blood cultures, bronchial lavage Conjunctival exudate Exudate Several exudates, prosthetic mesh Sinovial biopsy

139

M: Male, F: Female, AK: Amikacin, SXT: Cotrimoxazole, E: Erythromycin, CIP: Ciprofloxacin, CRO: Ceftriaxone, CZ: Cefazolin A/C: Amoxicillin/Clavulanic acid, TOB: Tobramycin, I: Isoniazid, R: Rifampin, and CLA: Clarithromycin.

Infections due to rapidly growing mycobacteria

Table 2

140

J. Esteban et al.

Figure 2 Dendrograms of the electrophoretic patterns obtained with the different primer sets (M. chelonae, M. fortuitum and M. peregrinum) (A) Primer OPA-2. (B) Primer OPA-18. (C) Primer INS-2. (D) Primer IS-986 FP.

Infections due to rapidly growing mycobacteria

Figure 2 (continued)

141

142

J. Esteban et al.

Figure 2 (continued)

Infections due to rapidly growing mycobacteria

Figure 2 (continued)

143

144 as cystic fibrosis,19 could be the reason of the absence of respiratory disease in our series. Only in 2 cases the mycobacteria cultures showed heavy growth and repeated isolation of bacteria in several sputum and bronchial washing samples. Both cases were from patients with tracheal prosthesis who had no symptoms of disease. The removal of the prosthesis leads to the disappearance of mycobacteria, and in one of the prosthesis that was sent to the laboratory, a heavy growth of mycobacteria was detected. This syndrome has been previously described,21 and could be the cause of misdiagnosis of tuberculosis in countries where this disease is prevalent, because the acid-fast stain of respiratory samples showed acid-fast bacilli, and this finding is usually diagnostic of tuberculosis in such countries. In our series, skin and soft tissue infections were clearly predominant. Of interest are the cases of folliculitis in young women with no predisposing factors except cold wax depilation.15 Infections secondary to several cosmetic procedures have been described previously,1,5,22e24 but in our cases there was no link between all of them, being the patients from different geographical areas, and go to different cosmetic centres to perform such procedure. Only 2 of our cases were surgical site infections, despite this syndrome has been described extensively in the literature.1,2,4,18 Both of our cases were late infections of prosthetic mesh used in hernia surgery.25 It is interesting that in all of our patients with infections associated with foreign bodies, removal of such prosthesis was needed to obtain the cure of the patients, being the antimicrobial therapy unable to eradicate the organism alone. This finding has been previously published in several infections, including cases extremely severe, such as prosthetic endocarditis.1,18 Treatment of our patients leads to cure them all. Patients with mild skin and soft tissue infections were mainly treated with only one antibiotic with good results, despite the fact that appearance of resistances has been previously described associated with monotherapy.26 The relatively low number of bacteria present in the patients and the efficacy of the drugs could be the explanation of the lack of failures due to selection of mutant strains. Epidemiology of rapidly growing mycobacteria is lesser known than clinical diseases or therapy. It has been stated that these organisms can be found in water systems,1 and this has been the source of several outbreaks and pseudo-outbreaks.2,24,27e29 Some of these outbreaks have been characterized by using molecular biology

J. Esteban et al. techniques, such as PFGE or RAPD.2 However, the molecular biology analysis has been performed only in one case with strains not included in an outbreak.11 In this study, strains of M. fortuitum were analyzed with PFGE and showed different profiles, and no relationship between the different strains was demonstrated. In our study, several issues must be commented. First, when we evaluated the number of cases, we detected an increase between 1995 and 1997 despite no changes in the protocols were performed during the study period. However, because this increase was due to several species, no outbreak was suspected. In our series, clinical cases of skin and soft tissue infection were predominant, and they were due to M. chelonae and M. fortuitum. M. abscessus has been isolated only in 2 cases, so it seems that this latter species is rarely found in our environment. RAPD analysis showed that no genetic relationship was detected in all the analyzed strains except two of them. These strains showed identity with all the primer sets and were epidemiologically related with the contamination of distilled water used for gringing of biopsies in the same day. Because no other biopsy was processed that day and we detected the contamination in the following day, no other sample was contaminated. We could not isolate the contaminant strain because the distilled water was discharged before it could be cultured for mycobacteria isolation. All other strains studied showed different profiles. Unfortunately, because of the retrospective nature of our study, not all strains were available for study, but classical epidemiology showed no relationship between patients, so it seems that no other outbreak or pseudo-outbreak has appeared among our cases. RAPD has been previously used to characterize other outbreaks or psudo-outbreaks.2,9,10 However, this technique has not been used for analysis of apparently unrelated strains. Because of the reproducibility problems of this technique,30 we have tried to minimize such problems by analyzing all the strains of the same species together in the same steps (i.e., culture, extraction of the DNA, PCR and gel electrophoresis), so the results could be properly evaluated. In conclusion, disease due to rapidly growing mycobacteria in our hospital is predominantly of skin and soft tissues. Although changes in the number of the isolates were detected, no relationship between the strains could be demonstrated neither by classical epidemiology studies nor by RAPD analysis, so other factors must exist to influence the incidence of these species.

Infections due to rapidly growing mycobacteria

145

Acknowledgments The work was partially presented in the 14th ECCMID (Prague, 2004) and in the 15th ECCMID (Copenhagen 2005). No conflict of interest exist for all authors. This study was funded by a grant from the Fondo de Investigaciones Sanitarias (FIS, PI 030146). Zamora N was funded by the Fundacio ´n Conchita Ra ´bago de Jime ´nez Dı´az.

13.

14.

15.

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