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1.
Hunninghake  GWCostabel  UAndo  M  et al.  ATS/ERS/WASOG statement on sarcoidosis Sarcoidosis Vasc Diffuse Lung Dis 1999;16149- 173
PubMed
2.
Du Bois  RMGoh  NMcGrath  DCullinan  P Is there a role for microorganisms in the pathogenesis of sarcoidosis? J Intern Med 2003;2534- 17
PubMedArticle
3.
Baughman  RPLower  EEdu Bois  RM Sarcoidosis Lancet 2003;3611111- 1118
PubMedArticle
4.
Saboor  SAJohnson  NMMcFadden  J Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction Lancet 1992;3391012- 1015
PubMedArticle
5.
Thakker  BBlack  MFoulis  AK Mycobacterial nucleic acids in sarcoid lesions Lancet 1992;3391537
PubMedArticle
6.
Bocart  DLecossier  Dde Lassence  AValeyre  DBattesti  JPHance  AJ A search for mycobacterial DNA in granulomatous tissues from patients with sarcoidosis using the polymerase chain reaction Am Rev Respir Dis 1992;1451142- 1148
PubMedArticle
7.
Gerdes  JRichter  ERusch-Gerdes  S Mycobacterial nucleic acids in sarcoid lesions Lancet 1992;3391536- 1537
PubMedArticle
8.
Lisby  GMilman  NJacobsen  GK Search for Mycobacterium paratuberculosis DNA in tissue from patients with sarcoidosis by enzymatic gene amplification APMIS 1993;101876- 878
PubMedArticle
9.
Fidler  HRook  GAJohnson  NMMcFadden  J Micobacterium tuberculosis DNA in tissue affected by sarcoidosis BMJ 1993;306546- 549
PubMedArticle
10.
Popper  HHWinter  WHofler  G DNA of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue in tuberculosis and sarcoidosis detected by polymerase chain reaction Am J Clin Pathol 1994;101738- 741
PubMed
11.
Ghossein  RARoss  DGSalomon  RNRabson  AR A search for mycobacterial DNA in sarcoidosis using polymerase chain reaction Am J Clin Pathol 1994;101733- 737
PubMed
12.
Richter  EGreinert  UKirsten  D Assessment of mycobacterial DNA in cells and tissues of mycobacterial and sarcoid lesions Am J Respir Crit Care Med 1996;153375- 380
PubMedArticle
13.
El-Zaatari  FANaser  SAMarkesich  DCKalter  DCEngstand  LGraham  DY Identification of Mycobacterium avium complex in sarcoidosis J Clin Microbiol 1996;342240- 2245
PubMed
14.
Vokurka  MLecossier  Ddu Bois  RM  et al.  Absence of DNA from mycobacteria of the M. tuberculosis complex in sarcoidosis Am J Respir Crit Care Med 1997;1561000- 1003
PubMedArticle
15.
Popper  HHKlemen  HHoefler  GWinter  E Presence of mycobacterial DNA in sarcoidosis Hum Pathol 1997;28796- 800
PubMedArticle
16.
Cannone  MVago  LPorini  G Detection of Mycobacterium tuberculosis DNA using nested polymerase chain reaction in lymph nodes with sarcoidosis, fixed in formalin and embedded in paraffin Pathologica 1997;89512- 516
PubMed
17.
Wilsher  MLMenzies  RECroxson  MC Mycobacterium tuberculosis DNA in tissues affected by sarcoidosis Thorax 1998;53871- 874
PubMedArticle
18.
Li  NBajoghi  AKubba  ABhawan  J Identification of mycobacterial DNA in cutaneous lesions of sarcoidosis J Cutan Pathol 1999;26271- 278
PubMedArticle
19.
Ishige  IUsui  YTakemura  TEishi  Y Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis Lancet 1999;354120- 123
PubMedArticle
20.
Ikonomopoulos  JAGorgoulis  VGZacharatos  PV  et al.  Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis Mod Pathol 1999;12854- 862
PubMed
21.
Grosser  MLuther  TMuller  J Detection of M. tuberculosis DNA in sarcoidosis: correlation with T-cell response Lab Invest 1999;79775- 784
PubMed
22.
Klemen  HHusain  ANCagle  PTGarrity  ERPopper  HH Mycobacterial DNA in recurrent sarcoidosis in the transplanted lung: a PCR-based study on four cases Virchows Arch 2000;436365- 369
PubMedArticle
23.
Eishi  YSuga  MIshige  I  et al.  Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis J Clin Microbiol 2002;40198- 204
PubMedArticle
24.
Drake  WPPei  ZPride  DTCollins  RDCover  TLBlaser  MJ Molecular analysis of sarcoidosis tissues for Mycobacterium species DNA Emerg Infect Dis 2002;81334- 1341
PubMedArticle
25.
Mitchell  ICTurk  JLMitchell  DN Detection of mycobacterial rRNA in sarcoidosis with liquid-phase hybridisation Lancet 1992;3391015- 1017
PubMedArticle
26.
Böddinghaus  BRogall  TFlohr  TBlöker  HBöttger  EC Detection and identification of mycobacteria by amplification of rRNA J Clin Microbiol 1990;281751- 1759
PubMed
27.
Degitz  K Detection of mycobacterial DNA in the skin: etiologic insights and diagnostic perspectives Arch Dermatol 1996;13271- 75
PubMedArticle
28.
Walsh  NMHanly  JGTremaine  RMurray  S Cutaneous sarcoidosis and foreign bodies Am J Dermatopathol 1993;15203- 207
PubMedArticle
29.
Val-Bernal  JFSanchez-Quevedo  MCCorral  JCampos  A Cutaneous sarcoidosis and foreign bodies: an electron probe roentgenographic microanalytic study Arch Pathol Lab Med 1995;119471- 474
PubMed
30.
Kim  YCTriffet  MKGibson  LE Foreign bodies in sarcoidosis Am J Dermatopathol 2000;22408- 412
PubMedArticle
31.
Marcoval  JMañá  JMoreno  AGallego  IFortuño  YPeyrí  J Foreign bodies in granulomatous cutaneous lesions of patients with systemic sarcoidosis Arch Dermatol 2001;137427- 430
PubMed
Study
January 2005

Absence of Ribosomal RNA of Mycobacterium tuberculosis Complex in Sarcoidosis

Author Affiliations

Author Affiliations: Departments of Dermatology (Dr Marcoval), Microbiology (Drs Benítez and Alcaide), and Internal Medicine (Dr Mañá), Hospital de Bellvitge, University of Barcelona, Barcelona, Spain.

Arch Dermatol. 2005;141(1):57-59. doi:10.1001/archderm.141.1.57
Abstract

Objective  To determine whether Mycobacterium tuberculosis ribosomal RNA (rRNA) is present in fresh tissue specimens from patients with sarcoidosis.

Design  A prospective study.

Setting  A university-based hospital.

Patients  Thirty-five patients diagnosed as having sarcoidosis at the University Hospital of Bellvitge, Barcelona, Spain, were included in the study. Fresh tissue samples with granulomatous inflammation were prospectively collected between 1997 and 2001 from all patients. For each sample tested, approximately 1 negative control was included.

Main Outcome Measures  Mycobacterium tuberculosis rRNA was detected using an isothermal enzymatic amplification system of target rRNA of M tuberculosis complex via DNA intermediates. Smears for acid-fast staining and mycobacteriological cultures were also obtained.

Results  A total of 78 biopsy specimens (57 skin, 10 lymph node, 3 lacrimal gland, 2 spleen, 2 lung, 2 muscle, 1 bone, and 1 nerve) collected from 74 patients (35 patients with sarcoidosis and 39 control patients) were included in the study. Stains for acid-fast bacilli and mycobacterial cultures were negative for organisms in all cases. Mycobacterium tuberculosis rRNA was not detected in the specimens from any patients with sarcoidosis or in those from control patients whose cultures were negative for organisms. Ribosomal RNA was detected in 6 tissue specimens from patients with cultures that were positive for M tuberculosis and that were processed in parallel to the samples included in the study.

Conclusions  Although previous studies have reported that mycobacterial antigens may play a role in granuloma formation in some patients with sarcoidosis, our results suggest that M tuberculosis cannot be considered to be the etiologic agent of the disease.

Sarcoidosis is a multisystemic disease that is defined by the formation of noncaseating granulomas in different organs. Although the etiology of sarcoidosis remains uncertain,1 the apparition of clusters of the disease in some communities and the existence of seasonal variations in incidence suggest that environmental or infectious agents may play a role in its development.2,3 Moreover, similarities in the clinical, pathologic, and immunologic abnormalities in patients with sarcoidosis and in certain patients with tuberculosis have raised the suspicion that mycobacterial infection could be associated with the pathogenesis of this disorder.2

In recent years, some studies have focused on the detection of mycobacterial DNA by polymerase chain reaction (PCR) in patients with sarcoidosis, with quite divergent results. Mycobacterial DNA has been found in approximately half of the samples in some studies, while it was not detected at all in other studies.424 Most of these studies were retrospective and were carried out in paraffin-embedded tissues. Only 1 study, which was performed using liquid-phase hybridization, looked for mycobacteria-specific rRNA in fresh samples from a small number patients with sarcoidosis (n=5).25

The purpose of the present study was to prospectively determine the presence of viable M tuberculosis organisms in fresh tissue samples from patients with sarcoidosis by means of smears for acid-fast staining, mycobacteriological cultures, and the detection of mycobacteria-specific rRNA.

METHODS
PATIENTS AND SAMPLES

Tissue specimens from patients with sarcoidosis were prospectively collected between 1997 and 2001 at the University Hospital of Bellvitge (a 1000-bed teaching institution in Barcelona, Spain). The diagnosis of systemic sarcoidosis was made according to the classic criteria: a compatible clinical and radiologic picture; histologic demonstration of noncaseating granulomas involving 1 or more tissues or a positive Kveim test result; and exclusion of other granulomatous diseases. Half of the specimen from each patient was fixed in 10% buffered formaldehyde and processed for histologic evaluation to determine whether granulomas were present in the samples.

CONTROLS

At least 1 negative control was included for each test sample. The negative control samples, which were analyzed in the same manner as the test samples, consisted of fresh specimens of normal tissue obtained from patients without evidence of sarcoidosis or tuberculosis. Most cutaneous specimens were obtained from redundant normal skin acquired during minor surgical procedures.

SPECIMEN PROCESSING, MICROSCOPY, AND CULTURE

All biopsy specimens were homogenized in 3 mL of 0.9% sodium chloride solution within 24 hours of specimen collection. Part of this suspension was used for the preparation of smears for acid-fast staining with auramine-rhodamine fluorochrome and for culture. The remaining suspension was stored at −80°C for subsequent use in the genetic amplification procedure. An equal volume (0.5 mL) of the processed specimens was inoculated in 2 culture media: a liquid medium bottle (MB/BacT; bioMérieux SA, Marcy l’Etoile, France) and a Löwenstein-Jensen slant (MAIM, Barcelona, Spain) as a solid medium. All cultures were incubated at 35°C to 37°C for up to 6 weeks. The bottles were registered and processed by means of a nonradiometric system for incubation and continuous monitoring of mycobacterium growth (BacT/ALERT 3-dimensional instrument; bioMérieux SA). The Löwenstein-Jensen media were incubated in 5% carbon dioxide and examined for colonies on the slant once a week.

EXTRACTION OF NUCLEIC ACIDS

The material was extracted with a commercially available kit (RNA/DNA Maxi Kit; Qiagen, Hilden, Germany) that allows rapid isolation of RNA and DNA from small numbers of bacteria in clinical samples.

GENETIC AMPLIFICATION PROCEDURE

A nucleic amplification test (AMDT; Gen-Probe Inc, San Diego, Calif) was used to detect M tuberculosis rRNA. This test is an isothermal amplification system based on the reverse transcription of mycobacteria-specific rRNA targets via DNA intermediates. It uses 2 enzymes (RNA polymerase and reverse transcriptase) and 2 primers, one of which contains a promoter sequence for RNA polymerase. Detection of RNA amplicons is achieved with an acridinium ester–labeled DNA probe. The entire process (amplification and detection) is performed in a single tube, which helps to reduce the risk of carryover contamination. In the present study, the amplification test was performed and the results were interpreted according to the manufacturer’s recommendations. Six clinical samples (3 lymph node specimens and 1 biopsy specimen each of spleen, liver, and lung) with cultures that were positive for M tuberculosis were used as quality control for extraction and amplification techniques.

RESULTS

A total of 78 specimens obtained from 74 patients (35 patients with sarcoidosis and 39 control patients) were included in the study. The 37 specimens obtained from specific granulomatous lesions of 35 patients with sarcoidosis were skin (n = 28), lymph node (n = 5), muscle (n = 2), lung (n = 1), and lacrimal gland (n = 1). The 41 specimens obtained from 39 control patients were skin (n = 29), lymph node (n = 5), spleen (n = 2), lacrimal gland (n = 2), lung (n = 1), bone (n = 1), and nerve (n = 1).

All the specimens included in the study were negative for acid-fast bacilli. Likewise, all the cultures were negative for M tuberculosis after at least 6 weeks of incubation.

The 37 samples from the 35 patients with sarcoidosis and the 41 specimens from the 39 subjects in the control group were all negative for M tuberculosis rRNA. However, rRNA was detected in 6 tissue specimens with cultures that were positive for M tuberculosis and that were processed in parallel to the samples included in the study.

COMMENT

In the present study, we were not able to detect the presence of M tuberculosis rRNA in fresh tissue samples of granulomatous lesions prospectively collected from patients with systemic sarcoidosis. The smears for acid-fast bacilli and mycobacteriological cultures were also negative for organisms.

Clinical and pathologic similarities to pulmonary tuberculosis have suggested a potential role of mycobacteria in the etiology of sarcoidosis. The histopathologic features of the granulomas and the occasional presence of caseation necrosis also indicate the possibility of a mycobacterial pathogenesis in sarcoidosis. However, the absence of caseation in most instances, the negativity of mycobacterial cultures, and the absence of a response to antituberculous treatment are factors that contradict this theory. Likewise, over the past century, while there has been a marked reduction in the incidence of tuberculosis, the incidence of sarcoidosis has not decreased.2

During the last decade, several studies have used PCR analysis in an attempt to detect mycobacterial DNA in a number of biopsy specimens from patients with sarcoidosis. The findings of these studies have been quite inconsistent, with positive results ranging from 0% to 80%.424 The surprising variability of these data may be explained by several technical factors, including the choice of target sequence and DNA-retrieval variability, depending on which technique is used to obtain the DNA.2 An alternative explanation for these divergent results is inadvertent contamination of samples in some series.14 It is noteworthy that the studies showing the highest proportion of positive results for samples from patients with sarcoidosis also reported a relatively high proportion of positive results for samples from control subjects and/or from individuals with prior tuberculous infections.14 Moreover, most studies about the detection of mycobacterial DNA using PCR in sarcoidosis were performed in formalin-fixed, paraffin-embedded specimens from retrospective pools of pathology departments. In such studies, there is a greater possibility of both contamination of the paraffin-embedded specimens and more DNA fragmentation. However, the present study was prospective and only fresh tissue was used. For each patient, half of the biopsy specimen was processed for routine histologic evaluation to confirm the presence of granulomas in the samples included in the study. The use of fresh tissue allowed us to apply a technique of rRNA amplification that has several advantages. It is more sensitive than DNA amplification because there are more than 2000 copies of rRNA for 1 DNA molecule.26 Another advantage of the nucleic acid amplification system that was used in the present study is the low level of cross contamination, because the entire process is performed in a single tube. The only study analyzing the presence of rRNA of mycobacteria on fresh samples of sarcoidosis included only 5 patients and used a technique in which the samples were more easily exposed to exogenous contamination (liquid-phase hybridization).25

To establish epidemiologically that an infective agent is the cause of disease requires consistent isolation of the agent from cases and production of disease in an organism when the agent is introduced. These points have not been demonstrated consistently to date.1,2 Polymerase chain reaction detection of mycobacterial DNA in sarcoidosis does not indicate whether the detected mycobacteria are still viable and susceptible to antituberculous therapy or whether they represent nonviable remnants of prior mycobacterial colonization.27 The presence of mycobacterial DNA in tissues involved with sarcoidosis only suggests that mycobacterial vestiges may be present in some lesions in a variable proportion of patients. However, the absence of mycobacterial rRNA, despite the higher sensitivity compared with DNA detection, and the negativity of the cultures rule out the presence of viable mycobacteria in the sarcoidosis lesions.

We suggest that vestiges of mycobacteria may act as a stimulus for granuloma formation in some patients who are developing sarcoidosis, as can occur when there are foreign bodies in the skin.2831 This hypothesis may explain why mycobacteria have never been recovered from sarcoidal tissue, why tuberculostatic therapy of sarcoidosis has generally not been successful, and why reactivation of tuberculosis has not been observed in sarcoidosis despite the use of corticosteroids or other immunosuppressive agents.

In conclusion, although mycobacterial antigens may be responsible for granuloma formation in some patients, our results suggest that M tuberculosis cannot be considered the etiologic agent of sarcoidosis.

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Article Information

Correspondence: Joaquim Marcoval, MD, Department of Dermatology, Hospital de Bellvitge, c/Feixa Llarga s/n, Hospitalet de Llobregat, 08907 Barcelona, Spain (jmarcoval@csub.scs.es).

Accepted for Publication: June 17, 2004.

Funding/Support: This study was supported by grant 98/774 from Fondo de Investigaciones Sanitarias (Dr Mañá).

Financial Disclosure: None.

References
1.
Hunninghake  GWCostabel  UAndo  M  et al.  ATS/ERS/WASOG statement on sarcoidosis Sarcoidosis Vasc Diffuse Lung Dis 1999;16149- 173
PubMed
2.
Du Bois  RMGoh  NMcGrath  DCullinan  P Is there a role for microorganisms in the pathogenesis of sarcoidosis? J Intern Med 2003;2534- 17
PubMedArticle
3.
Baughman  RPLower  EEdu Bois  RM Sarcoidosis Lancet 2003;3611111- 1118
PubMedArticle
4.
Saboor  SAJohnson  NMMcFadden  J Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction Lancet 1992;3391012- 1015
PubMedArticle
5.
Thakker  BBlack  MFoulis  AK Mycobacterial nucleic acids in sarcoid lesions Lancet 1992;3391537
PubMedArticle
6.
Bocart  DLecossier  Dde Lassence  AValeyre  DBattesti  JPHance  AJ A search for mycobacterial DNA in granulomatous tissues from patients with sarcoidosis using the polymerase chain reaction Am Rev Respir Dis 1992;1451142- 1148
PubMedArticle
7.
Gerdes  JRichter  ERusch-Gerdes  S Mycobacterial nucleic acids in sarcoid lesions Lancet 1992;3391536- 1537
PubMedArticle
8.
Lisby  GMilman  NJacobsen  GK Search for Mycobacterium paratuberculosis DNA in tissue from patients with sarcoidosis by enzymatic gene amplification APMIS 1993;101876- 878
PubMedArticle
9.
Fidler  HRook  GAJohnson  NMMcFadden  J Micobacterium tuberculosis DNA in tissue affected by sarcoidosis BMJ 1993;306546- 549
PubMedArticle
10.
Popper  HHWinter  WHofler  G DNA of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue in tuberculosis and sarcoidosis detected by polymerase chain reaction Am J Clin Pathol 1994;101738- 741
PubMed
11.
Ghossein  RARoss  DGSalomon  RNRabson  AR A search for mycobacterial DNA in sarcoidosis using polymerase chain reaction Am J Clin Pathol 1994;101733- 737
PubMed
12.
Richter  EGreinert  UKirsten  D Assessment of mycobacterial DNA in cells and tissues of mycobacterial and sarcoid lesions Am J Respir Crit Care Med 1996;153375- 380
PubMedArticle
13.
El-Zaatari  FANaser  SAMarkesich  DCKalter  DCEngstand  LGraham  DY Identification of Mycobacterium avium complex in sarcoidosis J Clin Microbiol 1996;342240- 2245
PubMed
14.
Vokurka  MLecossier  Ddu Bois  RM  et al.  Absence of DNA from mycobacteria of the M. tuberculosis complex in sarcoidosis Am J Respir Crit Care Med 1997;1561000- 1003
PubMedArticle
15.
Popper  HHKlemen  HHoefler  GWinter  E Presence of mycobacterial DNA in sarcoidosis Hum Pathol 1997;28796- 800
PubMedArticle
16.
Cannone  MVago  LPorini  G Detection of Mycobacterium tuberculosis DNA using nested polymerase chain reaction in lymph nodes with sarcoidosis, fixed in formalin and embedded in paraffin Pathologica 1997;89512- 516
PubMed
17.
Wilsher  MLMenzies  RECroxson  MC Mycobacterium tuberculosis DNA in tissues affected by sarcoidosis Thorax 1998;53871- 874
PubMedArticle
18.
Li  NBajoghi  AKubba  ABhawan  J Identification of mycobacterial DNA in cutaneous lesions of sarcoidosis J Cutan Pathol 1999;26271- 278
PubMedArticle
19.
Ishige  IUsui  YTakemura  TEishi  Y Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis Lancet 1999;354120- 123
PubMedArticle
20.
Ikonomopoulos  JAGorgoulis  VGZacharatos  PV  et al.  Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis Mod Pathol 1999;12854- 862
PubMed
21.
Grosser  MLuther  TMuller  J Detection of M. tuberculosis DNA in sarcoidosis: correlation with T-cell response Lab Invest 1999;79775- 784
PubMed
22.
Klemen  HHusain  ANCagle  PTGarrity  ERPopper  HH Mycobacterial DNA in recurrent sarcoidosis in the transplanted lung: a PCR-based study on four cases Virchows Arch 2000;436365- 369
PubMedArticle
23.
Eishi  YSuga  MIshige  I  et al.  Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis J Clin Microbiol 2002;40198- 204
PubMedArticle
24.
Drake  WPPei  ZPride  DTCollins  RDCover  TLBlaser  MJ Molecular analysis of sarcoidosis tissues for Mycobacterium species DNA Emerg Infect Dis 2002;81334- 1341
PubMedArticle
25.
Mitchell  ICTurk  JLMitchell  DN Detection of mycobacterial rRNA in sarcoidosis with liquid-phase hybridisation Lancet 1992;3391015- 1017
PubMedArticle
26.
Böddinghaus  BRogall  TFlohr  TBlöker  HBöttger  EC Detection and identification of mycobacteria by amplification of rRNA J Clin Microbiol 1990;281751- 1759
PubMed
27.
Degitz  K Detection of mycobacterial DNA in the skin: etiologic insights and diagnostic perspectives Arch Dermatol 1996;13271- 75
PubMedArticle
28.
Walsh  NMHanly  JGTremaine  RMurray  S Cutaneous sarcoidosis and foreign bodies Am J Dermatopathol 1993;15203- 207
PubMedArticle
29.
Val-Bernal  JFSanchez-Quevedo  MCCorral  JCampos  A Cutaneous sarcoidosis and foreign bodies: an electron probe roentgenographic microanalytic study Arch Pathol Lab Med 1995;119471- 474
PubMed
30.
Kim  YCTriffet  MKGibson  LE Foreign bodies in sarcoidosis Am J Dermatopathol 2000;22408- 412
PubMedArticle
31.
Marcoval  JMañá  JMoreno  AGallego  IFortuño  YPeyrí  J Foreign bodies in granulomatous cutaneous lesions of patients with systemic sarcoidosis Arch Dermatol 2001;137427- 430
PubMed
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