Molecular Diagnosis of a Mycobacterium chelonae Infection

Molecular Diagnosis of a Mycobacterium chelonae Infection

PATHOLOGY Teaching Case RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp Molecular Diagnosis of a Mycobacteri...

255KB Sizes 0 Downloads 245 Views

PATHOLOGY

Teaching Case

RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp

Molecular Diagnosis of a Mycobacterium chelonae Infection* Stephan Schulz1, Markus Kremer1, Antonello D. Cabras1, Andreas Lügering2, Matthias Seidel2, Heinz Höfler1 and Martin Werner1 1

Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, Klinikum rechts der Isar, München, Germany; 2 Medizinische Klinik und Poliklinik B, Westfälische Wilhelms-Universität Münster, Münster, Germany

Summary A 23-year-old female presented with enlarged cervical lymph nodes, and a diagnosis of nonspecific lymphadenitis with formation of pyogranulomas was rendered. Despite an initial oral antibiosis and subsequent long-term intravenous and oral antibiosis under hospitalized conditions, the symptoms progressed. The lymph nodes became larger and then affected the cervical region bilaterally. Her general condition worsened, and an exanthema of the extremities accompanied by a reactive arthritis occurred. Serological assays of various viral and bacterial markers and blood cultures were negative. Application of a polymerase chain reaction (PCR) protocol allowing specific amplification of mycobacterial DNA revealed DNA of Mycobacterium chelonea in formalin-fixed, paraffin-embedded lymph node tissue. Sequencing of the PCR product showed a 97% homology with the known Mycobacterium chelonae sequence. Modification of the antibiotic therapy with clarithromycin, imipenem and amikacin resulted in a rapid regression of the symptoms. The clinical course, in combination with the difficulties in detecting the infectious agent, supports the usefulness of molecular pathological analyses specific for nontuberculous mycobacteria (NTM). • This case was presented at the slide seminar of „Diagnostic Molecular Pathology“, 17th European Congress of Pathology, Barcelona / Spain, 22 of Sept. 1999 Pathol. Res. Pract. 197: 123–126 (2001)

Key words: Mycobacterium chelonae – Nontuberculous mycobacteria – Cervical lymphadenitis – Antituberculosis drugs – Molecular pathology

Introduction The clinical symptoms of infections with NTM are often nonspecific, weak and prolonged [1]. Classical diagnostic tools such as radiology, sonography, blood or sputum cultures and immunological analysis of serum and other body fluids fail to establish the correct diagnosis in most cases, particularly after the beginning of an antibiotic therapy. The morphological picture is often nonspecific, showing mild and diffuse histiocytic reactions, with few or no giant cells and mostly an absence of caseating granulomas [13]. Special staining for acid-fast bacilli is often negative [6]. Molecular pathological methods offer a chance to specifically detect mycobacterial DNA by polymerase chain reaction (PCR), even in specimens obtained from

Address for correspondence: Stephan Schulz, Institut für Allgemeine Pathologie und Pathologische Anatomie der Technischen Universität München, Klinikum rechts der Isar, Ismaningerstr. 22, 81675 München, Germany Fax: 0049/89/4140-6106, E-mail: [email protected] 0344-0338/2001/197/2-123 $15.00/0

124 · S. Schulz

formalin-fixed, paraffin-embedded tissue. Moreover, the different mycobacteria species can be characterized using restriction endonuclease digestion or direct sequencing of the PCR products [2 ,5].

em (i.v.) plus clarithromycin (oral) led to a rapid regression of the symptoms, resulting in a complete remission after long-term therapy. Histopathological findings

Case History A 23-year-old female presented with unilateral enlargement of the cervical lymph nodes. Despite an orally applied antibiosis her general condition worsened, and symptoms such as tiredness and strong headaches in the frontal region appeared. During the first phase of hospitalization, the laboratory parameters showed unspecific signs of acute inflammation. Under subsequent intravenous (i.v.) treatment with clindamycin and oral ciprofloxacin, a circumscribed papular rash appeared on the upper and lower extremeties. Progression of the right-sided lymph node swelling and enlargement of the contralateral cervical lymph nodes were noticed. A change in the antibiotic treatment, first using clarithromycin, then switching to oral doxycyclin, did not lead to a regression of the symptoms. The blood cultures, a tine-test and serological analysis for numerous different viral and bacterial agents were negative. One month after the onset, the symptoms progressed, with a fever up to 40 °C, strong headaches, arthritis of the ankle joints, and a sharply circumscribed maculopapular rash on the lower extremeties. None of the various clinical consultations contributed to rendering the exact diagnosis. Surgical exploration of the cervical region revealed massive conglomerates of the superficial and deep perivascular lymph nodes. The microbiological analysis of a lymph node biopsy did not show growth of bacteria. After the molecular pathological diagnosis of an infection caused by M. chelonae, the antibiotic therapy was modified. Treatment with amikacin and imipen-

Histological examination of a lymph node biopsy performed during surgical exploration showed ill-defined epitheloid granulomas with a small number of giant cells. Infiltration of neutrophilic leucocytes was observed in the center of the granulomas and appeared diffuse in the surrounding tissue. These so-called pyogranulomas did not show caseation necrosis (Fig. 1). Follicular lymphatic hyperplasia and sclerosis were seen especially in the subcapsular regions. Staining for acidfast bacteria was negative. The histomorphological picture was interpreted as lymphadenitis with abscess formation and granulomatous inflammation without necrosis. Cat scratch disease as well as tuberculous and mycotic infections were considered as differential diagnoses. Molecular pathological findings

DNA from formalin-fixed, paraffin-embedded material of the lymph node biopsy was extracted using an established protocol [7]. Two different PCR methods specific for amplification of the mycobacterial 65-kilodalton heat shock protein gene (hsp 65) were applied [8]. The less sensitive single-step PCR protocol from Pao et al. [5] generated a negative result (data not shown). In contrast, application of the nested PCR protocol of Cook et al. [2] resulted in a specific and intense signal (of 133 bp) from the analyzed material. The positive controls, consisting of DNA from Mycobacterium tuberculosis and Mycobacterium avium intracellulare, as well as the negative controls (template-free reaction mix with

Fig. 1. Lymph node biopsy showing microabscess formation in the center of an epitheloid granuloma (pyogranuloma) without caseation necrosis. H&E stain (200 ×).

Molecular Diagnosis of a Mycobacterium chelonae Infection · 125

deionized water) from internal and external PCR, were regular. Subsequent restriction fragment analysis using three different endonucleases [2] showed a restriction pattern specific for mycobacterium chelonae (Fig. 2a). To verify this result, the PCR product was sequenced using a cycle-sequencing procedure [7] and an automated sequencer (ABI Prism 377, Applied Biosystems, Perkin Elmer). A homology match, using the Blast Search program of the NCBI (National Center for Biotechnology Information, National Institutes of Health, USA), revealed a correspondence of 97% of the analyzed base pairs with the hsp65 gene of Mycobacterium chelonae (strain IP140420019, accession AF071141 of the NCBI BLASTN database), thereby confirming the diagnosis of a Mycobacterium chelonae infection (Fig. 2b).

Discussion

Fig. 2. Results of hsp65 amplification, restriction fragment analysis, and sequencing results. a: Gel-Control after nested PCR: The PCR product of the analyzed case (P) had the same length as the DNA of the two positive controls (Pos. Controls), Mycobacterium tuberculosis (M. tbc) and Mycobacterium avium intracellulare (MAI), whereas the negative controls (Neg. Controls) from external PCR (Ext.) and internal PCR (Int.) did not show any reaction products. MW (molecular weight marker). b: Specific mycobacterial PCR products and restriction fragment analysis of the analyzed case and the positive control. The restriction pattern of the analyzed case is comparable to that of M. chelonae (M. chel.), whereas the control shows the typical pattern of M. tuberculosis (M. tbc.). MW, molecular weight marker Hae 3; P, PCR product uncut; Hha1, BstU1 and Mbo1: applied restriction enzymes. c: Results of the homology matches: The obtained sequence (case) reveals the greatest homology (97%, 87/90 basepairs) with the known sequence from M. chelonae (M. chel., strain IP140420019, Blast search results from NCBI data library). Alignment of the case and the control sequence of M.tuberculosis revealed a lower homology of approximately 70-80%, confirming the diagnosis of M. chelonae infection. ( –: correspondence of the retrieved sequence from NCBI data library with case sequence)

Patients infected by M. chelonae are often young, i.e., children and young adults, or immunosuppressed patients typically presenting with cervical lymphadenitis or abscessing inflammation on the skin and subcutaneous soft tissue. In a few cases, osteomyelitis, systemic manifestations and corneal keratitis after keratectomy or trauma have been ascribed to M. chelonae. Chronic pulmonary disease may occur without showing cavitation. Arthritis and myalgia, as observed in our case, might rather be interpreted as nonspecific signs of acute inflammation. The histomorphological picture of M. chelonae infection is nonspecific, often presenting without granuloma formation, but rather showing an inflammatory picture of epitheloid cells intermingled with neutrophilic leucocytes. Standard diagnoses are reticulo-histiocytic lymphadenitis with abscess formation, suppurative inflammation

126 · S. Schulz

or pyogranuloma. Nevertheless, as for cervical lymphadenitis with microabscess formation, a significant correlation with NTM infections has been described in the literature [4]. Furthermore, the presence of ill-defined granulomas, as also seen in this case, are associated with NTM infections rather than with M. tuberculosis-complex infections, which usually show well-defined granulomas [4]. Differential diagnoses such as cat scratch disease and mycotic infections should always be considered, because special staining techniques for acidfast bacilli (Ziehl-Neelsen or auramine-O) give only positive results in less than one third of the cases [13]. NTM infections may be difficult to detect, even by molecular pathological methods. One reason may be severe DNA fragmentation caused by the formalin-fixation and paraffin-embedding of the histopathological routine material [12]. Furthermore, a previous application of an antibiotic therapy might also complicate the detection of mycobacterial DNA. There are great differences between the commonly used PCR-protocols for DNA detection of NTM. The PCR-method established by Cook et al [2]. has proven to be very sensitive. It is a further advantage of this method to include a restriction enzyme assay, allowing a fast subspecification of several mycobacteria species. Application of fragment analysis on an automated sequencer has proven to be a helpful and extremely sensitive addition to this method, even allowing a precise identification of the smallest fragment lengths [11]. Sequencing strategies followed by homology matches with large genomic databases should be applied in those cases not showing a clear fragment pattern after enzymatic digestion. PCR-methods amplifying the 16S rRNA, followed by a sequencing strategy, have also been shown to be highly efficient in detecting DNA of NTM (3). In contrast, amplification of the insertion sequence IS6110 is suitable only for detecting the DNA of the Mycobacterium tuberculosis complex [10]. Many of the NTM species are resistant to conventional antituberculosis drugs, and therefore microbiological susceptibility tests must be performed. Although M. chelonae is known to be a rapidly growing bacterium, microbiological detection in classical culture assays is a difficult procedure and often unsuccessful [13]. Unfortunately, the culture assays in this case were negative, thus preventing a susceptibility test. Nevertheless, a long-term triple therapy, applying imipenem and amikacin i.v. and clarithromycin orally, led to a continuous regression of the symptoms. The costs of routine molecular pathological diagnostics are higher than those of conventional staining or culture techniques. Nevertheless, PCR amplification of mycobacterial DNA has proven to be more sensitive than conventional detection methods [9], and the results can be obtained within two days. The rapid diagnosis of mycobacterial infection in combination with a more detailed specification of the mycobacteria subtypes by PCR allows for an earlier start for specific an-

tibiotic therapy regimens and therefore reduces the financial burden an public health systems. Acknowledgments: We thank Dr. H. Wegner for sending us the paraffin-embedded material, Mrs. B. Geist for her excellent technical support, and Dr. M. Atkinson for checking the English language.

References 1. 2. 3.

4.

5.

6.

7. 8. 9.

10.

11.

12. 13.

Connor DH, Chandler FW, Schwartz DA, Manz HJ, Lack EE (1997) Pathology of infectious diseases, p 384–5. Appleton & Lange, Stamford, Connecticut Cook SM, Bartos RE, Pierson CL, Frank TS (1994) Detection of atypical mycobacteria by the polymerase chain reaction. Diagn Mol Path 3: 53–58 Kirschner P, Springer B, Vogel U, Meier A, Wrede A, Kiekenbeck M, Bange FC, Böttger EC (1993) Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31: 2882–2889 Kraus M, Benharroch D, Kaplan D, Sion-Vardy N, Leibermann A, Dima H, Shoham I, Fliss DM (1999) Mycobacterial cervical lymphadenitis: the histological features of non-tuberculous mycobacterial infection. Histopathology 35: 534–538 Pao CC, Yen TSB, You JB, Maa JS, Fiss EH, Chang CH (1990) Detection and identification of mycobacterium tuberculosis by DNA amplification. J Clin Microbiol 28: 1877–80 Perosio PM, Frank TS (1993) Detection and species identification of mycobacteria in paraffin sections of lung biopsies by the polymerase chain reaction. Am J Clin Pathol 100: 643–7 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, p 7.46. Cold Spring Harbor Laboratory, New York Shinnick T (1987) The 65-Kilodalton Antigen of mycobacterium tuberculosis. J Bacteriol 169: 1080–88 Springer B, Stockman L, Teschner K, Roberts GD, Bottger EC (1996) Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol 34: 296–303 Vago L, Barberis M, Gori A, Scarpellini P, Sala E, Nebuloni M, Bonetto S, Cannone M, Marchetti G, Franzetti F, Costanzi G (1998) Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue with granulomatous diseases for rapid diagnosis of tuberculosis. Am J Clin Pathol 109: 411–415 Werner M, Schulz S, Kremer M, Cabras A, Höfler H (1999) Role of molecular pathology in tissue diagnostic of diseases of the gastrointestinal tract. Leber Magen Darm 29: 18–24 Wiegand P, Domhover J, Brinkmann B (1996) DNA degradation in formalin fixed tissues. Pathologe 17: 451–4 Woods GL (1993) Diagnostic pathology of infectious disease, p 382–93 Lea & Febiger, Philadelphia

Received: July 21, 2000 Accepted: October 20, 2000