Mycobacterium malmoense lymphadenitis in Spain: first two cases in immunocompetent patients
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- López-Calleja, A.I., Lezcano, M.A., Samper, S. et al. Eur J Clin Microbiol Infect Dis (2004) 23: 567. doi:10.1007/s10096-004-1142-4
Reported here are two cases of Mycobacterium malmoense lymphadenitis that occurred in two immunocompetent children in Spain. To the best of our knowledge, these are the first documented cases of extrapulmonary infection by M. malmoense in Spain. This report serves to draw attention to this emerging nontuberculous mycobacterium that is gaining increasing recognition as a pulmonary and extrapulmonary pathogen in different countries.
Mycobacterium malmoense is a slow-growing, non-photochromogenic mycobacterium that was first described by Schröder and Juhlin in 1977 . During recent years, it has been recognised with increasing frequency as a nontuberculous pathogen in countries of northern and north-western Europe [2, 3], and recently it has also been isolated from clinical samples in other areas [4, 5]. M. malmoense can cause pulmonary and extrapulmonary disease and disseminated infection. The most common extrapulmonary infection due to M. malmoense is cervical adenitis, which has been found to occur almost exclusively in children . In Spain, no cases of lymphadenitis due to M. malmoense have been documented. To the best of our knowledge, we report here the first two cases of cervical lymphadenitis due to M. malmoense occurring in two immunocompetent children in Spain.
Patient one was an 8-year-old boy with a history of epileptic seizures, who had been asymptomatic and without treatment for 3 years. He was referred for evaluation of an enlarged (2×2 cm), painless, superficial lymph node in the left cervical area, with no other local signs of inflammation. Physical and laboratory findings were unremarkable. The Mantoux test with 2 tuberculin units was negative. A fine-needle aspiration biopsy was performed to establish the diagnosis. The involved lymph node was surgically removed, and histological examination revealed a granulomatous inflammation with caseating necrosis. The child was discharged from hospital after 1 week and subsequently followed up in a paediatric practice. No antituberculosis treatment was given, and the wound healed leaving no fistula or extended scars.
Patient two was a 2-year-old previously healthy girl who was referred for evaluation of an enlarged (2×2 cm) lymph node in the right cervical area. She had been treated for 10 days with amoxicillin-clavulanate by her paediatrician without resolution of the lump. Physical examination was normal. The Mantoux test (2 tuberculin units) resulted in an 11-mm induration. Fine-needle aspiration was performed, and histological examination revealed necrotizing lymphadenitis, without granulomatous inflammation.
On clinical suspicion of tuberculous lymphadenitis, triple-drug therapy with isoniazid (75 mg/d), rifampicin (130 mg/d) and pyrazinamide (200 mg/d) was initiated. After the first week of treatment, the node drained spontaneously and a fistula developed. Excision was not possible due to the difficult location of the node. In addition, surgical resection was not recommended because of the spontaneous drainage. When microbiological results were available 2 months later, the antituberculous treatment was changed to rifabutin (75 mg/d), ethambutol (200 mg/d) and clarithromycin (200 mg/d). However, ethambutol was suspended after 1 month because of the risk of ocular toxicity and the lack of patient co-operation at ophthalmologic examinations. A double-drug regimen with rifabutin and clarithromycin was continued for 3 months, amounting to a 6-month total course of treatment. The wound swelled for a long time, and a bacterial infection occurred 5 months after the presentation of the node, which was topically treated. In this case the development of a fistula, scarring, and bacterial infection of the wound prolonged the patient’s recovery period. The patient is still under paediatric supervision, but no recurrence of the mycobacterial infection has been detected, and the evolution of the wound has been satisfactory.
Specimens were decontaminated with N-acetyl-L-cysteine/NaOH and seeded onto Löwenstein–Jensen slants and into a liquid system (BacT/Alert MP; bioMérieux, Durham, NC, USA). Acid-fast bacilli were detected by auramine and Ziehl–Neelsen staining. Subcultures on Löwenstein–Jensen and Coletsos (a pyruvate-containing medium) slants were inoculated from positive cultures onto liquid media. All cultures were incubated at 37°C.
Species identification was achieved using polymerase chain reaction and restriction enzyme pattern analysis (PRA). The PRA technique includes the amplification of a 439 bp fragment of the heat shock protein gene (hsp65) and cleavage with HaeIII and BstEII using the methodology described previously . Direct sequence determination of the hypervariable region within the 16S rRNA gene in which mycobacterial species are characterised by species-specific nucleotide sequences was also performed. The amplification products of the hypervariable region were sequenced, as described by Kirschner et al. . The sequences obtained were compared using the nucleotide–nucleotide BLAST (blastn) software from the National Center for Biotechnology Information (NCBI) (Bethesda, Maryland, USA) against the GenBank database, an annotated collection of all publicly available nucleotide and amino acid sequences, provided by the NCBI.
Microscopic examination of the fine-needle aspiration biopsy specimens yielded few acid-fast bacilli in both cases. Cultures in liquid medium grew mycobacteria after 72 days in the first case and 36 days in the second. Primary cultures on Löwenstein–Jensen were observed for 75 days, and in both cases they remained negative. The excised lymph node obtained from patient 1 showed neither acid-fast bacilli nor mycobacterial growth.
Organisms isolated from culture were confirmed as slow-growing mycobacteria that were non-photochromogenic and did not hybridise with Mycobacterium tuberculosis or Mycobacterium avium-intracellulare probes (Accu-Probe; Gen-Probe, San Diego, CA, USA) Subcultures from positive liquid media grew extremely slowly, but better results were seen in Coletsos medium than in Löwenstein–Jensen medium.
In both cases M. malmoense was identified by PRA and on analysis of the 16S rRNA gene. The size fragments (bp) obtained with BstEII restriction were 240/120/100 for the first isolate and 240/210 for the second. The size fragments (bp) obtained with HaeIII were 145/105/80 for both isolates. Both patterns are described on the website http://www.hospvd.ch/prasite (provided by the Swiss National Center for Mycobacteria, Les Hospices Cantonaux, Lausanne, Switzerland and Institute Pasteur, Paris, France) as belonging to M. malmoense type I and type II, respectively . Analysis of the hypervariable region within the 16S rRNA gene revealed 100% homology of both sequences to that of M. malmoense isolate 29571A (accession number AY215281) of the NCBI nucleotide databases.
Unilateral cervical lymphadenitis is the most frequent extrapulmonary infection due to M. malmoense, and it predominantly affects children . In Spain, no case of lymphadenitis due to M. malmoense has been documented previously. The four infections due to this microorganism that have been reported from Spain were all pulmonary infections; three occurring in HIV-infected patients [9–11] and one in a 66-year-old woman .
Cervical lymphadenitis is eradicated most reliably by surgical excision of infected lymph nodes [6, 13, 14]. Chemotherapy is required if the infected lymph node is drained but not totally excised to avoid relapses caused by the mycobacteria left at the site of infection . Occasionally, the affected lymph node is not well-suited for surgical resection because of potential damage to branches of the facial nerve ; this was the case with the second patient reported here, for whom excision was not recommended.
The conventional methods developed to recover Mycobacterium tuberculosis are adequate for the growth and identification of most nontuberculous mycobacterial species; however, to ensure a sensitive primary isolation of fastidious pathogens such as M. malmoense, it is crucial to carefully choose culture media and conditions . In the two cases reported here, we were unable to recover M. malmoense on primary Löwenstein–Jensen slants; positive cultures were only detected with BacT/Alert MP liquid media, and only after a long incubation period in the first case (possibly due to a low inoculum of mycobacteria). Hoffner et al.  previously demonstrated that broth culture is significantly more sensitive and rapid for the detection of M. malmoense in tissue samples than solid culture on egg medium. Currently, it is generally accepted that the use of a liquid medium in combination with at least one solid medium is essential for good laboratory practice in the isolation of mycobacteria . Furthermore, use of pyruvate-or glycerol-containing media of pH 6–6.5 and incubation for at least 7 weeks at both 30 and 37°C could improve the detection of M. malmoense on solid cultures . In our case, cultures were only incubated at 37°C, which could be a reason for the absence of growth on solid cultures.
For a long time, reports of the recovery of M. malmoense from clinical specimens have been restricted to a handful of northern European countries [2, 3], but new cases have been reported increasingly from other countries like Italy , France , Germany , Canada  and the USA . Reports of new clinical isolates from different countries and isolation of the organism from the environment in places like Zaire, Finland and Louisiana  indicate that M. malmoense is not exclusively limited to specific zones. Probably, like most of the nontuberculous mycobacteria, M. malmoense is present in many geographic areas and may colonise or cause infections in humans and animals. At present, there are no clues about the origin or route of infection by M. malmoense in humans  and reasons for the continuous increase in incidence are still uncertain [5, 19].
With this report of two cases of M. malmoense lymphadenitis in immunocompetent children in Spain, we want to draw the attention of microbiologists and clinicians to this emerging pathogen that should be added to the list of nontuberculous mycobacteria responsible for disease in immunocompetent patients. M. malmoense has fastidious growth requirements and is difficult to isolate and identify. Prolonged culture in liquid medium followed by DNA-based analysis will improve the recovery and identification of this microorganism, and direct detection from clinical specimens by PCR will shorten the time to diagnosis. Improved detection methods that will avoid misdiagnosis of the organism could help establish a true estimation of the prevalence of M. malmoense in humans.
The authors thank the Mycobacteria Genetic Group of the University of Zaragoza for the molecular identification of the strains. The molecular diagnosis was supported by FIS grant 01/3088.