NEW MICROBES IN HUMANSCervicofacial lymphadenitis due to
Mycobacterium mantenii: rapid and
reliable identification by
MALDI-TOF MST. Nebreda Mayoral1, A. G. Andrés Andrés2,
S. Fuentes Carretero3, R. Calleja Fernández1 and
M. S. Jiménez Pajares4
1) Servicio de Microbiología del Complejo Asistencial Universitario de
León, 2) Servicio de Pediatría del Complejo Asistencial Universitario de
León, 3) Servicio de Cirugía Pediátrica del Complejo Asistencial universitario
de León, León and 4) Centro Nacional de Microbiología, Instituto Carlos III,
Majadahonda, Madrid, Spain
Keywords: Cervicofacial lymphadenitis, malditof mass
spectrotrometry, Mycobacterium mantenii
Original Submission: 11 November 2017; Revised Submis-
sion: 29 November 2017; Accepted: 7 December 2017
Article published online: 20 December 2017Corresponding author: T. Nebreda Mayoral, Servicio de
Microbiología, Complejo Asistencial Universitario de León, Avenida
Altos de Nava s/n, León, Spain
E-mail: tnebreda@saludcastillayleon.esMycobacterium mantenii is a scotochromogenic non-
tuberculous mycobacterium (NTM). Van Ingen et al. [1]
described this bacterium for the first time in 2009 from the
cervical lymph node samples from two immunocompetent
children, respiratory samples from two adults and a water
sample from Zambia. Since then, only two cases of dissemi-
nated M. mantenii infection have been described in immuno-
compromised patients [2,3]. This mycobacterium has also been
isolated in environmental samples from Ghana [4] and the
Czech Republic [5].
In this report, two new cases involving immunocompetent
girls with cervicofacial lymphadenitis due to M. mantenii are
presented, and the reliability of matrix assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS)
for identifying M. mantenii is evaluated.This is an open access artiPatients and methodsCase 1
A 5-year-old girl referred to University Hospital of León
(CAULE) for preauricular adenitis with 1 month of evolution
had previously been treated unsatisfactorily with amoxicillin–
clavulanic acid. The patient had no relevant history. On physical
examination, right preauricular adenopathy was observed. A
tuberculin skin test and a QuantiFERON test were both
negative. Ultrasonography showed a 2-cm adenopathy with
preserved fatty hilum. The patient was otherwise asymptom-
atic. Curettage of the adenopathy yielded abundant necrotic
material for microbiological and histological examination. Ana-
tomopathological assessment revealed necrotizing granuloma-
tous inflammation. Empirical treatment with ciprofloxacin and
clarithromycin was initiated; ciprofloxacin was discontinued
when the microbiology results had been obtained. Three
months of treatment with clarithromycin was completed,
resulting in partial response. At 6 months follow up, the lesion
had decreased slightly in size, but there remained a preauricular
adenopathy with a diameter of approximately 1 cm, a gummy
consistency and overlying erythematous, violaceous skin
(Fig. 1).
Case 2
A 3-year-old girl was referred to CAULE for left submandibular
adenitis with 1 month of evolution, associated catarrh at onset
and fever on the first day. She received amoxicillin–clavulanic
acid treatment for 15 days without improvement. In a physical
examination, a conglomerate of mobile left submandibular
adenopathies and laterocervical submandibular micro-
adenopathies were detected. A tuberculin skin test was 13 mm
and a QuantiFERON test was negative. A chest X-ray produced
normal findings. In an ultrasound examination, multiple altered
lymph nodes were observed in both laterocervical chains. Fine-
needle aspiration yielded abundant purulent liquid that was sent
for microbiological study. Based on a suspicion of NTM adenitis,
empiric treatment with azithromycin and ciprofloxacin was
initiated, but ciprofloxacin was suspended when the microbi-
ology results were obtained. The evolution of the patient’s
condition was suggestive of spontaneous scrofula. Antibiotic
treatment was continued for 4 months. Initially, the post-
scrofula scar exhibited a thickened, irregular and erythema-
tous appearance with progressive improvement at 9 months of
follow up.New Microbe and New Infect 2018; 22: 1–3
© 2017 The Author(s). Published by Elsevier Ltd
cle under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
https://doi.org/10.1016/j.nmni.2017.12.003
FIG. 1.
2 New Microbes and New Infections, Volume 22 Number C, March 2018 NMNIFor both patients, basic analytic results were normal and
there were negative serological findings for cytomegalovirus,
Epstein–Barr virus, Toxoplasma, human immunodeficiency virus
and Bartonella.
The microbiological study of both samples included PCR for
Mycobacterium tuberculosis complex (Xpert1 MTB/Rif PCR,
Cepheid AB, Solna, Sweden), auramine staining and culturing in
mycobacteria growth indicator tube (MGIT, Becton-Dickinson
and Company, Sparks, USA) medium (Becton-Dickinson, Ox-
ford, United Kingdom) and Löwenstein– Jensen medium (Bec-
ton Dickinson). In both cases, PCR results were negative.
Auramine staining was positive for the first patient and negative
for the second patient. MGIT cultures for the first and second
patients were positive at 12 and 30 days of incubation,
respectively. Strains were subcultured in Löwenstein– Jensen
medium. The strains were yellow (scotochromogenic). Identi-
fication via MALDI-TOF MS (Bruker Daltonics Inc., Bremen,
Germany) indicated that these strains were M. mantenii, with
scores 
2.08. Both strains were sent to the National Centre
for Mycobacteria (Instituto Carlos III, Majadahonda, Madrid) to
confirm their identification using phenotypic and genotypic
approaches and to perform an antibiotic sensitivity study. PCR-
restriction fragment length polymorphism of the hsp65 gene
with the restriction enzyme BstEII resulted in two bands (235
and 210 bp), and digestion with the enzyme HaeIII yielded three© 2017 The Author(s). Published by Elsevier Ltd, NMNI, 22, 1–3
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licebands (130, 105 and 60 bp). Because the chromogenic strain did
not match the Mycobacterium avium pattern, a fragment of the
1470 bp 16S rRNA gene was sequenced. The obtained se-
quences were compared with those of the GenBank database,
which indicated 100% agreement with M. mantenii
NLA000800224 (FJ232521) sequences. The profiles of the two
strains of M. mantenii obtained by random amplified poly-
morphic DNA–restriction fragment length polymorphism with
IS986 and enterobacterial repetitive intergenic consensus PCR
showed different patterns.
Drug susceptibility on MGIT 960 was made following the
manufacturer’s recommendations. The strain from the first
patient was sensitive to cycloserine (minimum inhibitory con-
centrations (MIC) <20 μg/ml) and clarithromycin (MIC <2 mg/
L), and the strain from the second patient was only sensitive to
clarithromycin (MIC < 2 mg/L).DiscussionTo our knowledge, the aforementioned cases are the second
group of published cases involving lymphadenitis caused by
M. mantenii. Both cases fit the typical description of this disease
in that they involved 1- to 5-year-old immunocompetent girls
with unilateral lymphadenopathy and preauricular or subman-
dibular localization. Cervical lymphadenitis is the most common
NTM infection in immunocompetent children. The incidence of
this condition is low, which contributes to delays in diagnosis;
diagnosis typically follows several therapeutic failures with
systemic antibiotics [6–9], as occurred in the cases described
here. The majority of cases are caused by M. avium complex
[6–9], although the spectrum of disease-causing NTM species is
broad [10–13]. NTMs are ubiquitous, but a direct link between
their environmental prevalence and their involvement in
mycobacterial lymphadenitis has not been established [8].
The optimal treatment of NTM lymphadenitis is controversial.
The gold standard is the complete removal of the affected nodes,
although complications and recurrence can occur [14–16].
Certain authors believe that in the absence of large, randomized
studies, treatment should be individualized [17]. Our patients
progressed satisfactorily after curettage or spontaneous drainage
combined with treatment with an antimycobacterial agent.
New geneticmethods have allowed for the identification of new
NTM species [10], some of which are closely related both
phenotypically and phylogenetically.Mycobacteriummantenii shares
99% identity for the 16S rRNA gene with Mycobacterium scrof-
ulaceum, Mycobacterium seoulense and Mycobacterium saskatch-
ewanense [1]. Moreover, it is misidentified by the INNO-LiPA
Mycobacteria test and the GenoType Mycobacterium CM assay as
a member of theM. avium–M. intracellulare–M. scrofulaceum groupnses/by-nc-nd/4.0/).
NMNI Nebreda Mayoral et al. Mycobacterium mantenii cervicofacial lymphadenitis 3and as M. intracellulare, respectively. This similarity may have
contributed to the underdiagnosis of M. mantenii infections and
the overestimation of M. scrofulaceum and M. avium complex in
cervical lymphadenitis [18].
There was 100% agreement between MALDI-TOF MS and
16S rRNA sequencing results with respect to the identification
of the two strains of M. mantenii in this study. Hence, MALDI-
TOF MS is a reliable, rapid and cost-effective diagnostic tool for
the identification of M. mantenii and may therefore be useful for
providing guidance regarding the antibiotic treatment of choice
for this NTM, which is resistant to most antibiotics.Conflict of interestThe authors declare no conflicts of interest.References[1] Van Ingen J, Lindeboom JA, Hartwig NG, de Zwaan R, Tortoli E,
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