ARTICLE
Auteur(s) : Hongsheng Wang, Peiying Jin, Qinxue Wu
Institute of Dermatology, Chinese Academy of Medical Sciences
and Peking, Union Medical College, Jiangwangmiao Road 14#, Nanjing
210042, China
accepté le 18 Janvier 2008
Cutaneous infections with rapidly growing pathogenic
mycobacteria, especially in immunosuppressed individuals, have
become increasingly common [1, 2]. Mycobacterium abscessus is
ubiquitous in water, soil and dust [3], and is commonly associated
with localized cutaneous disease [4]. Infection with M. abscessus
is usually caused by injections of substances contaminated with the
bacterium or through invasive medical procedures employing
contaminated equipment or material. Infection can also be
life-threatening in immunosuppressed patients due to the risk of
dissemination with a potentially fatal outcome [3].
Our case report describes a disseminated cutaneous infection
with Mycobacterium abscessus in a patient with low levels of CD4+ T
cells in the peripheral blood cells and successful treatment
combining antibiotics with immunomodulatory remedies. The infection
with NTM was diagnosed by culture of acid-fast bacilli (AFB), but
accurate mycobacterial species identification was confirmed by
PCR-RFLP analysis and sequencing of the hsp65 and 16SrRNA genes
[5].
Case report
A 20-year-old woman was admitted to our hospital with a
disseminated cutaneous infection. The lesions had started as small
inflammatory papules on her face 2 years previously and gradually
developed tender, red nodules, erythema and erythematous
infiltrated plaques with brown crusts, symmetrically (figure 1). Her disease had
been refractory to conventional antibiotic therapy and the lesions
on her neck, trunk and extremities continued to develop and slowly
progressed in size and quantity. It was variously diagnosed as
seborrheic dermatitis, SLE and cutaneous tuberculosis. Various
topical and oral drug treatments produced no improvement and the
lesions continued to develop. Her general condition was good, and
no active pulmonary disease was detected by chest X-ray.
Routine laboratory tests showed no noteworthy abnormalities. The
results of HIV antibody detection were negative (twice in 3
months). The levels of CMI (cell-mediated immunity) in peripheral
blood cells from the patient were detected by flow cytometer, and
showed that the count of CD4+T cells in the peripheral blood cells
was lower than normal, while other cells counts were normal.
Specific stains (periodic acid – Schiff, Giemsa) for
microorganisms were negative. However, the Ziehl-Neelsen stain was
positive. Tissue cultures showed that colonies without pigment grew
on L-J medium at 37 °C and 32 °C for 3-5 days. Fungal and
other standard bacterial cultures were negative.
A skin biopsy of the lesion on the right cheek showed a nearly
normal epidermis and two marked dermal inflammatory infiltrates in
which lymphocytes and epithelioid cells predominated and no
caseation necrosis formed. Multinucleated giant cells were also
observed, appearing in a tuberculoid pattern (figure 2).
Considering CD4+ T cell counts in the peripheral blood cells
were lower than normal, we treated the patient by combining the
antibiotics (as mentioned above) with thymosin. After 6 months of
therapy, the skin lesions were greatly improved (figures 1A and 1B)
and the CD4+ T cell count in the peripheral blood cells became
normal.
Materials and methods
Identification of mycobacterial isolate by acid-fast staining
and conventional procedures
Samples of skin lesion were inoculated on L-J medium and incubated
at 37 °C. Ziehl-Neelsen staining was used to confirm the
cultured organisms as AFB.
Preparation of template DNA
One loop of bacteria on an L-J medium slope was harvested and
suspended in 2 mL of sterile distilled water. Samples were
then frozen in liquid nitrogen and transferred to boiling water
five times, to release mycobacterial DNA. After centrifugation to
pellet non-soluble debris, the supernatant was used as the DNA
template for PCR. DNA abstracted from a Mycobacterium abscessus
reference strain was also subjected to PCR as a positive control.
Polymerase chain reaction
Two oligonucleotide primers Tb11 (5’-ACCAACGATGGTGTGTCCAT-3’) and
Tb12 (5’-CTTGTCGAACCGCATACCCT-3’) were used to amplify a 439-bp
fragment of the mycobacterial hsp65 gene.6 Another two
oligonucleotide primers (forward, 5’-GAGATACTCGAGTGGCGAAC -3’ and
reverse, 5’-GGCCGGCTACCCGTGGTC-3’) were used to amplify a 208-bp
fragment of the mycobacterial 16S rDNA sequences [6]. PCR was
carried out in a 50 μL reaction volume containing 10 μL
of DNA template, 1.25 U of Taq polymerase (Promega, Madison, WI,
USA), a 10 × reaction buffer as supplied by the manufacturer, 200
μmol L–1 deoxyribonucleoside triphosphates, 2.5 mmol
L–1 MgCl2 and 1 μmol L–1 of
each primer. The thermal profile for the amplification of the
mycobacterial hsp65 gene involved initial denaturation at 94 °C for
5 min, and 45 cycles of 94 °C for 1 min, 60 °C for
1 min and then 72 °C for 1 min, followed by 10 min
of extension at 72 °C. The PCR conditions for the amplification of
the mycobacterial 16S rDNA sequences were PCR mixtures were
incubated for 10 min at 40 °C, 40 cycles of 94 °C for
1.5 min, 65 °C for 2 min, and 72 °C for 3 min, After
the last PCR cycle, the vials were maintained at 72 °C for
10 min. The presence of amplified products was confirmed by
agarose gel electrophoresis.
Restriction fragment length polymorphism analysis and DNA
sequencing
Ten microlitres of the amplification products were then
respectively incubated with BstEII (5 U) and HaeIII (5 U)
endonucleases (MBI Fermentas Inc., Burlington, ON, USA) at 37 °C
for 3 h. The digestion products (10 μL) were analysed by
electrophoresis on 2% Metaphore agarose gel (FMC BioProducts,
Rockland, ME, USA) and stained with ethidium bromide. A photograph
of the restriction patterns was taken; this was then analysed
visually to determine the number of fragments present, and the
sizes of the fragments were compared with the molecular size
standard (100-bp ladder; MBI Fermentas). Restriction fragments
shorter than 60 bp were not taken into account as they were
suspected of being primer or primer dimer bands. The data were
analysed based on those reported by Ena P et al. [7]. The PCR
products of hsp65 and 16S rDNA genes were sequenced and the BLAST
program was used to compare the sequences of the isolated strain
with those of other mycobacterial species in GenBank.
Results
Rapid growth of smooth colonies without pigment was noted after 4
days of incubation at 32 °C and 37 °C. Ziehl-Neelsen staining
confirmed the cultured organisms to be acid-fast bacilli (AFB). The
isolated AFB belonged to Runyon’s group IV. A fragment of 439 bp,
encoding mycobacterial 65-kDa heat shock protein, was amplified in
both the standard strain of M. abscessus and the strain isolated
from the patient. Digestion of the PCR product from M. abscessus
yielded two fragments of 245/220 bp with BstEII, and two
fragments of 160 bp/60 bp with HaeIII. The RFLP pattern
of the isolated AFB was identical with M. abscessus. No bands were
observed in negative controls (figure 3).
Sequencing of the hsp65 gene and 16s rDNA showed respectively
99.70% and 100% similarity with M. abscessus.
Discussion
Rapidly growing mycobacteria (RGM) are ubiquitous in soil and water
and are known as “rapid growers” because of their ability to
produce mature growth on solid media after 3-7 days with an optimal
incubation temperature ranging from 25 °C - 40 °C. RGM
can cause a variety of cutaneous and systemic diseases. The
causative organisms are typically Mycobacterium fortuitum,
Mycobacterium chelonae and M. abscessus [8]. M. abscessus is a
rapidly growing mycobacterium and cutaneous infection is the most
common presentation. The presentation depends on the
immunocompetence of the infected individual with either
disseminated lesions in immunosuppressed patients, particularly
those on oral corticosteroids, or as a localized cutaneous lesions
in immunocompetent patients, where the organism gains entry via a
penetrating wound. The lesions are often nodules or pustules, but
infection can also present as draining abscesses, ulcers,
cellulitis and regional lymphadenopathy [9]. In this case, the
levels of CMI were detected by flow cytometer, and showed that the
CD4+T cell count (25.7 percent of total T cells) in the peripheral
blood cells was lower than normal. Firstly, we considered that the
patient might be infected with HIV, but the results of HIV antibody
detection were negative. In addition, whether the patient suffered
from idiopathic CD4+ T lymphocytopenia or not should also be taken
into account. However, idiopathic CD4+ T lymphocytopenia is defined
as a CD4+ T cell count < 0.3 × 109/L or < 20% of
the total T-cell count on two occasions in the absence of any
immunodeficiency disorder or therapy associated with reduced CD4 +
T cell count [10]. The CD4+ T cell count was 25.7 percent of total
T cells in our patient and as the count was in the normal value
range after the first detection, we could not confirm that the
patient suffered from idiopathic CD4+ T-lymphocytopenia. Because of
the nonspecific appearance of the patient’s skin lesions, it was
difficult to make a definite diagnosis. For the erroneous diagnosis
in another hospital, some inappropriate therapies such as oral
triamcinolone, terbinafine, and topical Chinese herbs had been used
for a long period. So we thought the low level of CD4+T cells at
the first count might have been caused by the misuse of drugs.
Detection and accurate identification of mycobacteria is a
critical step in patient management as this will affect both the
diagnosis and the choice of proper treatment [11]. Culture,
dermatopathology and identification of the organism in suspected
infected tissue should be done. Rapid growth of smooth colonies was
noted after 4 days of incubation at 32 °C and 37 °C in
this case. Histopathological findings showed discrete non-caseating
granulomas, which is usual for the infections with non-tuberculous
mycobacteria. Although a negative Ziehl-Neelsen stain is common, we
got a positive result from the patient’s tissue sample. It is
usually difficult to obtain a definite conclusion by these
procedures, because the results can be significantly influenced by
variations of cultural conditions, and the interpretation may also
be variable due to the technicians’ experience [12, 13].
In recent years, molecular methods such as PCR-based techniques
and genetic sequencing have gradually become useful tools for
mycobacterial differentiation [14]. PCR-RFLP restriction patterns
have the advantage of less methodological variations or
misinterpretations than conventional procedures, so they have the
potential to provide more accurate identification [15]. In our
case, the strain isolated from the skin lesions was a rapidly
growing mycobacterium. Based on the phenotypic, the PCR-RFLP
restriction patterns and sequencing of the hsp65 gene and 16s rDNA
from the isolated organism, we could predict it was M. abscessus.
Because the patient is a beautician and she herself often does
facials, we surmise the source of the M. abscessus infection in
this case might be the skin care products such as facial cleanser,
toner, facial mist and facial scrub, to which the imperceptible
wounds on her face were exposed. M. abscessus is usually sensitive
to amikacin, cefoxitin and clarithromycin [16]. Because of worries
about long term treatment by muscular injection or injection in the
veins and of an adverse reaction of amikacin and cefoxitin, the
patient refused the therapy of amikacin and cefoxitin. In addition,
the patient had a low level of CD4+ T cells in the peripheral blood
and thymosin can lead to an augmentation of T lymphocyte function,
including modulation of interleukin-2, induction of T lymphocyte
and natural killer cells and stimulation of thymopoiesis [17]. We
treated the patient combining rifampin, isoniazid, ofloxacin, and
clarithromycin with thymosin. After 6 months of therapy, the skin
lesions were greatly improved and the CD4+ T cell count in the
peripheral blood cells became normal. Surgical excision or drainage
may be appropriate for localized infections. The surgical option
was not chosen in our patient, due to the disseminated cutaneous
lesions.
Acknowledgements
This work was supported by grant BK 2006015 from Natural Funds of
Jiangsu province of PR. China. Conflict of interest: none.
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