skin and soft tissue infections due to nontuberculous mycobacteria · 2019-06-17 · skin, soft...

SKIN, SOFT TISSUE, BONE AND JOINT INFECTIONS (N SAFDAR AND A POP-VICAS, SECTION EDITORS)

Skin and Soft Tissue Infections Due to Nontuberculous Mycobacteria

Elizabeth Ann Misch1& Christopher Saddler2 & James Muse Davis3

# Springer Science+Business Media, LLC, part of Springer Nature 2018

AbstractPurpose of Review This review describes recent trends in the epidemiology of nontuberculous mycobacteria (NTM), emergingpathogens, new insights into NTM pathogenesis, and advances in diagnosis and treatment.Recent Findings Emerging pathogens include Mycobacterium chimaera and drug-resistant subspecies of Mycobacteriumabscessus. Important virulence mechanisms of pathogenic NTM include the ability to alter the macrophage’s permis-siveness to intracellular bacterial growth. New diagnostic tools consist of DNA probes, gene sequencing, and matrix-assisted laser desorption ionization-time of flight. These methods allow rapid speciation of NTM species, in some casesdirectly from patient samples. There are few novel agents available to treat NTM, although some repurposed drugs showexcellent activity.Summary The incidence of NTM infections appears to be increasing in a number of regions around the world. Molecularmethods are now the diagnostic tools of choice. Discovery of novel effective agents and/or drug combinations with greaterlikelihood of cure, shorter treatment duration, and fewer side effects are research priorities.

Keywords Nontuberculousmycobacteria .M.chimaera .Skinandsoft tissue .Rapidlygrowingmycobacteria (RGM) .Moleculardiagnostics

Introduction and Background

Nontuberculous mycobacteria (NTM) encompass allmycobacteria species other than those in the Mycobacterium

tuberculosis complex1 and Mycobacterium leprae, the agentof leprosy. More than 190 species have been identified(http://www.bacterio.net/mycobacterium.html) [1], ofwhich approximately 40 species are considered patho-genic [2]. Mycobacteria are ubiquitous in the naturaland constructed environment. They can be found in allregions of the world in soil, natural or treated water(including tap and shower water), and in associationwith plants, birds, fish, and other animals [3•, 4, 5, 6].A few species, including Mycobacterium haemophilumand Mycobacterium ulcerans, are rarely isolated fromthe environment [7], but this fact may reflect specialgrowth requirements (M. haemophilum), or the extended in-cubation periods (M. ulcerans) needed for successful culture.

In humans, NTM are facultative intracellular pathogens.They may be present as colonizers or pathogens in the respi-ratory tract. Asymptomatic infection is probably far more

1 M. tuberculosis complex members: M. africanum, M. bovis, M. cannetti,M. caprae, M. microti, M. pinnepedii, andM. tuberculosis.

This article is part of the Topical Collection on Skin, Soft Tissue, Bone andJoint Infections

* Elizabeth Ann [email protected]

1 Division of Allergy and Infectious Disease, Department ofMedicine,School of Medicine and Public Health, University of Wisconsin,Medical Foundation Centennial Building, 1685 Highland Avenue,5th floor, Madison, WI 53705-2281, USA

2 Division of Allergy and Infectious Disease, Department ofMedicine,School of Medicine and Public Health, University of Wisconsin,Madison, WI, USA

3 Division of Infectious Disease, Department of Pediatrics, School ofMedicine and Public Health, University of Wisconsin, Madison, WI,USA

Current Infectious Disease Reports (2018) 20:6 https://doi.org/10.1007/s11908-018-0611-3

frequent than disease. Antibodies against lipoarabinomannan,a glycolipid abundant in the cell wall of mycobacteria, aredetected in increasing numbers of healthy children after theage of 1 [8]. In the USA, skin test reactivity toMycobacteriumavium sensitin has been reported in 16% of adults who other-wise would have been misclassified as having latent tubercu-losis infection (due to cross-reactivity of the immune responseto environmental mycobacteria and purified protein derivative(PPD)) [9]. In one survey, 33% of adults showed evidence ofprior, presumably subclinical, infection with M. avium [10].

In adults, NTM are most frequently associated with lung dis-ease. In children, lymphadenitis is the most common diseasemanifestation and comprises 75 to 85%percent of total infections[11, 12]. Other disease presentations include skin and soft tissueinfection, tenosynovitis, septic arthritis, osteomyelitis, or keratitis[3•, 13, 14]. Disseminated disease involving the blood, centralnervous system, or other dispersed sites can occur, particularlyformore virulent species, such asM. avium andM. abscessus [5].

Immune-compromised hosts, including patients with ad-vanced HIV infection, organ or stem cell transplantation, andindividuals with Mendelian susceptibility to mycobacterialdisease (MSMD), are also at greater risk of disseminated dis-ease due to NTM [5, 15]. The latter individuals carry autoso-mal or X-linkedmutations in genes within the interferon-gam-ma/IL-12 immune response pathway [15]. Several naturallyoccurring polymorphism in TLR1 and TLR2, invariant recep-tors on the surface of immune cells that recognize bacteriallipopeptides, have also been associated with altered suscepti-bility to M. tuberculosis or M. leprae in human populations[16–20]. These data suggest that there are likely other com-mon variants in immune response genes that modulate humansusceptibility to clinically important NTMs via complex in-heritance (non-Mendelian) patterns.

Skin and soft tissue infections are the most common pre-sentation for the rapid-growing species Mycobacteriumfortuitum, M. abscessus, and M. chelonae [3•]. Certain slow-growing species of mycobacteria, namely Mycobacteriummarinum, M. ulcerans, M. chimaera, and M. haemophilum,are also more frequently associated with skin disease [3•,21–23]. However, virtually any species can localize to the skinor soft tissues. Clinically, lesions may appear as papules, pus-tules, nodules, abscesses, panniculitis, folliculitis, or plaques.They are typically erythematous or violaceous, but not warm.Lesions may later become ulcerated [7]. Infection may presentwith a “sporotrichoid” pattern of spread, tracking along sub-cutaneous lymphatics from the site of inoculation proximally.This pattern is particularly classical forM.marinum cutaneousdisease [21, 24]. Skin and soft tissue infection (SSTI) mayfollow minor trauma and inadvertent inoculation, such as oc-curs when the skin is punctured by wood splinters, fish spines,or needles [21]. Infections can also arise through accidentalcontamination of surgical or open wounds [24–26]. Finally,skin involvement may result from hematogenous

dissemination in immune-compromised hosts [27]. There islittle evidence of person-to-person transmission of NTM, withthe possible exception of M. abscessus, which has been im-plicated in outbreaks of clonally related strains in cystic fibro-sis patients at two different centers [3•, 28, 29].

Mycobacteria elaborate a lipid-rich, waxy cell wall thatfunctions as a hydrophobic biofilm and allows organisms toadhere to hard surfaces, including pipes, drains, and tubing.Biofilms inside pipes resist being physically dislodged despitehigh flow rates. Because of their biofilm-forming ability, en-vironmental mycobacteria are highly resistant to decontami-nation with standard antiseptics and biocides, including chlor-hexidine and glutaraldehyde [30–34]. As a result, mycobacte-rial species are frequently found even in hospital environ-ments and have been associated with outbreaks of nosocomialinfections following surgery or cosmetic procedures [35, 36,37•, 38, 39, 40]. Clustered cases have also been reported fromnon-hospital settings, including tattoo parlors (M. chelonae,M. fortuitum); fish markets (M. marinum); and nail salons(M. chelonae, M. fortuitum, M. abscessus) [41–46].

In contrast toM. tuberculosis, treatment regimens for NTMinfections have not been extensively studied in clinical trials.For most species, there are no standard regimens and ourunderstanding of the role of antibiotic susceptibility testingis incomplete [47]. Treatment decisions are thus largely basedon case studies and expert opinion published in guidelines [3•,48–52]. Some emerging pathogens, such asM. abscessus, arehighly drug-resistant and cannot be treated with typicalagents, which include macrolides, fluoroquinolones, amino-glycosides, cefoxitin, imipenem, sulfamethoxazole, and tetra-cyclines (other than tigecycline) [53]. Table 1 summarizes theclinical presentation, notable features, and treatment of select-ed NTMs that cause SSTIs.

Recent Epidemiology

An accurate estimate of the burden of NTM disease in thegeneral population is limited by the fact that infections arenot reportable in most countries, including the USA [3•, 51].Available evidence suggests, however, that NTM pulmonarydisease is increasing in North America, much of Europe, NewZealand, Australia, and tertiary centers in East Asia [81•, 82].Data fromChina, India, and Africa are far from complete [81•,83, 84]. In the USA, the annual rate of pulmonary disease iscurrently 5–10/100,000 [81•]. Several US studies have shownincreases in the frequency of NTM infection [85•, 86, 87•].

In the past, 75 to 80% of reported NTM disease representedpulmonary infection [3•]. It is unclear whether this distributionis stable or changing. One single center series from a universityhospital in France, for example, reported that pulmonary infec-tions made up only 54% of 170 total NTM infections between2003 and 2010. Skin infections comprised 23% of the total

6 of 17 Curr Infect Dis Rep (2018) 20:6

Table1

Selected

nontuberculous

Mycobacteriaassociated

with

skin

andsofttissueinfections

Species

Clin

icalpresentatio

nDistin

guishing

features

Treatmentb

Rapidly

grow

ing

Mycobacteria

(RGM)

•Variablelesion

appearance:tender,

erythematousnodules,papules,pustules,

plaques,or

abscesses

•Frequently

reported

ascomplicationof

surgery,cosm

eticprocedures,tattooing,

pedicures

•Disseminated

diseasein

immune-

comprom

ised

hostscanoccur

•Optim

alincubatio

n:28–30°C

[3•,54]

•Depends

onspecies

M.a

bscessus

Subspecies

abscessus

Subspecies

massiliense

Subspecies

bolletii

(rare)

•Sk

ininfections

less

frequent

than

pulm

onary

disease,buto

ccur

in(non-H

IV)im

mune-

comprom

ised

hosts

•Alsoreported

innorm

alhostsin

health

care

settingsandaftercosm

eticprocedures

orenvironm

entalexposure[25]

•Presence

oftheerm(41)

gene

inM.a

bscessus

subspp.

abscessusandbolletii

confersmacrolideresistance

•Not

standardized;atleasttwoagents,often

for

4–6months.Longerdurationfordissem

inated

disease

•Macrolide-basedtherapyifsusceptible

•In

vitroactivity

:macrolid

es(clarithromycin,

azith

romycin)(iferm(41)-negative),amikacin,

cefoxitin

,imipenem

,tigecycline(a

gylcylcycline)

M.chelonae

•Sk

in,bone,softtissue,cornealo

rdissem

inated

infection

•Contactlens

keratitis

•Normalor

in(non-H

IV)im

mune-comprom

ised

hosts

•Reportedaftercosmetic,ocularand

othersurgical

procedures

ortattoos

•So

mestrainsgrow

between28

and33

°C[3•,54]

•Lacks

erm(41)

gene

•Not

standardized;o

ften

twoagentsfor4–6months.

Longerduratio

nfordissem

inated

disease.

•In

vitroactivity

:macrolides,tetracyclines

(doxycyclin

eor

minocycline),linezolid,fluoroquinolones

(ciprofloxacin

ormoxifloxacin),imipenem

,tobramycin

•Asingleagent(fluoroquinoloneor

clarithromycin)may

beused

asforless

severe

disease,although

resistance

toclarith

romycin

hasbeen

reported

whenused

asmonotherapy

[55]

M.fortuitu

m•Localinfectionaftertraumaor

inadvertent

inoculationduring

surgery,tattooing,or

pedicure

•Containserm(39)

gene,w

hich

encodesinducible

macrolid

eresistance

[56]

•Manyoptions;atleasttwoagents4–6months

•Often

susceptib

leto

quinolones,tetracyclines,

sulfonam

ides,cefoxitin,im

ipenem

,amikacin

•Evenwith

documentedin

vitrosusceptib

ility,m

acrolides

should

beused

with

cautiondueto

possibleinducible

resistance

[57–59]

•In

vitrosusceptibility

predictsclinicalresponse

[3•]

Slow

lygrow

ing

Mycobacteria

•Variablelesion

appearance:tender,erythematous

nodules,papules,pustules,plaques,or

abscesses

•OnlyM.chimaera

reported

innosocomial

outbreaks

•Disseminated

diseasein

immune-comprom

ised

hostscanoccur

•Fo

rmostS

GM

species(exceptio

nsnotedbelow),

grow

thoccursat35–37°C

[3•,54]

•Depends

onspecies

M.chimaera

•Globalo

utbreaklin

kedto

contam

inated

heater-coolerunits

used

incardiacsurgerya

•Presentatio

ncanbe

delayedby

monthsto

years

•Su

rgicalsiteinfection,sometim

eswith

dissem

inationandendocarditis[60]

•Will

grow

onstandard

mycobacterialcultu

remedia

at37

°C•Not

standardized;Ifclarith

romycin-susceptible,m

ayuse

regimen

ofclarithromycin,etham

butoland

rifampin

with

orwith

outanam

inoglycoside,asforMAC[60]

Curr Infect Dis Rep (2018) 20:6 of 17 6

Tab

le1

(contin

ued)

Species

Clin

icalpresentatio

nDistin

guishing

features

Treatmentb

M.h

aemophiliu

m•Sk

inandsofttissueinfection,lymphadenitis,

occasionally

dissem

inated

disease[61–63]

•HIV

ornon-HIV

immunecomprom

ise[62–64]

•Optim

algrow

th:2

8–32

°C•Culturegrow

threquiresmediasupplementationwith

iron

(hem

in,hem

oglobin,or

ferricam

monium

citrate)[62]

•Nostandardized

susceptibility

methods

•In

vitroactivity

:amikacin,m

acrolid

es,quinolones,rifampin/rifabutin

,sometim

esdoxycyclineandsulfonam

ides

•Uniform

lyresistanttoethambutol

•Treatmentd

urations

usually

>6months[3•,62]

M.m

arinum

•Sk

inandsofttissueinfection,tenosynovitis,

synovitis;o

ccasionally

dissem

inates

tobone

•“Fishtank”or

“swimmingpool”granulom

awith

asporotrichoidpattern

ofnodules[21,24]

•Outbreaks

reportedinfishmarketw

orkers[44]

•Optim

algrow

th:2

8–30

°C[3•,54]

•M.m

arinum

infectioncanproducefalse-positive

results

oninterferon-gam

mareleaseassays

used

todetectlatenttuberculosisinfection(QuantiFERON®-TB

Goldor

T-SP

OT®.TBtest)

•Nostandardized

regimens

•Activeagents:clarithromycin,tetracyclines,rifam

pin,

ethambutol,sulfonam

ides,amikacin

[65–67]

•Recom

mendedtreatm

ent:1–2agentsfor1–2months

beyond

clinicalresolutio

n[3•]

M.u

lcerans

•Initially,a

nontendernodule,plaqueor

edem

a•Su

bsequently,the

originalarea

ulceratesand

form

sdisfiguringscar

[68–70]

•Seen

predom

inantly

inchild

ren

•So

meform

smay

have

anassociationwith

HIV,

butthisiscontroversial(seereferences

citedin

[71])

•PC

Rof

IS2404

from

directclinicalspecimens(punch

biopsy

fornonulcerated

lesions;sw

absforulcerated

lesions)isdiagnosticmethodology

ofchoice

[70,72–75]

•Diagnosisin

resource-lim

itedsettings:aloop-m

ediated

isotherm

alam

plificationmethodthatdoes

notrequirea

cold

chainhasbeen

reported

[76];a

dryreagent-based

qPCRmethodcanbe

used

byunskilled

staff[77]

•Thinlayerchromatographyto

detectmycolactone

may

beafuture

fielddiagnostictechnique[78]

•Laboratorycultivation:9–12

weeks

onLow

enstein-Jensen

mediaincubatedattemperaturesof

28–32°C

[70]

•Culturesused

forepidem

iologicalinvestigation,relapses,

detectionof

drug

resistance

[70]

•WHO:(1)

rifampicin(10mg/kg

once

daily

)and

streptom

ycin

(15mg/kg

once

daily

)or

(2)rifampicin

(10mg/kg

once

daily

)andclarithromycin

(7.5

mg/kg

twicedaily

)for8weeks

c

•Australia:rifam

picin(10mg/kg

once

daily

)and

moxifloxacin(400

mgonce

daily)for8weeks

c[79]

•Sh

orterregimensmay

bepossible:rifam

pin/rifampicinwith

ciprofloxacinor

rifampin/rifampicinwith

clarith

romycin

for29

days

[80]

IS2404,insertio

nsequence

2404

MACMycobacterium

aviumcomplex,P

CRpolymerasechainreactio

n,qP

CRquantitativepolymerasechainreactio

n,WHOWorld

Health

Organization

aSeetext

fordiscussion

andadditio

nalreferences

bTreatmentregim

ensforlung

ordissem

inated

infectionaregenerally

longer,relyon

aninitialphaseof

intravenoustherapy,andmay

involvethreeor

moreagents

chttp://www.who.int/m

ediacentre/factsheets/fs199/en.


[88]. In a Taiwanese university hospital, the annual incidence(per 100,000 patients) of overall NTM disease rose from 8.6 in1997 to 16.55 in 2003 [14]. The rate of skin and soft tissueinfection also rose, from 1.67 to 6.72 per 100,000 patients overthe same period. Approximately 14% of these patients hadSSTI or osteomyelitis. M. marinum was isolated in 28%,M. abscessus in 26%, andM. kansasii in 12% of those individ-uals [14]. In a report from Taiwan of 1105 NTM patients, asharp rise in the rate of pulmonary disease was observed be-tween 2000 and 2008. Extra pulmonary disease also increasedover this time period [89•]. Over the 9-year study period, 11.4%of patients had a skin and soft tissue infection. M. abscessuswas the most frequent cause of SSTI (37.7% of cases) [89•].

In the USA, a recent study of NTMs isolated in inpatientand outpatient laboratories in three counties in North Carolinafound that 0.2 isolates per 100,000 persons (15 in total) werefrom dermal sites. The most frequent strains from dermal siteswere M. abscessus (10/15), M. marinum (1/15) ,M. immunogenum (1/15), and M. avium (1/15) [86]. A studyin the state of Oregon, where reporting of NTM infectionbecame mandatory in 2005–2006, found that skin and softtissues represented the second most common site of NTMdisease, with a reported incidence of 0.9 cases per 100,000persons [85•]. M. marinum, followed by M. abscessus andM. chelonae, were the most often isolated [85•]. A retrospec-tive series of 40 patients who presented with cutaneous NTMdisease to a tertiary center in Minnesota between 1980 and2009 found that the incidence of skin infection had increasedfrom 0.7 per 100,000 person-years in the period 1980–1999 to2.0 per 100,000 person-years in the period 2000–2009.Infections due to M. marinum declined from 64% of casesin the earlier era to 33% in the more recent era, while those dueto M. chelonae or M. abscessus rose from 7% to 46% ofSSSTI cases [87•].

Pediatric Population

There is no definite evidence of an increase in the incidence ofNTM infections among children. However, the absence of thisobserved trend may reflect inadequate data collection [12, 90].Based on inpatient surveys, the incidence of total NTM infec-tions in children ranges from 0.6 to 1.6 cases per 100,000, ofwhich 2–18% involve skin and soft tissues [90–94]. Skin in-fections comprise a higher proportion of cases in countriessuch as Australia, where M. ulcerans is endemic.

In North America, three species of rapid growers,M. chelonae, M. abscessus, and M. fortuitum accounted for74% (17/23) of pediatric infections, according to one study ofhospitalized cancer patients. Most of the children in this studyhad catheter-related infections [92]. In contrast, two otherstudies of 28 inpatient children in Australia reported thatM. marinum andM. ulceranswere the most common species,representing 16 out of 28 infections.Members of theM. avium

complex (MAC), not found in the North American study, werealso found in three SSTIs in these studies [12, 91]. Finally, astudy from Singapore reported eight cases of skin and softtissue infection, five with M. abscessus, two withM. haemophilium, and one with M. kansasii, out of a total of67 positive cultures reviewed [94]. Together, these four re-ports represent 50 years of mostly tertiary center inpatientexperience and yet describe only 59 cases of pediatric skinand soft tissue NTM infections, underscoring how little epi-demiological data is available [12, 91, 92, 94]. The incidenceof NTM infections in children in the outpatient setting, forexample, is unknown.

Emerging Pathogen: M. chimaera

Mycobacterium chimaera was once considered a rare patho-gen. However, since 2015, this species has been identified innosocomial outbreaks of surgical site infections. The infec-tions have been epidemiologically linked to a specific brandof heater-cooler units (HCUs), which are stand-alone devicesused during surgeries requiring cardiopulmonary bypass. Firstreported in Switzerland by Sax et al., M. chimaera-contami-nated HCUs on three different continents have been associatedwith surgical site infections that frequently disseminateand cause significant morbidity and mortality [95]. HCUs regu-late the temperature of patient blood and cardioplegia solutionvia a water circuit without direct contact between fluids. TheHCUs are not airtight and utilize fans to dissipate heat moreefficiently. Fans within the LivaNova 3T HCUs aerosolizeM. chimaera, which can then deposit on the operative field,contaminating wounds and prosthetic material. Theaerosol-dispersion mechanism has been proven using smoketests, sedimentation plates, and particle counters. Strains se-quenced from infected patients have also matched those isolatedfrom the HCUs [95, 96•, 97, 98•].

In most cases, contamination of the HCU seems to haveoccurred at the manufacturer, given the similarity of isolatesfound in the manufacturing plant to clinical isolates fromAustralia, New Zealand, the USA, and UK [99, 100, 101•].Nonetheless, the risk of local contamination also exists.Studies have shown that a large variety of NTMs colonize thehealth care and home environment [101•, 102]. Van Ingen et al.identified at least one clinical strain ofM. chimaerawhose genesequence was more homologous to local isolates than the out-break strains [101•]. A separate single-center outbreak ofM. abscessus was felt to have arisen from two local strainsspread via HCUs [103]. Extracorporeal membrane oxygenationmachines may also become colonized, although none so farhave been implicated in this outbreak, perhaps due to theirairtight, closed-system construction [104].

Despite the extensive use of the LivaNova 3T HCUs andprevalence of cardiac surgery worldwide, rates ofM. chimaera infection have been low. In the UK, 0.17 to 3


cases per 1000 procedures (0.39 per 10,000 patient years)were reported, similar to other cohorts [101•]. The majorityof infections have involved chest surgery (requiring an HCU)and either prosthetic material or heart transplant. For example,of 24 cases identified in a US investigation, 46% had heartsurgery for a prosthetic valve or valve ring, 29% received avascular graft, 21% had a left ventricular assist device im-planted, 13% underwent heart transplant, and 4% had coro-nary artery bypass grafts [36]. Prosthetic material is a signif-icant risk factor for deep or disseminated infection and pooroutcomes. In one series, endocarditis was seen in 28%, endo-carditis with aortic root abscess in 17%, disseminated infec-tion in 17%, sternal osteomyelitis in 11%, anterior mediastinalabscess in 6%, and spinal osteomyelitis or diskitis in 6%[101•]. Ophthalmologic involvement occurs and may reflectdisease burden [106]. The time to presentation has variedwidely, ranging from 3 months to 5.1 years. It has been rec-ommended that a diagnosis of M. chimaera surgical site in-fection be considered as late as 6 years following surgery [95,101•]. Identification of cases requires a high index of suspi-cion. Signs and symptoms are often vague, but may includefatigue (90%), fever (75%), sweats (60%), dyspnea (60%),weight loss (60%), and cough (50%) [36]. Laboratory findingsare nonspecific. Anemia, lymphopenia, and thrombocytope-nia are common. Most cases are diagnosed by positive bloodcultures. Thus, blood cultures for acid-fast organisms are rec-ommended in suspected cases. Bone marrow, tissue biopsy, oroperative samples may also be cultured. Histopathologicalsamples are frequently negative on acid-fast staining; granu-lomatous changes in such samples have led to misdiagnosiswith sarcoidosis.

There are no formal treatment guidelines for M. chimaerainfections. Until 2004, M. chimaera was classified within theM. avium complex (MAC) [107]. Therefore, treatment regi-mens similar to those for MAC have been used. Macrolide,rifamycin, and ethambutol form the backbone of therapy, of-ten with the addition of moxifloxacin or amikacin. Linezolidor clofazimine has been added in rare cases [35, 95, 106].Similar to MAC, there is no evidence that susceptibility test-ing of drugs other than macrolides correlates with treatmentresponse [108–110].

Unfortunately, treatment responses are often poor.Mortality has reached 50–60% in the largest series, with morethan half of patients experiencing breakthrough on therapy.The disease is very difficult to cure, and most patients whosurvive report feeling unwell on therapy. Extensive operativeinterventions, including valve replacement, endovascular graftexcision, and serial debridement are often required, even forpatients on appropriate therapy [95, 101•].

To date, the best infection control measures involve removalof colonized HCUs from the operating theater or engineering anairtight structure that surrounds the HCU and connects directlyto the ventilation system [38, 97]. The CDC recommends

following the manufacturer’s evolving guidelines for cleaningand decontamination. Some published protocols have demon-strated clearance of cultures from HCUs for up to 6 months[111]. Other authors using a similar decontamination regimendocumented the re-emergence ofM chimaera growth; althoughin this study, tubing was not replaced [38]. The manufacturer’smost recent decontamination protocols include initial cleaningwith peracetic acid prior to clinical use, daily cleaning with(2 μm) filtered tap water containing 3% hydrogen peroxide,and regular (i.e., weekly) full decontamination with peraceticacid. While there has been continued evolution of decontami-nation procedures, it remains unclear whether colonized HCUscan be decontaminated and remain sterile over the long-term.Despite these issues, the LivaNova 3T heater-cooler units arestill in use in many hospitals.

General Features of Mycobacterial Virulenceand the Immune Response

As a class, NTM possess two features important for virulence:the ability to elaborate an antibiotic-impervious biofilm andthe presence of a cell wall rich in glycolipids, such aslipoarabinomannan (LAM). Microbial biofilm may modifyimmune responses, by sequestering microbes from immunecells and effector molecules, thereby preventing immune cellrecognition [112]. Separately, biofilm allows the physical per-sistence of organisms on hardware and other materials, such asnon-biologic vascular grafts. Environmental mycobacteria re-siding in biofilms around faucets, drains, and pipes, or onmedical equipment are resistant to most conventional disin-fectants [33, 112]. As discussed above, environmentalmycobacteria have been associated with disease outbreaksepidemiologically linked to water systems and medical sup-plies and equipment [37•, 102, 113]. The second virulencefactor shared among mycobacteria is a thick and waxy cellwall, frequently decorated with LAM and other closely relatedglycolipids. LAM and similar molecular motifs have beenassociated with altered differentiation and cytokine expressionof the host cell (reviewed in [114, 115]). Bacterially mediatedimmunemodulation is both species- and strain-dependent. Forexample, inM. marinum, LAM elongation and branching en-ables virulence in zebrafish and disruption of this processleads to attenuated disease in this host [116]. Whether similarmechanisms extend to all pathogenic NTM is not yet known.

New Insights into NTM Pathogenesis

Rapidly Growing NTM

Rapidly growing NTM are species that grow on solid mediawithin 7 days. Overall, this group of mycobacteria is consid-ered less pathogenic than the slow-growing species. Some


recent data suggest, for example, that M. abscessus andM. fortuitum are less efficient at infecting cells thanM. tuberculosis or M. celatum. This reduced infection effi-ciency may allow the host to mount a more successful im-mune response [117].

M. abscessus

Mycobacterium abscessus is the most pathogenic and clinical-ly challenging of the rapid-growing mycobacteria.M. abscessus causes entrenched pulmonary infections in pa-tients with cystic fibrosis or lung transplantation, and is oftenassociated with broad antimicrobial resistance. There are threesubspecies in the M. abscessus group: M. abscessus subsp.abscessus , M . abscessus subsp. massil iense , andM. abscessus subsp. bolletii [118, 119]. M. abscessus subsp.massiliense lacks the intrinsic antimicrobial resistance (nota-bly, the erm(41) gene conferring macrolide resistance) of theother members [120].

During pulmonary infection, M. abscessus changes from asmooth to a rough colony phenotype, through loss of surfaceglycopeptolipids (GPLs). The smoothmorphotype of this speciespredominates in skin and soft tissue infections [121]. The smoothvariant is less virulent and survives well in biofilms, while therough variant is more virulent and pro-inflammatory and growsaggressively in the host by forming cords that impede phagocy-tosis [122–124]. The surface GPLs of the smooth colony variantappear to shield other cell wall molecules from interactions withToll-like receptor 2 [125]. Only the rough morphotype inducesapoptosis and autophagy, events that have the potential to en-hance virulence, in macrophages [124]. Absence of phenolicglycolipid (PGL) on the surface of the rough variant appearscentral to this cell death-inducing phenotype, since apopto-sis is not induced when cultures of rough variantmycobacteria are supplementedwith PGLs. Interestingly, onlythe less virulent smooth form ofM. abscessus induces breaksin the phagosome membrane [124], a process associated withenhanced virulence in other mycobacteria.

One proposed explanation for the particular virulence ofM. abscessus in lung infections is the recently discoveredputative phospholipase C (PLC), a gene which is absent inother rapidly growing species [126]. This enzyme is cytotoxicto mouse macrophages and shows similar activity to PLC-Nof Pseudomonas aeruginosa, an organism that causes chronicinfections in cystic fibrosis patients [127]. The PLC gene isexpressed in smooth colony variants ofM. abscessuswhen thebacteria are residing within amoebae. When pre-cultured inamoebae, M. abscessus is more infective in mouse lungs[127]. This finding suggests that environmental amoebaemay be important for pulmonary infection of mammalianhosts. In this model of virulence, smooth variant bacteria re-siding within amoebae are primed to establish infection in thelungs. After infection, the bacteria transition to a rough colony

phenotype. Epidemiological evidence for this mechanism,however, is weak. Survival within free-living amoebae indrinking water has not been demonstrated for M. abscessus,although it has for other mycobacteria [128]. This proposedvirulence mechanism also does not have relevance for skinand soft tissue infections due to M. abscessus.

M. chelonae

Mycobacterium chelonae is more likely than M. fortuitum tocause disseminated rather than localized skin infection. Thisspecies is also less closely related toM. fortuitum than it is toM. abscessus [129]. However,M. chelonae lacks the erm(41)gene that confers macrolide resistance in M. abscessus [120].It also differs fromM. abscessus in having a greater predilec-tion for skin than lung infections. In the environment,M. chelonae has been found within free-living amoebae[128]. In zebrafish, experimental infectivity is enhanced whenthe bacteria are associated with the amoeba Parameciumcaudatum [130]. There is a single case report of M. chelonaeliving within a protozoan colonizing the nasal mucosa of anHIV patient [131]. Other evidence that protozoa enhance thevirulence ofM. chelonae during natural infection, however, isso far lacking.

M. fortuitum

Mycobacterium fortuitum is a frequent cause of localized skinand soft tissue infections [132]. Relatively little is knownabout the immune pathogenesis of infections due to this spe-cies. M. fortuitum infection of murine macrophages inducesboth pro-and anti-inflammatory responses that are mediatedby Toll-like receptor 2 [133]. A 2016 study in zebrafish mac-rophages demonstrated that M. fortuitum inducescaspase-mediated apoptosis in macrophages via a calciuminflux-dependent mechanism, thus enhancing infection [134].

Slowly Growing NontuberculousMycobacteria

M. marinum

In nature, M. marinum is found in freshwater, brackish water,and salt water. Infection with the organism produces a fataltuberculosis-like, granulomatous disease in fish. In humans,M.marinum causes “fish-tank” or “swimming pool” granuloma,producing single or multiple papulo-nodular skin lesions, oftenon the hands, which may spread proximately in a sporotrichoidpattern [21, 24].M. marinum has been exploited experimentallyto model tuberculosis in zebrafish embryos, which are conve-niently transparent. The zebrafish model has yielded major in-sights into the earliest stages of mycobacterial pathogenesis


[135–137]. Two recent studies demonstrated that M. marinumuses specific membrane lipids (phenolic glycolipids) to recruitmore permissive macrophage types to the site of infection[138•]. The bacilli then transfer from their original macrophagehost cells, which restrict intracellular growth, to the more per-missive new arrivals, thus surviving and disseminating [139].

Like M. tuberculosis, the M. marinum genome contains anESX-1 locus that encodes major virulence factors ([140];reviewed in [141•]). The ESX-1 locus had previously beenshown to contribute to lysis of the phagosome membrane(and the consequent escape of bacteria to the cytosol) and gran-uloma formation [137, 142]. More recent studies have elucidat-ed the mechanism of phagosome membrane lysis [143] andidentified ESX-1-encoded proteins that modulate macrophagefunction to promote intracellular survival [144]. A paralogouslocus, ESX-5, has been shown to influence autophagy (usingsimultaneous activation and repression of its mechanisms) toenhance intracellular bacterial persistence [145].

M. ulcerans

Mycobacterium ulcerans causes an ulcerating skin infection,known as Buruli or Bairnsdale ulcer, which may bore throughoverlying soft tissue to bone. Worldwide, it is the third mostcommonly reported mycobacterial infection, after tuberculo-sis and leprosy [68, 146]. Buruli ulcer, a neglected tropicaldisease, is encountered in Africa, Australia, Southeast Asia,and Central and South America. The majority of cases comefrom West Africa and the Democratic Republic of Congo(90% of global cases reported in 2016) and Australia (10%of global cases) [147].

Mycobacterium ulcerans is most closely related toM. marinum, and similarly prefers lower temperatures forgrowth. It is unique among the NTM species in being a pre-dominantly extracellular pathogen [69]. In 1999, the factorresponsible for the unique pathogenesis of M. ulcerans wasfound to be mycolactone, a highly diffusible, cytopathic lipidtoxin [148]. In the last 5 years, two direct mechanisms ofaction for this toxin have been elucidated (reviewed in[149]). First, mycolactone directly binds the Wiscott-Aldrichsyndrome protein (WASP), leading to uncontrolled actin as-sembly in the cytoplasm and defective cell adhesion and mi-gration [150••]. Second, mycolactone directly inhibits theSec61 translocon, which, under normal circumstances, facili-tates transit of secretory proteins to the endoplasmic reticulum[151••]. Mycolactone blockade thus broadly inhibits translo-cation (and therefore secretion) of immune response proteins,including Cox-2, TNF and IL-6 [149, 151••]. These new find-ings help to explain the pleiotropic effects of a toxin thatmakes M. ulcerans so clinically distinct from other NTMspathogenic to humans. However, another fundamental aspectof M. ulcerans pathogenesis—how the infection istransmitted—remains unsolved. Various mechanisms have

been proposed, including superinfection of minor skin abra-sions, inoculation by mosquitoes or specific species of wet-land insects, and via amoebae [152], but there is no currentconsensus. A common theme appears to be microbial accessto the microvasculature, which is achieved through puncturesor vector inoculation, rather than superficial abrasion.

Newer Diagnostic Tools

Traditionally, identification of NTMs in the laboratory reliedheavily upon phenotypic characteristics such as biochemicaltests, growth rate, and colony pigmentation and morphology.More advanced recent diagnostic tools have included high-performance liquid chromatography and molecular diagnostictests [153, 154•, 155]. DNA hybridization, DNA sequencingof 16S ribosomal RNA and other gene regions have permittedthe identification of many new species. Molecular methodsare now routinely used in reference labs and academic centersRecently, Pranada and colleagues were successful in discrim-inatingMycobacterium intracellulare fromM. chimaera, twospecies whose 16S RNA gene sequences differ by a singlebase pair, using MALDI-TOF [156]. Table 2 summarizesthese important new diagnostic tools.

Advances in Antibiotic Treatmentof Nontuberculous Mycobacteria

Novel Anti-tuberculosis Agents

Most of the recent advances in the treatment of NTMs havecome from repurposing antibiotics used against other bacteria.However, two new agents recently developed for tuberculosis,delamanid and bedaquiline, have some activity againstnontuberculous mycobacteria. A recent study reported thatthe mean inhibitory concentrations (MICs) of delamanid, adihydro-nitroimidazooxazole derivative, were quite low for20 strains of M. avium and M. intracellulare (MAC).Nineteen of the 20 strains had MICs ≤ 0.2 ng/ml. Includedamong these strains were some with resistance toclarithromycin, the mainstay of MAC therapy [169]. Theanti-tuberculosis drug bedaquiline is an ATP synthase inhibi-tor with bactericidal activity againstM. tuberculosis [170] andis FDA-approved for multi-drug-resistant and extremely drugresistant tuberculosis. Bedaquiline also has in vitro activityagainst M. avium complex, M. abscessus, M. ulcerans, andM. smegmatis [171–176].

Unlike delamanid, bedaquiline has been studied in severaldifferent animal models. In a mouse footpad model ofM. ulcerans infection, bedaquiline monotherapy has shownsome bactericidal activity [175, 177]. In this model, a regimencontaining rifamycin and bedaquiline was comparable to


rifamycin and streptomycin or rifamycin and amikacin, asmeasured by lesion size and bacteria recovered from treatedlesions [175]. In one study utilizing nude (T cell-deficient)micewith disseminatedM. abscessus, bedaquiline did not havesignificant activity, as assessed bymortality, lesion burden, andbacteria recovered from the spleen and lung [178]. In a separatestudy, in gamma interferon knockout (GKO) mice and B- andT-cell-deficient severe combined immune deficiency (SCID;spontaneous mutation Prkdcscid) mice, bedaquiline alone andin combination with clofazimine reduced bacterial burden inthe lung, spleen, and liver [179]. Experimental infections ofmice with M. abscessus have generally utilized immune-deficient mouse strains, without a clear consensus as to whichstrain makes the ideal model. Thus, both methods and resultshave been conflicting. In zebrafish larvae, bedaquiline treat-ment was associated with improved survival, fewer abscessesand the inhibition of in vivo cord formation [174].

The human clinical experience with bedaquiline for NTMdisease so far involves two uncontrolled case studies of pa-tients with lung infection [105, 180]. In the first, 9 of 10patients with refractory pulmonary mycobacterial disease (6with MAC, 4 withM. abscessus infection), experienced earlysymptomatic improvement. Four out of 10 also had radio-graphic improvement at 4 months. None had sustained micro-biologic clearance [180]. In the second, 13 patients receivedbedaquiline as part of multi-drug salvage regimen for pulmo-nary M. intracellulare, with 8 initially responding to treat-ment. However, all eight subsequently had microbiologic re-lapse and developed resistant (mmpT5) variants with two- toeightfold higher bedaquiline MICs [105]. Of note, six of theeight patients who relapsed were also receiving rifampin,which is known to decrease exposure to bedaquiline due tocytochrome P 3A4 induction [181]. Given the difficulty oftreating multi-drug-resistant strains of M. abscessus,bedaquiline could prove useful, although more human clinicaldata are needed.

Oxazolidinones

Linezolid and tedizolid both have activity against NTMs, buttedizolid is emerging as a more tolerable alternative to linez-olid, with equivalent or lowerMICs in vitro for both slow- andrapid-growing mycobacteria [182]. There is at least one casereport of the successful use of tedizolid, as part of a multi-drugregimen for pulmonary for NTM disease, following linezolidtoxicity [183]. Although more experience is needed to firmlyestablish efficacy and tolerability, tedizolid may become avery useful addition to the NTM armamentarium.

ß-Lactams

The role of ß-lactams in the treatment of NTMs is evolving,especially in the case of M. abscessus. Kumar et al. recently

demonstrated that the majority of linkages in the cell wallpeptidoglycan of M. abscessus are synthesized byL,D-t ranspept idases , ra ther than the t radi t ionalD,D-transpeptidases [184••]. The authors cloned, expressed,and purified two different L,D-transpeptidases and then mea-sured the ability of various ß-lactam antibiotics to inhibit theseenzymes. Cephalosporins and carbapenems, but not penicillins,were able to cause inhibition. While each of the two L,D-transpeptidases had a different inhibition profile, cefdinir-doripenem was the most effective combination for both [184••].

Resistance to ß-lactams inM. abscessus is mediated in partthrough the chromosomally encoded Ambler class A ß-lactamase and BlaMab, which hydrolyzes both ß-lactams andthe ß-lactamase inhibitors clavulanate, sulbactam, and tazo-bactam [185]. Deletion of the BlaMab gene in human macro-phages and zebrafish restores in vitro susceptibility to mostpenicillins, cephalosporins, and carbapenems (with the excep-tion of aztreonam and ceftazidime) and rescues susceptibilityto amoxicillin [186]. The novel ß-lactamase inhibitor,avibactam, is not hydrolyzed by BlaMab and can restore sus-ceptibility to ß-lactams in BlaMab-positive M. abscessusstrains, similar to strains with BlaMab gene deletion [186].The addition of avibactam to the carbapenems imipenem,faropenem, biapenem, doripenem, meropenem, panipenem,ertapenem, and tebipenem resulted in heightened activity forall penems against 28 clinical isolates of M. abscessus, someof which were highly resistant. This effect is likely due to bothcarbapenem inhibition of L,D-transpeptidase and efficacyagainst BlaMab [187]. Studies in human macrophages andzebrafish further support the efficacy of avibactam when usedwith a carbapenem (imipenem) [188••].

Although more data is needed, the combination ofavibactam and other carbapenems may allow clinicians addi-tional options for the treatment of drug-resistant and refractoryNTM infections, particularly M. abscessus. Currently, in theUSA, avibactam is only available in co-formulation with cef-tazidime, which has no activity against M. abscessus.Tebipenem also exists in an oral formulation, but is not yetavailable in the USA.

Conclusion

Although systemic surveillance data are lacking, the incidenceof NTM infections, including those involving skin and softtissues, is rising in many regions. M. chimaera is an emergingnosocomial pathogen associated with skin and soft tissue infec-tions following cardiac surgery. In some series, rapid-growing mycobacteria, including the multi-drug-resistant spe-cies M. abscessus, have surpassed M. marinum as the mostfrequently isolated pathogen in patients with skin and soft tissuedisease. M. ulcerans continues to cause major morbidity inAfrica. The most reliable diagnostic approaches for NTM


Table 2 Molecular methods for the identification of nontuberculous mycobacteria

Assay name or type RNA/DNA target and methods Species identified Disadvantages/advantages

GenoType Mycobacterium CM(Hain Lifescience, Nehren,Germany)

Target: 230 bp fragment of 23SrRNA gene

Method: PCR of a 23S rRNA generegion, followed by reversehybridization and line probetechnology [157]

> 20 species of NTM andM. tuberculosis

• Requires growth on culture• 37 species identifiable.• Tsukamurella may be

misidentified as mycobacterialspp. [158]

• M. chimaera misidentified asM. intracellulare [158]

• M. terrae and M.nonchromogenicum notcovered

• Commercially available• Highly sensitive and specific

GenoType Mycobacterium AS(Hain Lifescience, Nehren,

Germany)

Same as Mycobacterium CM [157] 19 less common NTM species

GenoType NTM-DR(Hain Lifescience, Nehren,

Germany)

Species identification: same asMycobacterium CM

drug resistance targets: erm41, rrl,and rrs

• Detects M. avium,M. intracellulare, M. chimaera,M. chelonae and species andM. abscessus subspeciesmassiliense, bolletti, abscessus

• Requires growth on culture• Distinguishes M. chimaera from

M. intracellulare• Detects drug resistance mutations:—macrolides (erm(41), rrl genes)—amikacin/tobramycin (rrs gene)• Sensitivity: 79% for

clarithromycin resistance, 71%for aminoglycoside resistance[159]

Genotype CMdirect(Hain Lifescience, Nehren,

Germany)

reverse hybridization line probeassay (DNA)

> 20 species of NTM and M.tuberculosis

• May be used directly ondecontaminated sputum

• Commercially available

Gen-Probe AccuProbe®(Hologic, Marlborough, MA, USA)

Target: rRNASingle-stranded DNA probe

hybridizes to a ribosomal RNAtarget

Individual assays for each species:M. avium, M. intracellare, M.gordonae, andM. kansasi

• Requires growth on culture• Lower sensitivity for detection of

M. fortuitum strains thanGenoType [158]

• Commercially available• Not multiplexed

INNO LiPA® Mycobacteriav2 (Fujirebio, Tokyo, Japan)

Target: 16S–23S rRNA intergenicspacer (ITS)

DNA hybridization line probe assay

16 species of NTM • Requires growth on culture• 87–100% sensitivity; 100%

specificity [160, 161]• Commercially available

Restriction enzyme pattern analysis(hsp65 gene)

Target: segment of the 65-kDa heatshock protein gene (hsp 65).

DNA segment is amplified by PCRand the product is digested byrestriction enzymes, withfragments separated by agarosegel electrophoresis. The resultingrestriction patterns are analyzedby comparison to those of knownreference strains [161]

Often used to compare a handful ofspecies

• Requires growth on culture• hsp gene encodes sequences that

are unique and common –somespecies may not have uniquerestriction patterns [161]

• Time-intensive, research orreference lab method

16S rRNA, rpoB, or hsp65 genesequencing [158, 162, 163]commercial platform: MicroSeq500 16S rDNA microbialidentification system (AppliedBiosystems, Foster City,California)

Molecular target: hsp65, 16S rRNAgenes

DNA segment is amplified andsequenced using broad rangeprimers applicable to allmycobacterial species. Nucleotidesequences are compared to publicdatabase (GenBank or others*)[158, 162, 163]

• In theory, many species areindividually identifiable

• In practice, closely related speciesmay not be resolved (clearlydistinguished) due to negligiblesequence differences

• May be done on direct patientsample/specimen, if sterile

• Available at reference centers


Table 2 (continued)

Assay name or type RNA/DNA target and methods Species identified Disadvantages/advantages

Multiplex SNaPshot method Target: polymorphic sites in 16SrRNA and hsp 65 genes

Genus-specific primers amplify354-bp fragment of hsp65 and a436-bp fragment of 16S rRNA.Forward or reverse extensionprimers are designed to annealadjacent to polymorphic sites in16S rRNA gene (positions 125,141, 231, 264, 471) or hsp65 gene(positions 163, 235, 265) presentin the PCR products. Single baseextension reactions withfluorescently labeled ddNTPs areperformed for multiple SNP sitessimultaneously. Resultantfragments are resolved byelectrophoresis and SNP allelesare identified by fluorescenceemission (red, green, blue, black)after laser excitation [164]

• Simultaneous analysis of 8 singlenucleotide polymorphisms(SNPs) in 16S rRNA and hsp 65genes allowing discrimination of 5NTM species (M. avium,M. intracellulare,M. chelonae,M. kansasii,M. gordonae) andM. tuberculosis

• Requires growth on culture• Research method but utilizescommercially available kit (ABI

Prism SNaPshot Multiplex Kit),capillary electrophoresis on anABI Prism 3130 × 1 geneticAnalyzer (Applied Biosystems),and genetic analysis usingcommercial software(GeneMapper, version 4.0,software, Applied Biosystems)[164]

Denaturing high-performance liquidchromatography (DHPLC)

Target: 16S–23S rRNA ITS generegion or 16S rRNA region[165, 166]

Amplification of the target DNA,followed by denaturinghigh-performance liquidchromatography

Many NTM species identifiable • Amplification of the 16S–23SrRNA ITS region allowsdifferentiation of M. chimaerafrom M. intracellulare [165]

• Applicable to M. chimaeraoutbreak settings

• Requires growth on culture• Much more sensitive than culture

[166]• Rapid, high through-put• Not widely available

Matrix-assisted laser desorptionionization time-of-flight massspectrometry (MALDI-TOF MS)

Mycobacterial cultures are treatedwith formic acid and acetonitrileto break down cell walls andextract protein. The resultantsamples (analytes) are applied to asteel target plate and coated with amatrix solution (e.g.,α-cyano-4-hydroxycinnamicacid). The plate is irradiated with apulsed laser and energy istransferred from the matrix to theanalyte, which undergoesdesorption (removal into the gasphase) and ionization. The ionizedparticles are subjected to anelectric potential that propels themthrough a flight tube into the massspectrometer, where they areseparated by mass and charge.Different bacterial species displaydistinct spectral fingerprints(stereotypical pattern of peaks),and are identified by reference to alarge database [156, 167, 168].

• In theory, unlimited• In practice, limited by size ofspectral library

• Requires growth on culture• Rapid method• M. abscessus subspecies may not

be well separated• M. chimaera andM.

intracellulare may be conflated.However, one report of a modelthat distinguishesM chimaeraandM. intracellulare [156]

*Other databases include the DNA Data Bank of Japan (DDBJ), the European Molecular Biology Laboratory (EMBL), and the RibosomalDifferentiation of Medical Microsystems database (RIDOM)


species are molecular, and involve DNA probes, gene sequenc-ing (of 16S ribosomal RNA, hsp65, rpoB, or the 16-23Sintergenic spacer region) and MALDI-TOF. Research into thebasic mechanisms of virulence of the more pathogenic NTMs,particularlyM. marinum andM. abscessus, has the potential tooffer biologically based therapeutics. There is an unmet demandfor such novel agents, especially for drug-resistant species, suchas M. abscessus, which causes significant morbidity and mor-tality in cystic fibrosis and lung transplant patients. Study of theactivity of available and new antibiotics, both singly and incombination, against NTM species lags behind the develop-ment of new drugs for tuberculosis and is urgently needed.

Compliance with Ethical Standards

Conflict of Interest Each author reports no conflicts of interest.

Human and Animal Rights and Informed Consent This article does notcontain any studies with human or animal subjects performed by any ofthe authors.

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skin and soft tissue infections due to nontuberculous mycobacteria · 2019-06-17 · skin, soft...

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