Short Communication
High prevalence of non-tuberculous
mycobacterial disease among non-HIV
infected individuals in a TB endemic country –
experience from a tertiary center in Delhi,
India
Sarika Jain1, Manimuthu M. Sankar2, Navneet Sharma3, Sarman Singh2, T. D.
Chugh3
1
Department of Microbiology, VMMC and Safdarjung Hospital, New Delhi, India, 2Department of Laboratory
Medicine, All India Institute of Medical Sciences, New Delhi, India, 3Department of Microbiology, BLK
Superspeciality Hospital, Pusa Road, New Delhi, India
Non-tuberculous mycobacteria, MGIT, Multiplex
PCRIntroduction
Non-tuberculous mycobacteria (NTM) considered
mostly as colonizers or ignored as environmental
contaminants in the past, are now increasingly
recognized as important pulmonary pathogens in
both immunocompromised and immunocompetent
population.1 Specific risk factors identified are HIV
infection, cystic fibrosis, underlying chronic lung
disease, previous tuberculosis (TB), and work in the
mining industry.1 These are also reported to cause
surgical-site infections, post-injection abscesses,
osteomyelitis, catheter-related blood-stream infections, and central nervous system infections.2 Nontuberculous mycobacteria rates of infection and
disease has significantly increased in recent years
and rates vary widely depending on population and
geographic location.1 Most reports are from developed countries that have low rates of TB. However,
in countries with high burden of TB, including India,
NTM pulmonary disease often goes unrecognized
and is misdiagnosed as pulmonary TB because
clinical presentation of NTM and Mycobacterium
tuberculosis (MTB) diseases are indistinguishable
from each other. Prevalence of NTM is unknown in
India as NTM disease is not a reportable condition
and there is lack of awareness among clinicians
coupled with lack of laboratory capacity to diagnose
these infections.3 Among few reports available, NTM
isolation rates are reported to range from 0.5 to 8.6%
in India.4 A recent study from central India reported
Correspondence to: Sarika Jain, Department of Microbiology, VMMC and
Safdarjung Hospital, New Delhi, Delhi, India. Email: drsarika6@gmail.com
118
ß W. S. Maney & Son Ltd 2014
DOI 10.1179/2047773214Y.0000000133
prevalence of NTM increased from 1.0% in 2005 to
3.5% in 2008 and 88.6% of the NTM isolated were
clinically relevant.4 Identification of NTM is of
clinical relevance as most of the NTM are notably
resistant or only partially susceptible to the standard
anti-tubercular drugs and the treatment strategies
and the duration of these infections differ from MTB.
We conducted retrospective review of isolation
rates of Mycobacterium tuberculosis complex (MTC)
and NTM, the species of NTM characterized and
their clinical significance during January 2011–June
2012 at our tertiary care hospital (700 bedded) in
Delhi, India. The study was approved by the ethical
committee of our institute. A total of 436 clinical
specimens were processed for mycobacteria culture,
237 from pulmonary (sputum, bronchoalveolar
lavage, bronchial wash, and endotracheal aspirates)
and 199 from extrapulmonary sites (urine, pus,
peritoneal fluid, lymph node aspirate, synovial fluid,
endometrial biopsy, CSF). Specimens were digested
and decontaminated by the standard N-acetyl-Lcysteine–NaOH method and inoculated into
BACTEC mycobacteria growth indicator tube
(MGIT) 960 vials (BD Diagnostics, Sparks, MD,
USA) and Löwenstein–Jensen (LJ) medium slants
(Hi Media laboratories, Mumbai, India). Smear
microscopic examination was performed using Zeihl
Neelsen (ZN) and fluorochrome stains. All positive
MGIT vials were subjected to identification of MTC,
by p-nitrobenzoic acid (PNBA) assay on MGIT 960
as described elsewhere5 and SD TB MPT64 antigen
rapid assay by immunochromatographic method
(Standard Diagnostics, Seoul, South Korea). pNitrobenzoic acid resistant and/or MPB 64 antigen
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Jain et al.
negative isolates (suspected to be NTM) were
subjected to a multiplex PCR using primers for
mycobacterium genus (targeting hsp65), MTC (targeting ESAT6), and Mycobacterium avium complex
(targeting MAC) specific genes.3 Confirmed NTM
isolates were further speciated by pigment production, growth on MacConkey agar, rate of growth,
nitrate reduction test, tellurite reduction, urease
production, tween-80 hydrolysis, arylsulfatase, heatstable catalase, iron uptake, and tolerance to 5%
NaCl.6 Drug-susceptibility testing was performed for
MTC for PSIRE (pyrazinamide, streptomycin, isoniazid, rifampicin, and ethambutol) by BACTEC
MGIT 960 (1% proportional method).
Of 436 specimens, 109 (25.6%) were positive for acidfast bacilli by smear microscopy (ZN and/or fluorescent) and 131 (30.7%) specimens were culture positive.
Of 131 positive cultures, 118 (90.1%) isolates were
identified as MTC (71.2% from pulmonary and 28.8%
from extrapulmonary samples) and 13 (9.9%) isolates as
NTM species. The NTM were isolated from pulmonary
specimens in nine (69.2%) patients, and four (30.8%)
were isolated from non-pulmonary specimens, one each
from urine, ascitic fluid, bone, and blood specimens.
The most common NTM species characterized was
Mycobacterium kansasii (4) followed by Mycobacterium
chelonae (3), Mycobacterium xenopi (2), Mycobacterium
scrofulaceum (1), M. avium (1), Mycobacterium asiaticum (1), and Mycobacterium fortuitum (1). Clinical
details and speciation of the NTMs isolated are shown
in Table 1. Fluorescent staining was more sensitive as it
detected 12 additional culture positive cases that were
negative by ZN stain (of these, four were NTM). As
NTM are ubiquitous in nature, NTM pulmonary
infections consistent with the American thoracic society
(ATS) and British thoracic society (BTS) guidelines
(defined in Table 1) were considered clinically relevant.7,8
Of nine patients with pulmonary NTM isolated,
seven (77.9%) had definite NTM disease and two
(15.4%) had probable NTM disease; among four
patients with extrapulmonary specimens, two (50%)
had definite NTM disease, one (25%) had probable
NTM disease, and one (25%) had possible disease or
colonization (Table 1). Majority of patients were males
(69.2%) and 46.1% (6/13) were older in age
(§60 years). The main underlying risk factors found
to be associated with NTM infections were pre-existing
pulmonary disease (54%), chronic obstructive pulmonary disease (COPD; 23.1%), past and/or present history
of TB (30.8%, two pulmonary TB and one Pott’s spine
cases were diagnosed by both smear for AFB and
culture positivity for MTB and a case of osteomyelitis
was smear positive for AFB), chronic smoking (15.4%),
diabetes (23.1%), steroids (7.6%), and malignancy
(7.6%). No predisposing condition was detected in
three patients (23.1%). All 13 patients with NTM
Non-tuberculous mycobacterial disease in non-HIV infected
disease were HIV seronegative (ARCHITECTH HIV
Ag/Ab Combo Assay, Abbott diagnostics).
Between PNBA and MPB64 antigen assay results,
100% concordance was observed. All mycobacteria
grew both in MGIT and LJ medium; however mean
isolation time was much shorter with MGIT than with
LJ (11 vs 28 days). Furthermore, mean recovery time
of MTC from pulmonary specimens was lesser than
that of non-pulmonary specimens (10 vs 16 days) by
MGIT culture. Among NTMs, slow-growing mycobacteria grew in mean time of 12.9 days. Rapidly
growing mycobacteria (RGM) bloodstream infection
due to M. chelonae in one of the patient could be
detected by MGIT in 2.2 days. Of the MTC, 67% were
sensitive to all tested drugs, 14.7% were multidrugresistant (MDR), and 17.6% were mono-resistant.
Treatment details of the patients were not retrieved.
While clinical diagnosis of NTM pulmonary
disease is challenging, reliance only on positive smear
microscopy for the initiation of treatment for
suspected TB in most TB endemic countries and
not on Mycobacteria culture, species identification,
and drug-resistance testing results, is unfortunate as
acid-fast bacilli visualized in smears may not necessarily be MTB. Moreover, NTM co-infections with
MTB disease are not infrequent, though rarely
diagnosed.10 As NTM strains exhibits high drugresistance to first-line anti-tubercular drugs, many of
NTM infections would be considered TB treatment
failures, and subsequently treated for multidrugresistant TB (MDR-TB) disease. The same was
highlighted in a recent report from China, where
NTM accounted for 30% of suspected MDR-TB
cases and 4% of retreatment TB cases.11 In another
study from New Delhi, India, detected NTM were
detected by multiplex PCR in 17.6% of the suspected
MDR-pulmonary TB cases and in 12.4% of the
suspected extrapulmonary TB cases.3 These findings
highlight the necessity of laboratory speciation of
mycobacteria and initiation of treatment for mycobacterial infections based on in vitro susceptibility
testing. In the present study, 12 of the 13 NTM
infections (92.3%) were provisionally diagnosed as
pulmonary or extrapulmonary TB based on clinical
presentation and smear microscopy findings. Of
these, three patients (23.1%) having past history of
TB were considered for retreatment (MDR-TB
suspected) and one patient was found to be coinfected with MTB (pulmonary NTM and Pott’s
spine). None of the patient in this series was HIV
seropositive. In a recent 3 years study from India,
42% of AFB cultures were positive and approximately 2% of these were NTM. Pulmonary infections
represented 81% of all NTM cases and the major
predisposing condition was underlying lung disease
(54%) including 40% patients with a past or present
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119
NTM species
Age
(years)
/sex
Site of
infection
Other underlying
disease
Specimen
(number
of culture
positive)
Pulmonary
COPD, past history
of pulmonary TB,
chronic smoker
Sputum (2)
84/M
Pulmonary
COPD
Sputum (2)
59/M
Pulmonary
Sputum (2)
76/F
Pulmonary
Ankylosing
spondylitis,
on steroids
Diabetes mellitus
Mycobacterium
scrofulaceum
(n 5 1)
Mycobacterium
fortuitum (n 5 1)
65/M
Pulmonary
COPD, chronic
smoker
Sputum (1)
56/M
Pulmonary
Bronchoalveolar
lavage (1)
Mycobacterium
avium (n 5 1)
60/M
Pulmonary
Diabetes mellitus,
concurrent Pott’s
spine
None
Mycobacterium
asiaticum (n 5 1)
53/M
Pulmonary
Mycobacterium
chelonae (n 5 1)
3/M
Pulmonary
61/M
Osteomyelitis
Past history of
pulmonary TB
51/F
Abdomen
None
2014
Pulmonary (n 5 9)
71/M
Mycobacterium
xenopi (n 5 2)
VOL .
Mycobacterium
kansasii (n 5 2)
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NO .
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Extrapulmonary
(n 5 4)
M. kansasii
(n 5 2)
Diabetes mellitus with
neuropathy, pulmonary
TB defaulter
Post-operative case
of tracheoesophageal
fistula
Sputum (2)
Sputum (2)
Sputum (2)
Bronchoalveolar
lavage (1)
Synovial fluid,
granulation
tissue (1)**
Ascitic fluid{
Smear microscopy
Radiological
findings
Fibrocalcific (old TB)
lesions in upper lobe,
consolidation
in right middle zone
Bilateral pulmonary
infiltrates
Bilateral pulmonary
infiltrates
Consolidation in
left middle
and lower zone
Consolidation in
bilateral upper
and middle lobe
Consolidation in
right upper
and middle zone
Right hilar and
mediastinal
lymphadenopathy
Consolidation in
left middle
and lower zone
Diffuse bilateral
nodular lesions
and large nodule
in upper right lobe
Lytic lesion in
medial condyle
of right knee
ND
ZN Stain
Fluorescent
stain
Growth on
MGIT
culture
(days)
Multiplex
PCR
Clinical
significance
of NTM isolated
(as per ATS)
Negative
5 fluorescing bacilli
in 200 fields
7
hsp65 z
ESAT6-MAC-
Definite NTM
disease
Negative
Negative
9
2z
20 bacilli/HPF
10
hsp65 z
ESAT6-MAChsp65-*
ESAT6 z MAC-
Definite NTM
disease
Definite NTM
disease
1z
2–4 fluorescing
bacilli/HPF
16.5
hsp65-*
ESAT6 z MAC-
Probable NTM
disease
Negative
0–2 fluorescing
bacilli/HPF
15
hsp65 z
ESAT6-MAC-
Probable NTM
disease
Negative
6 fluorescing bacilli
in 200 fields
hsp65 z
ESAT6-MAC-
Definite NTM
disease
2z
.50 fluorescing
bacilli/HPF
16 and 14
(2 sputum
samples)
5
hsp65 z
ESAT6-MACz
Definite NTM
disease
3z
.100 fluorescing
bacilli/HPF
18
hsp65 z
ESAT6-MAC-
Definite NTM
disease
Scanty
7 fluorescing bacilli
in 200 fields
5
hsp65 z
ESAT6-MAC-
Definite NTM
disease
Scanty
2–3 fluorescing
bacilli/HPF
13
hsp65*ESAT6 z MAC-
Definite NTM
disease
Negative
7 fluorescing
bacilli in 200 fields
23
hsp65*ESAT6 z MAC-
Probable NTM
disease
Non-tuberculous mycobacterial disease in non-HIV infected
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Table 1 Clinical and microbiological features of non-tuberculous mycobacteria (NTM) isolated
Table 1 Continued
NTM species
M. chelonae
(n 5 2)
Age
(years)
/sex
Site of
infection
Other underlying
disease
Smear microscopy
Specimen
(number
of culture
positive)
Radiological
findings
Fluorescent
stain
ZN Stain
Growth on
MGIT
culture
(days)
Multiplex
PCR
29/F
Urinary tract
None
Midstream urine
(1 of 3 samples)
ND
Negative
Negative
6
hsp65 z
ESAT6-MAC-
30/F
Central venous
catheter-related
mycobacteremia9
Ovarian carcinoma,
on chemotherapy
Blood from
catheter (4),
1peripheral (1),
catheter tip
ND
ND
ND
2.2
hsp65 z
ESAT6-MAC-
Clinical
significance
of NTM isolated
(as per ATS)
Possible NTM
disease/
contamination
Definite NTM
disease
Jain et al.
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Non-tuberculous mycobacterial disease in non-HIV infected
Pathogens and Global Health
Case Definitions:
Definite NTM lung disease (criteria by American Thoracic Society):7 at least two sputum samples positive for culture with NTM (or a single isolate in the case of bronchoscopy specimens) coexistent with
appropriate radiographic findings and clinical symptoms.
Definite Extrapulmonary NTM disease (British thoracic Society, BTS guidelines):8 one isolation of NTM from sterile site, repeated isolation (.2 times) from a non-sterile source, and histological evidence of tissue
invasion with compatible radiological and/or clinical features.
Catheter-related blood-stream infections: typical symptoms (exit site or tunnel site erythema, purulence, fever) and positive blood culture.
We defined probable NTM disease as one positive culture for NTM from sterile or non-sterile sources with compatible radiological and/or clinical findings but no evidence of tissue invasion or positive blood
culture.
Possible NTM disease/colonization: patients who did not fulfill the criteria for definite or probable NTM disease with single isolation of NTM from clinical specimens were considered to have possible NTM
disease or colonization.
* M. kansasii produces double band for ESAT6 primer, so it hinders the amplification of hsp65.
**Histopathological examination of biopsy showed granulomatous inflammation with necrosis and chronic inflammatory changes.
{Ascitic fluid cytology showed lymphocytic infiltrate with macrophages, leukocyte count: 200 cells/mm3 (12% neutrophils, 83% lymphocytes).
TB: tuberculosis; ZN: Zeihl Neelsen; ATS: American thoracic society; COPD: chronic obstructive pulmonary disease; MGIT: mycobacteria growth indicator tube; HPF: high-power field.
Jain et al.
Non-tuberculous mycobacterial disease in non-HIV infected
history of pulmonary TB while only 2% patients were
known HIV-positive.12
High prevalence of NTM infections observed among
non-HIV seropositive individuals in a TB endemic
country in this report, underscores the need for
increased awareness of these emerging human pathogens and importance of mycobacteria speciation to
reduce morbidity and mortality resulting from these
diseases. Use of liquid culture medium and molecular
methods in clinical laboratories can significantly reduce
turn-around time particularly for diagnosis of infections
due to slow-growing NTM and MDR-TB. However,
with improved facilities for recovery of NTMs that may
result in increased frequency of isolation of these
organisms, NTM disease requires clinical correlation
and differentiation from colonization.
Disclaimer Statements
Contributors Sarika Jain contributed to concept and
designing of the study, searching literature, analysing
data and preparing the manuscript. Manimuthu
Sankar performed conventional and molecular tests
for identification of NTM isolates and contributed in
analysing the data. Navneet Sharma contributed in
data acquisition and data analysis. Sarman Singh
contributed in manuscript preparation and editing. T.
D. Chugh contributed in the concept of study,
searching literature and manuscript editing.
Funding None.
Conflicts of interest None.
Ethics approval Ethical approval was not required.
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