|Year : 2020 | Volume
| Issue : 2 | Page : 169-175
Epidemiology and risk factors associated with NTM pulmonary and extrapulmonary infections in a high tuberculosis endemic Region
Sanam Rasool Wani, Chand Wattal, Reena Raveendran
Department of Clinical Microbiology and Immunology, Sir Ganga Ram Hospital, New Delhi, India
|Date of Submission||22-Jun-2020|
|Date of Decision||29-Jun-2020|
|Date of Acceptance||13-Jul-2020|
|Date of Web Publication||29-Aug-2020|
Dr. Chand Wattal
Department of Clinical Microbiology and Immunology, Sir Gangaram Hospital, New Delhi
Source of Support: None, Conflict of Interest: None
Introduction: Non-tuberculous mycobacteria, although identified as pathogenic to humans long time ago, are emerging as the new threat in the past two decades. Even in tuberculosis endemic country such as India, they are being isolated from the clinical specimens more often than previously. This change in trend is of concern, because they are often misdiagnosed as Mycobacterium tuberculosis or even as drug-resistant tuberculosis. Objectives: A prospective, observational study was planned to identify the frequency and risk factors associated with pulmonary and extrapulmonary non-tuberculous mycobacterial (NTM) infections. Agreement between two commercially available molecular systems, namely GenoType Mycobacteria CM assay and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) used in the identification of mycobacterial species is also analysed. Materials and Methods: NTM isolated from pulmonary and extrapulmonary clinical specimens over a period of 1½ year was included in the study. Patient demographics were collected, and the risk factors associated with NTM infections were analyzed. NTM grown on culture was speciated using GenoType Mycobacteria CM assay (HAIN Life Sciences, Germany) and MALDI TOF MS (bioMerieux, France). Drug-susceptibility tests were done for rapid-growing NTM using E-test (bioMerieux, France). Results: Eight hundred and fifty-four mycobacteria were isolated from 5009 specimens processed during the study period. Out of the mycobacteria grown, 74 (8.7%) were NTM and 780 (91.3%) were Mycobacterium tuberculosis complex. The NTM isolated from pulmonary specimens were 46 (62.16%) and from extrapulmonary sources were 28 (37.84%). The most common species isolated from pulmonary specimens was Mycobacterium intracellulare and from extrapulmonary specimens was Mycobacterium abscessus. Concordance between the two commercial assays used for the identification was 96.49%. The most common risk factor associated with pulmonary NTM was previous lung pathology, while with extrapulmonary NTM infection was previous surgical intervention. Drug-susceptibility tests for rapid growers showed amikacin and clarithromycin as the most active drugs in vitro. Conclusions: NTM plays a significant role in causing pulmonary and extrapulmonary infections even in our part of the country with high endemicity of tuberculosis. NTM has emerged as important pathogens even in the immunocompetent patients. There is a need for rapid diagnosis and susceptibility testing of NTM to aid physicians administer timely and appropriate treatment to the patients.
Keywords: E-test, extrapulmonary, matrix-assisted laser desorption/ionisation time of flight mass spectrometry, Mycobacterium CM assay, non-tuberculous mycobacteria, pulmonary
|How to cite this article:|
Wani SR, Wattal C, Raveendran R. Epidemiology and risk factors associated with NTM pulmonary and extrapulmonary infections in a high tuberculosis endemic Region. Indian J Med Microbiol 2020;38:169-75
|How to cite this URL:|
Wani SR, Wattal C, Raveendran R. Epidemiology and risk factors associated with NTM pulmonary and extrapulmonary infections in a high tuberculosis endemic Region. Indian J Med Microbiol [serial online] 2020 [cited 2020 Sep 18];38:169-75. Available from: http://www.ijmm.org/text.asp?2020/38/2/169/293906
| ~ Introduction|| |
Non-tuberculous mycobacteria (NTM), although recognised as pathogens way back in 1930's, have seen a rising trend in laboratory isolation over the last couple of decades. This increase can be attributed both to increased disease awareness and improved identification techniques or there may be a true increase in NTM prevalence. Found ubiquitously in the environment, NTM has been reported from soil, water and hospital environment as well. They cause a range of infections, with pulmonary infections being the most common followed by other infections such as lymphadenitis, soft-tissue infections, infections of joints/bones, skin ulcers and disseminated disease., The rise in NTM isolation is of great concern as these are difficult to diagnose as well as to treat the treatment depends on the species involved, the site of disease and drug-susceptibility profile. Because of the NTM species being resistant to most of the drugs used in the first-line antitubercular treatment, it is important to speciate them to prevent them being misdiagnosed as multidrug-resistant tuberculosis. The present study aimed at finding the most common NTM species causing pulmonary and extrapulmonary infections to find out the risk factors associated with these infections and to find out the drug-susceptibility pattern of rapidly growing NTM species. We also tried to find out the agreement between two commercially available molecular systems, namely Genotype Mycobacteria CM assay (HAIN CM, HAIN Life Science, Germany) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS, bioMerieux, France) used in the identification of mycobacterial species.
| ~ Materials and Methods|| |
Ours was a prospective study done in the Department of Clinical Microbiology and Immunology at a tertiary care hospital in New Delhi, India, over a period of 1½ years from December 2015 to May 2016. The study was approved by the research and ethics committee of the hospital (EC/01/15/768). Both pulmonary and extrapulmonary specimens were included in the study. All the samples received in the Department of Clinical Microbiology for mycobacterial culture were subjected to fluorescent microscopy (Nikon, Japan) by auramine-rhodamine (HiMedia, Dindori, Nashik, Maharashtra) staining and culture in BacT/Alert 3D system (bioMerieux, Durham, NC) as well as on Lowenstein Jensen medium (LJ) (HiMedia, Mumbai, India).
All respiratory samples, urine and tissue samples were subjected to digestion and decontamination by N-acetyl-L-cystine (NALC), sodium hydroxide (NAOH) method. Other samples from sterile sites were subjected to decontamination only if a Gram stain or routine bacterial culture showed the presence of any organism, otherwise we proceeded directly.
All the samples were subjected to culture by BacT/ALERT and LJ media. The isolates which grew on the solid or liquid media were subjected to Accuprobe identification test (Gen-Probe Inc., San Diego, CA, USA) for MTB complex initially. Those negative for the Accuprobe MTB complex identification test were subjected to HAIN CM Assay as well as MALDI-TOF MS subsequently. As HAIN CM assay is a molecular assay detecting DNA in comparison to MALDI TOF MS which identifies organisms based on the proteomics of the organisms, we have tentatively taken HAIN CM as the gold standard. The NTM isolates were reported in accordance to the American Thoracic Society guidelines for respiratory isolates  and after correlating with clinical, radiological and histopathological evidences for both pulmonary and extrapulmonary samples and repeat isolation of the same organism wherever possible. All clinically significant NTM isolates were further evaluated for patient demographics and risk factors from the hospital records. Rapidly growing mycobacteria were subjected to drug-susceptibility using E-test (bioMerieux, France). Algorithm of work flow is shown in [Figure 1].
Geno Type Mycobacteria CM, HAIN Life Science
Based on the DNA-strip technology, this assay permits the simultaneous molecular genetic identification of the M. tuberculosis complex and 14 of the most common NTM species (Mycobacterium avium sp, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium interjectum, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium peregrinum, Mycobacterium marinum/Mycobacterium ulcerans and Mycobacterium xenopi) from the cultivated samples. The Genotype Mycobacteria CM assay was performed as per the recommendations of the manufacturers. Briefly, the amplification mixture consisted of 35 μl primer nucleotide mix, 5 μl of PCR buffer with, 2 μl of 25 mM MgCl2, 1 U of HotStar Taq DNA polymerase from Qiagen, 3 μl of molecular biology grade water, and 5 μl DNA supernatant in a final volume of 50 μl. Amplification was done in a thermal cycler (MyCycler, Bio-Rad Laboratories, USA) using the amplification profile: denaturation of 15 min at 95°C, followed by 10 cycles of 30 s at 95°C and 2 min at 58°C, 20 cycles of 25 s at 95°C, 40 s at 53°C and 40 s at 70°C and the extension step of 8 min at 70°C. Hybridisation was performed using a pre-programmed TwinCubator (Hain Life Science GmbH, Nehren, Germany). After denaturation, the biotin labelled amplicons were hybridised to the single-stranded membrane-bound probes. After a stringent washing, as streptavidin-alkaline phosphate conjugate was added to the strips, an alkaline phosphatase-mediated staining reaction was observed as bands where the amplicon and the probe hybridised.
Matrix assisted laser desorption/ionisation time of flight mass spectrometer
Microbial specimens are analysed based on the unique spectral fingerprints produced by extracted proteins. The resulting spectrum of mass distribution was interpreted according to a knowledge database developed by bioMerieux. It was performed as per the recommendations of the manufacturer. Briefly, a fresh subculture of each isolate was used as the starting material. After sufficient incubation to obtain adequate biomass (3–5 days for rapid growers and up to 3 weeks for slow growers), 1 μl loopful of growth suspended in 500 μl of 70% ethanol, bead beaten for 5 min and left to inactivate for 10 min, vortexed and suspension transferred to empty vial, then centrifuged and 10 μl of 70% formic acid added to the sediment after the removal of ethanol supernatant, vortexed, then 10 μl of acetonitrile added, centrifuged, and finally, 1 μl of this suspension inoculated on target slide followed by 1 μl of HCCA matrix on target slide and tested in VITEK MS.
All the rapidly growing mycobacterial isolates (M. abscessus, M. fortuitum and M. chelonae) were subjected to drug susceptibility testing by E-test. Amikacin, doxycycline, co-trimoxazole, ciprofloxacin, imipenem and clarithromycin were tested for MIC using E-test. All antibiotics were read at complete inhibition of growth, except co-trimoxazole which was read at 80% inhibition., MIC results were interpreted as per the CLSI guidelines for rapidly growing mycobacteria.
Descriptive statistics were analysed with the SPSS Inc.IBM statistics for Windows, version 17.0. Chicago: SPSS Inc. Continuous variables were presented as mean standard deviation. Categorical variables were expressed as frequencies and percentages. Nominal categorical data between the groups were compared using the Chi-square test or Fisher's exact test as appropriate. P < 0.05 was taken to indicate a significant difference.
| ~ Results|| |
A total of 5009 samples were processed during the study period in the mycobacteriology laboratory with 2132 (42.6%) pulmonary and 2877 (57.4%) extrapulmonary samples. A total of 854 mycobacteria were isolated over a period of 1½ years from both pulmonary and extrapulmonary samples, out of which 780 (91.3%) were M. tuberculosis complex and 74 (8.7%) were NTM.
Out of the 74 NTM, 46 (62.16%) were isolated from the pulmonary specimens as compared to 28 (37.84%) from extrapulmonary specimens, even though the samples tested were higher in the extrapulmonary category. The direct fluorescent staining report was positive in 38 (51.3%) of the 74 NTM cases. The mean age of patients in our study was 48.7 years; 49 (66.2%) patients were above 40 years of age. Nearly 47.3% of cases were males and 52.7% were females in our study the majority of pulmonary diseases were caused by M. intracellulare in 17 (36.96%) cases followed by M. kansasii in 13 (28.26%) cases. Other NTM isolates were M. abscessus in 10 (21.74%) cases, M. chelonae in 2 (4.35%), M. fortuitum in 2 (4.35%), M. gordonae in 1 (2.17%) and M. interjectum in 1 (2.17%) cases [Figure 2]. In extrapulmonary cases, a major chunk of infections was caused by M. abscessus followed by M. fortuitum, M. chelonae and M. gordonae, as shown in [Figure 3].
The most common risk factor in pulmonary cases was an underlying lung pathology in 29 (63%) of cases [Figure 4]. Among the lung pathologies, previous Kochs' disease was the most common with 14 (30.43%) cases followed by chronic obstructive pulmonary disease in 5 (10.87%), pleural effusion in 4 (8.7%), pneumonia in 3 (6.52%), bronchogenic carcinoma in 2 (4.35%) and bronchiectasis in 1 (2.17%) patient. Other risk factors associated with respiratory diseases in our study were smoking in 15 (32.6%) followed by diabetes mellitus in 8 (17.39%) cases. There was no obvious risk factor in 32.6% of pulmonary cases. A significant risk factor that we found in our extrapulmonary infection was a previous surgical exposure [Figure 5]. The surgeries were conducted in various other hospitals in Delhi and neighbouring states. The group comprised of patients following cholecystectomy, nephrectomy, hysterectomy, laparoscopic cystectomy, knee replacement surgery and thoracostomy. Diabetes was the risk factor found commonly associated with extrapulmonary isolates; however, 10.7% of extrapulmonary cases did not have any evident risk factor.
In the present study, out of the 74 NTM isolates identified by Genotype Mycobacteria CM by HAIN life sciences, 60 isolates were subjected to MALDI TOF MS. In our study, MALDI-TOF MS performed well, and there was 96.5% concordance between GenoType Mycobacteria CM and MALDI TOF MS [Table 1].
|Table 1: Correlation non-tuberculous mycobacterial species as identified by the two assays (MALDI TOF MS and GenoType Mycobacteria CM)|
Click here to view
Identification by HAIN CM is used as the reference standard in our study and was used for patient reporting. When 60/74 isolates were subjected to MALDI TOF MS subsequently, there were two discrepant isolates, both of them were identified by HAIN CM as M. chelonae, but were identified as M. abscessus by MALDI TOF MS. Taking HAIN CM as the gold standard, there were two discordant results by MALDI TOF MS. DNA sequencing could not be done to resolve discrepancy. There were 3 more isolates, 1 each of M. chelonae, M. interjectum and M. gordonae that were unidentified by MALDI TOF MS (M. interjectum is not in the data base of MALDI TOF MS but M. chelonae and M. gordonae are).
The susceptibility testing results of rapidly growing mycobacteria are shown in [Table 2] with amikacin and ciprofloxacin being the best options.
|Table 2: Drug susceptibility pattern of rapidly growing non-tuberculous mycobacterial (n=41)|
Click here to view
| ~ Discussion|| |
Although in a country like India, tuberculosis is still more common when mycobacterial disease related burden is considered, NTM diseases are showing a rising trend recently. For treating the patient appropriately, it gets important to identify mycobacteria to species level and not only rely on positive microscopy which cannot differentiate Mycobacterium tuberculosis from NTM. If not done, it only leads to diagnostic and clinical dilemmas to the disadvantage of the patient.
Out of the 5009 samples processed in the present study, 780 samples grew M. tuberculosis complex and 74 grew NTM which comprised 1.48% of the total mycobacterial isolates. There are not many studies on NTM prevalence from India. Among the available data from our country, a study by Jesudason andGladstone conducted in Vellore, Tamil Nadu, M. tuberculosis comprised 96.1% of the acid-fast bacilli and NTM were 3.9%. In yet another study from India, NTM constituted 9.9% of the total 131 culture-positive mycobacteria. In our study, pulmonary specimens comprised 62.16% (n = 46) and extrapulmonary comprised 37.84% (n = 28) of total 74 NTM isolated. In another study from India by Shenai et al., similar results were observed, 81% of the total NTM isolates were from pulmonary sources and 19.5% were from extrapulmonary sources. Jain et al. also found pulmonary isolates constituted 69.2% as against 30.8% of non-pulmonary specimens of the total NTM isolated. However, there are studies where extrapulmonary NTM isolates outnumber pulmonary ones like the one conducted in Vellore India where pus and biopsy specimens yielded maximum number of NTM followed by respiratory specimens unlike our study.
It has been reported by various studies that the sensitivity of direct fluorescent staining is lower for NTM as compared to MTB like the one reported by P W Wright et al. wherein the sensitivity of MTB was higher (63%) as compared to NTM (56%). Lower sensitivity in case of NTM may be because of the paucibacillary nature of NTM diseases. In the current study of NTM, we also found the direct fluorescent positivity in 38 (51.3%) cases.
Males comprised 47.3% of cases in our study and females comprised 52.7%. In most of the studies on NTM infections males outnumber females. Similar to our result, Jesudason et al. found NTM to be more prevalent in women as compared to men. In our pulmonary isolates, M. intracellulare and M. kansasii were the main pathogens. These species have been found to outnumber others in a study by Shenai et al. also wherein pulmonary infections were mostly caused by M. intracellulare followed by M. simiae and M. abscessus. Although in another study mentioned earlier from India, the most common NTM isolated from respiratory specimens were M. fortuitum and M. chelonae which is at variance from our finding, instead majority of our extrapulmonary infections were caused by M. abscessus followed by M. fortuitum and M. chelonae.
It has long back been accepted that NTM diseases are associated most of the times with underlying risk factors. In our study, we found underlying lung pathology to be associated with pulmonary NTM diseases in 29 (63%) of cases [Figure 4]. Among the lung pathologies, previous Kochs disease lead the group with 14 (30.43%) followed by chronic obstructive pulmonary disease, pleural effusion, pneumonia, bronchogenic carcinoma and bronchiectasis. Such risk factors have also been documented in earlier studies as well., In a study conducted in African gold miners by Sonnenberg et al. to find risk factors associated with pulmonary tuberculosis and NTM infections, NTM infected were more likely to have had a previous tuberculosis treatment. Other risk factors associated with respiratory diseases in our study were smoking in 15 (32.6%) followed by diabetes mellitus in 8 (17.39%) cases. In a study conducted in South London in non-HIV infected pulmonary NTM infections predisposing factors were smoking (70%), alcohol abuse (28%) and chronic obstructive pulmonary disease (37%). We had one patient who was simultaneously detected as a case of bronchogenic carcinoma. In our study, we had a significant number of patients who did not have any apparent risk factors. Jain et al. in their study also had 23.1% of cases who did not have any apparent risk factor.
Notoriously known for their resistance to the commonly used disinfectants, the rapidly growing mycobacteria pose a serious threat of infection in the post-operative patients. As we found in our study, a very high number of extrapulmonary cases, 20 (71.43%) were from post-operative surgical wounds. The surgeries were conducted at various different hospitals in and around Delhi. These results compare favourably with a study by Kannaiyan et al. from south India where they analyzed 19 patients presenting with painful subcutaneous draining nodules from the surgical sites. Pus samples of all these patients grew rapidly growing mycobacteria M. fortuitum and M. chelonae. Addressing the problem of port site infections in laparoscopic surgeries, Sasmal et al. have given 10 commandments for the prevention of port site infections. Such observations question the sterility precautions taken during surgeries. Other risk factors found commonly associated with our extrapulmonary isolates were diabetes. Diabetes is known to predispose a person to NTM infections. Only three NTM isolates were from blood specimens which could possibly be a disseminated infection. Two patients were HIV negative and HIV status of third patient was not known.
Phenotypic methods for speciating NTM are cumbersome and not very reliable. In the present study, NTM isolates were speciated using the molecular methods such as HAIN CM and MALDI TOF MS. Many studies have already shown that Geno Type Mycobacteria CM assay to correctly identify 89.3%–100% of mycobacterial isolates.,,, Mycobacterium CM assay has an advantage of rapid and accurate identification of a range of mycobacteria as compared to phenotypic methods and an additional advantage over other molecular methods is the less turnaround time. Out of the 74 NTM isolates in our study, all were subjected to GenoType HAIN CM, and only 60 isolates were subjected to MALDI TOF MS. In our study, MALDI-TOF MS performed well, and there was 96.5% concordance between HAIN CM and MALDI TOF MS results. The difference in the results of the two assays was statistically insignificant [Table 1]. This is consistent with the results of Mediavilla-Gradolph et al. where they found 96.9% concordance between the results of these two assays. There were only two discrepancies between the results of two assays. Both the discrepancies were between M. abscessus and M. chelonae. There were three isolates which were not identified by MALDI TOF MS, one isolate each of M. gordonae, M. chelonae and M. interjectum. These results are similar to a study by Sanchez et al. where they found discrepancy between M. abscessus and M. chelonae between the two assays and in their study also M. gordonae was not identified by MALDI TOF MS. M. abscessus and M. chelonae are two closely related species that are often not distinguished by clinical laboratories. They are so closely related species that, prior to 1992, M. abscessus was considered as a subspecies of M. chelonae.
The American thoracic society recommends drug-susceptibility tests to be done for rapidly growing mycobacteria as the sensitivity pattern varies from the organism to organism. In our study, Clarithromycin and amikacin were the most potent drugs in vitro. Ciprofloxacin and imipenem were showing some activity, whereas cotrimoxazole and doxy cycline were resistant in most of the cases [Table 2]. These observations are similar to a study by Hui et al. where 31 anti-microbial agents were tested. Aminoglycosides and fluoroquinolones displayed a range of activities against the rapidly growing mycobacteria isolates. Amikacin demonstrated the highest activity (72/73, 98.63%), while moxifloxacin showed a range of activities (57/73, 78.08%). Tigecycline (70/73, 95.89%) had much higher activity against the isolates than minocycline (30/73, 41.10%) and doxycycline (25/73, 34.25%). Clarithromycin (48/73, 65.75%), azithromycin (53/73, 72.60%) and roxithromycin (48/73, 65.75%) showed different in vitro activities against the rapidly growing mycobacterial isolates. Meropenem (52/73, 71.23%) exhibited good activity against the strains tested. In another study from our centre, the drug-susceptibility pattern of rapidly growing mycobacteria showed a similar pattern. M. fortuitum and M. chelonae showed 100% sensitivity to amikacin and M. chelonae and M. abscessus showed 100% sensitivity to clarithromycin.
There were two major limitations in our study: HAIN CM which was used to identify NTM can detect only 14 most common mycobacterial species causing human infections. However, we could identify all NTM isolates in this study fortunately. Molecular gene sequencing could not be done in cases where there was a discrepancy between HAIN CM assay results and MALDI-TOF MS results, to resolve the discrepancy.
| ~ Conclusions|| |
Even in a TB endemic country like India, we should not ignore the possibility of acid-fast bacilli seen in microscopy as being an NTM, especially in cases with pre-existing pathologies, culture and identification are highly recommended in such settings. Speciation of NTM should always be done as different species have different managements.
As most of our extrapulmonary cases presented with post-operative infection, we recommend infection control measures should be strictly enforced and adequate care should be ensured in areas such as reprocessing of endoscopes and hospital water supply.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]