|Year : 2013 | Volume
| Issue : 2 | Page : 117-122
Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry
A Panda1, S Kurapati1, JC Samantaray1, VP Myneedu2, A Verma2, A Srinivasan3, H Ahmad1, D Behera4, UB Singh1
1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, L. R. S. Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
3 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
4 Department of Respiratory Medicine, L. R. S. Institute of Tuberculosis and Respiratory Diseases, New Delhi, India
|Date of Submission||07-Aug-2012|
|Date of Acceptance||03-Apr-2013|
|Date of Web Publication||19-Jul-2013|
J C Samantaray
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: The work was supported by the All India Institute of Medical Sciences and the L. R. S. Institute of Tuberculosis and Respiratory Diseases, New Delhi, Conflict of Interest: None
Purpose: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Materials and Methods: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. Results: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score) >2.0) and conventional methods established it to be non-tuberculous Mycobacterium. Conclusions: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.
Keywords: Clinical, identification, matrix assisted laser desorption ionization-time of flight mass spectrometry, mycobacterial species, rapid
|How to cite this article:|
Panda A, Kurapati S, Samantaray J C, Myneedu V P, Verma A, Srinivasan A, Ahmad H, Behera D, Singh U B. Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry. Indian J Med Microbiol 2013;31:117-22
|How to cite this URL:|
Panda A, Kurapati S, Samantaray J C, Myneedu V P, Verma A, Srinivasan A, Ahmad H, Behera D, Singh U B. Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry. Indian J Med Microbiol [serial online] 2013 [cited 2017 Dec 14];31:117-22. Available from: http://www.ijmm.org/text.asp?2013/31/2/117/115217
| ~ Introduction|| |
Among the infectious diseases that are prevalent throughout the developing world, tuberculosis (TB) continues to be a major public health issue. The WHO report on Global Tuberculosis Control (2011) states that about 8.8 million new TB cases occurred in 2010 world-wide with India being the highest TB burdened country in the world, accounting for a quarter (26%) of all TB cases worldwide. 
TB control has been a challenge for health-care providers due to ambiguous, imprecise diagnosis and long duration of treatment. , Diagnosis of active tuberculosis is labour intensive and time consuming process, requiring 6-8 weeks for identification after culture positivity. With the introduction of broth culture systems for the isolation of mycobacteria, there has been a decline in the time required for identification substantially. However, species identification by conventional phenotypic traits is still lengthy and may frequently result in erroneous identification.  The current approaches for early identification of Mycobacterium tuberculosis in culture include polymerase chain reaction (PCR)  and the analysis of bacterial cell wall components by high performance liquid chromatography. , However, these methods are expensive and technically demanding and therefore, inaccessible to the lower strata of the society. To optimize patient care, therapeutic management and timely intervention for infection control, there is an urgent need for rapid, accurate, simple and economical technique for mycobacterial identification.
As an alternative to chromatographic and DNA-dependent methods, mass spectral analysis and identification of micro-organisms has become increasingly recognized. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for accurate and rapid identification of various microorganisms, , such as gram-positive bacteria, ,,,,, Enterobacteriaceae,  yeast, , nonfermenting bacteria ,,, and mycobacteria. ,,,,,, This technique is based upon the detection of highly abundant proteins in a mass range between 2 and 20 kDa by computing their mass (m) to charge (z), m/z values. Thus, a typical spectrum is generated for each microorganism, which is used for comparison with the stored reference spectra and thereby providing identification for the sample.
In this initial study from India, the applicability of MALDI-TOF MS for identifying clinical mycobacterial isolates was evaluated. This approach was further validated by conventional, morphological as well as biochemical identification.
| ~ Materials and Methods|| |
A total of 42 smear positive acid fast specimens were collected from clinically diagnosed pulmonary tuberculosis patients. Out of the 42 cases, 28 were male and 14 were female. Informed consent was taken from all the patients. The age group of the study population varied from 11 years to 65 years with the median age being 31 years. 38 isolates were grown from sputum specimens, 3 isolates from pus specimens and 1 from lymph node aspirate. These samples were further cultivated for a period of 2-8 weeks on Lowenstein-Jensen slants in duplicate. The isolated colonies in Lowenstein-Jensen slant were confirmed by Ziehl-Neelsen staining. The smear confirmed mycobacterium growths were subjected to biochemical test for species identification by established standard protocols.
Mycobacterial preparation for MALDI-TOF MS analysis
Trifluoroacetic acid (TFA)/acetonitrile (ACN) extraction procedure was followed. Isolated colonies grown on Lowenstein-Jensen slants were taken by scraping and the intact colonies were (5-10 mg) transferred into a 1.5 ml "eppendorf tube" with the help of an inoculation loop. To this biological material, 50 μl of 80% TFA was added. This was followed by resuspension of the mixture by pipetting until the complete dissolution of the biological material. The denatured mixture was then incubated at room temperature for 10-30 min. Post-incubation, three volumes of distilled water (150 μl) was added; an equal volume of absolute ACN (200 μl) was added to it and mixed using a vortex device. Centrifugation of the mixture was carried out at 13,000 rpm for 2 min. The MALDI-TOF MS target plate was thus charged with 2 μl of the supernatant. Once dried, it was overlaid with 2 μl of matrix solution (cinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid). Freshly prepared Bruker bacterial test standard ( Escherichia More Details coli) (#255343) and M. tuberculosis H37RV (reference strain obtained from Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases (LRSH)) were used as controls and were also pipetted onto the plate and overlaid with the matrix. The samples as well as controls were allowed to dry completely at room temperature. The MALDI-TOF MS target was subsequently introduced into the MALDI-TOF MS for automated measurement and data interpretation. For identification, all the samples were blinded and run in duplicates.
The samples were analysed using a Microflex LT MALDI-TOF MS instrument (Bruker Daltonik GmbH, Bremen, Germany). The spectra were recorded in the linear positive mode at a laser frequency of 20 Hz within a mass range from 2000 to 20,000 Da. Parameter settings for Microflex instrument were ion source 1 at 20 kV, ion source 2 at 18.5 kV, lens at 8.5 kV, pulsed ion extraction of 250 ns and no gating.
Initial manual/visual estimation of the mass spectra was performed using the FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany). For automated data analysis, raw spectra were processed using the MALDI BioTyper 1.1 software (Bruker Daltonik GmbH, Germany) with default settings. The smoothing, normalization, baseline subtraction and peak picking was carried out by the software, thereby creating a list of the most significant peaks of a spectrum (m/z values with a given intensity). The generated peak lists derived from the mycobacterial MALDI-TOF profile mass spectra were compared with each entry of the MALDI Biotyper database, which currently contains 3287 references (16 mycobacterial references), using the standard parameters of the pattern-matching algorithms. These algorithms have different mathematical approaches, which have already been described. ,, The results of the pattern-matching process were expressed as log (score) values, computed by comparison of the peak list for an unknown isolate with the reference main spectral pattern (MSP) in the database. The log (score) value ranged from 0 to 3, a log (score) value ≥1.7 is indicative of a close relationship (i.e., at the genus level) and a log (score) value ≥2.0 is the set threshold for a match at the species level. The highest log (score) of a match against the score in the database was used for species identification.
| ~ Results|| |
In this study, a total of 42 clinical mycobacterial isolates and 2 controls were analysed by MALDI-TOF MS. It was found that the two controls gave log (score) values of >2.30 which indicate "highly probable species identification." Among the total 42 clinical isolates while 41/42 (97.61%) samples were identified as Mycobacterium isolates, 1/42 (2.38%) could not be identified as "no peaks" were detected. Out of these 41, 36 (87.80%) were recognised as M. tuberculosis isolates while the rest 5 (12.19%) were deemed "not reliable identification" or "NRI" as their log (score) values were too low (<1.70). M. tuberculosis H37RV and M. tuberculosis Ly_67PGM strains were observed in these 36 isolates [Table 1].
|Table 1: Summary of sample identifi cation by conventional technique and MALDI-TOF MS|
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In order to validate our results, the conventional tests were performed on the same culture samples separately. All the 42 samples were identified till the genus level as Mycobacterium isolates. Furthermore on performing the speciation, it was observed that 38 (90.47%) isolates were M. tuberculosis, 3 (7.14%) were classified as "non-tuberculous Mycobacterium (NTM)" and 1 (2.38%) was M. triplex [Table 1].
On comparing the results obtained by MALDI-TOF MS with those from conventional diagnostic techniques, it was noticed that out of 41 isolates which were identified as Mycobacterium (till genus level) by MALDI-TOF MS, 35 (85.36%) isolates were identified accurately to the species level and 5 (12.19%) had inaccurate species identification. 1 (2.43%) isolate was declared "discordant" because MALDI-TOF MS established it as M. tuberculosis (log (score) >2.0) and conventional methods established it to be NTM [Table 1]. Among the 5 inaccurately identified isolates (at species level) that were designated as NRI by MALDI-TOF MS, 2 were identified as M. tuberculosis, 2 as NTM and 1 as Mycobacterium triplex by the conventional techniques [Table 2]. Certain signals ranging from 3 to 10 kDa were common for all the 35 isolates that were identified as M. tuberculosis by MALDI-TOF MS. These "species-specific biomarkers" at m/z 5519, 5700, 7100, 8336, 9270, 10,662, and 11,376 are all present in each isolate [Figure 1].
|Figure 1: (a) Matrix assisted laser desorption ionization-time of fl ight mass spectrometry (MALDI-TOF MS) of bacterial test standard. (b) MALDI-TOF MS of Mycobacterium tuberculosis H37RV. (c) MALDI-TOF MS of M. tuberculosis Ly_67PGM|
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|Table 2: Overview of samples identifi ed as "NRI" by MALDI-TOF MS versus conventional identifi cation|
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| ~ Discussion|| |
Although there have been advancements in the diagnostics for the identification of tuberculosis, none has substantially reduced the time lag between diagnosis and accurate treatment. In this initial study from the subcontinent, we evaluated the application of MALDI-TOF MS as a potential alternative tool for diagnosis of TB. MALDI-TOF MS could accurately identify all the controls. Similar results were also obtained in other studies. ,, The results obtained from our study suggest that identification of M. tuberculosis and the different strains within, is possible using MALDI-TOF MS. Since, our study was a blinded one with the samples being processed and run in duplicates, the reproducibility of the instrument was tested and was subsequently found to be consistent for all the samples. The reproducibility is in accordance with previous evaluations of MALDI-TOF MS. ,,,
On comparing with the conventional tests, the results yielded by MALDI-TOF MS corroborated on most of the accounts at the genus level except one isolate implying that the MALDI-TOF MS displayed 97.61% precision. Furthermore, it was noted that majority of the samples were confirmed at the species level thereby exhibiting an accuracy of 85.36%, indicating the ability of MALDI-TOF MS to precisely recognise M. tuberculosis. Similar conclusions were drawn by other researchers ,, also; it reiterates the fact that MALDI-TOF MS has the capacity to distinguish between various genera and species. The assignment of NRI status could be attributed to an incorrect species allocation due to the non-availability of taxonomic reference for the given spectrum. This accounts for the marginally lower species level identification when weighed against the genus level accuracy in our study. In addition to this, one isolate which was considered discordant in the study could be ascribed to a possible mixed or contaminated culture.
In previous studies, investigators have demonstrated the applicability of MALDI-TOF MS for identification of various mycobacterial species, but only a fraction have concentrated on clinical isolates.  In the present study, the prime focus was on clinical isolates of M. tuberculosis along with multiple strains of the same at a tertiary care hospital. On carrying out an extensive analysis of the conventionally verified isolates, a consistent spectral pattern among all the isolates at the species level was revealed. Since, a majority of the protein signals occurred between 2 and 13 m/z, it could imply that a significant portion of the signals were derived from ribosomal proteins which range from 2000 to 20,000 Da.  Within the observed mass range, a few unique signals were conserved across all 35 isolates similar to the studies conducted by Hettick et al.,  El Khéchine et al.  and Saleeb et al.  on different M. tuberculosis strains thereby highlighting these signals to be potential "species-specific biomarkers." Furthermore, it was observed that there was a difference in the peak height ratio across the spectra of strains within a species [Figure 1]. This could be attributed to the differential expression of the same protein among different strains. Such conclusions were reached by other researchers as well. 
The method could analyse samples rapidly within minutes and thereby facilitate high throughput outcome. In addition to this, the simple extraction procedure, low running cost (consumable costs less than Rs. 4 per sample) and the non-requirement of high technical expertise provide MALDI-TOF MS an edge over other methods for identification. The limitations like the application of excessive supernatant while charging the plate and cross-contamination induced by liquid smears between neighbouring spots leading to poor quality of results, can be dealt with care.
| ~ Conclusions|| |
Our study demonstrated that owing to its rapid and accurate nature, MADLI-TOF MS could be a possible alternate diagnostic tool for identification and differentiation of clinical mycobacterial isolates. It may be validated with larger sample numbers in a multicentric study.
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[Table 1], [Table 2]