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Method- Freshly grown mycobacterial isolates on Lowenstein-Jensen medium were taken; Acetonitrile/Trifluoroacetic Acid extraction procedure was carried out, followed by charging of MALDI target plate with the extract and overlaying with Cinnamic acid matrix solution. Identification was performed using the MALDI-TOF MS.
Results- A comparative analysis of 42 clinical mycobacterial isolates using MALDI -TOF MS against conventional techniques was carried out. Amongst these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. However, the rest were not in accord with the conventional assays, including 1 discrepant sample. Furthermore, we observed a few signals recurrent in all mycobacterial isolates at species level.
Conclusion- In this first study from India, MALDI TOF MS was found to be an accurate, rapid, cost effective, highly versatile and robust system for identification of Mycobacterium tuberculosis. This innovative approach holds promise for earlier therapeutic intervention leading to better patient care and is an identification technology of the future.
Key Words: MALDI-TOF MS, Mycobacterium tuberculosis, India, identification.
Among the infectious diseases that are prevalent throughout the developing world, Tuberculosis (TB) continues to be a major public health issue. The updated WHO report (2009) states that about 9.4 million new TB cases occurred in 2008 worldwide with India being the highest TB burdened country in the world, accounting for 21% of the global incidence. In the year 2008, the incidence of tuberculosis was reported to be 1.982 million with prevalence of 2.186 million with mortality due to TB being 276,512 (1).
TB control has been a challenge for health care providers due to ambiguous, imprecise diagnosis and long duration of treatment (2, 3). Diagnosis of active tuberculosis is labor intensive and time consuming process, requiring 6 to 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 (4). The current approaches for early identification of Mycobacterium tuberculosis in culture include PCR (5) and the analysis of bacterial cell wall components by HPLC (6, 7). But 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 (8, 9, 10, 11, 12, and 13), Enterobacteriaceae (14), yeast (15, and 16), nonfermenting bacteria (17, 18, 19, and 20) and mycobacteria (21, 22, and 23). 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 first 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:
This study was carried out at the All India Institute of Medical Sciences (AIIMS), India, in collaboration with LRS Institute of Tuberculosis and Respiratory Diseases (LRSH), India, for a period 3 months from May, 2010 to July, 2010. All strains were clinical mycobacterial isolates obtained by cultivation of sputum, pus and lymph node aspirate specimens from different patients.
A total of 42 samples were taken and cultivated for a period of 2-8 weeks. Out of the 42 samples, 28 were male and rest 14 were female, informed consent was taken from all the patients. The age group of the study population varied from 11years to 65years with the median age being 31years. 38 isolates were grown from sputum specimens, 3 isolates from pus specimens and 1 from lymph node aspirate and all the specimens obtained were clinically diagnosed as pulmonary tuberculosis (Table 1).
Culture Conditions and Sampling:
The various samples received in the Microbiology laboratory of LRSH were cultured in 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 Âµl of 80% TFA was added. This was followed by resuspension of the mixture by pipetting until the complete dissolution/denaturation of the biological material. The denatured mixture was then incubated at room temperature for 10-30 minutes. Post incubation, 3 volumes of distilled water (150 Âµl) was added; an equal volume of absolute ACN (200 Âµl) was added to it and mixed using a vortex device. Centrifugation of the mixture was carried out at 13,000 rpm for 2minutes. The MALDI-TOF MS target plate was thus charged with 2 Âµl of the supernatant. Once dried, it was overlaid with 2 Âµl of matrix solution (Cinnamic acid in 50% Acetonitrile and 2.5% Trifluoroacetic acid). Along with the samples, Bacterial Test Standard and Mycobacterium tuberculosis H37RV (reference strain), 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 mass spectrometer for automated measurement and data interpretation. For identification, all the samples were blinded and run in duplicates.
The samples were analyzed 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 2,000 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 (PSI) of 250 ns, and no gating.
Initial manual/visual estimation of the mass spectra was performed using 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 done 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 (24, 25 and 26). 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 MSP in the database. The log (score) value ranged from 0 to 3, a log (score) value â‰¥1.7 is indicative of a close relationship (i.e. at the genus level) and a log (score) value â‰¥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.
During this study, a total of 42 clinical mycobacterial isolates and 2 controls were analyzed by MALDI-TOF MS. It was found that all the 2 controls gave log score values of >2.30 which indicate 'highly probable species identification'. Among the total 42 clinical isolates, while 97.61% (41/42) samples were identified as Mycobacterium isolates, 2.38% (1/42) could not be identified and was declared as 'no peaks' since the system could not find any signals. Out of these 41, 36 (87.80%) were recognised as Mycobacterium 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) according to the thresholds of the MALDI Biotyper software. Two strains were observed in these 36 isolates, namely Mycobacterium tuberculosis H37RV and Mycobacterium tuberculosis Ly_67.
In order to validate our results, the conventional tests were performed on the same culture samples separately. Through these tests, it was found that 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 Mycobacterium tuberculosis, 3 (7.14%) were classified as 'Non-Tuberculous Mycobacterium (NTM)' and 1 (2.38%) was Mycobacterium triplex.
On comparing the results obtained by MALDI-TOF MS with those from conventional diagnostic techniques, it was noticed that 41 isolates which were identified as Mycobacterium (till genus level) by MALDI-TOF MS were confirmed positively by the conventional methods. In addition to this, while 35 (85.36%) were declared accurate till the species level, the rest 5 (12.19%) did not agree with the conventional result. Furthermore, there was 1 (2%) isolate that was declared 'discordant' because although MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0), it was stated as NTM by conventional methods. All the 5 isolates (NRI) were correctly identified to the genus level by MALDI-TOF MS but had inaccurate species identification when compared to the reference methods. (Table.2). Amongst 5 isolates that were established as NRI by MALDI-TOF MS, 2 were identified as Mycobacterium tuberculosis, 2 as NTM and 1 as Mycobacterium triplex by the standard techniques.
On further analysis, it was seen that certain signals ranging from 3 to 10 kDa were common for all the 35 isolates. These "species-specific biomarkers" at m/z 5519, 5700, 7100, 8336, 9270, 10662 and 11376 are all present in each isolate. Moreover, it was observed that there were some signals that were typical of each strain namely Mycobacterium tuberculosis H37RV and Mycobacterium tuberculosis Ly_67. The signals at m/z â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. were restricted to Mycobacterium tuberculosis H37RV where as those at m/zâ€¦â€¦â€¦â€¦â€¦â€¦â€¦were unique to Mycobacterium tuberculosis Ly_67 .
Although there have been advancements in the diagnostics required for the identification of tuberculosis, none have substantially reduced the time lag between diagnosis and accurate treatment. In this first 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 and similar results were also obtained in other studies (14, 15, and16). The results obtained from our study suggest that identification of Mycobacterium 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 (8, 14, 15, and 23).
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 Mycobacterium tuberculosis. Such parallel conclusions were drawn by other researchers (21, 22, and 23) also thus reiterating 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, 1 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 (21). In the present study, the prime focus was on clinical isolates of Mycobacterium 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 (Fig. 1). Since a majority of the protein signals occurred between 2 to 13 m/z, it could imply that a significant portion of the signals were derived from ribosomal proteins (27) which range from 2000 to 20,000 Daltons.
Within the observed mass range, a few unique signals were conserved across all 35 isolates. In a similar study conducted by Hettick et al (23) on Mycobacterium tuberculosis, potential 'species-specific biomarkers' at m/z 4747, 5002, 5043 and 9083 Da were seen. In our study, in addition to such species-specific biomarkers, we found distinctive signals at m/z â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. Furthermore, previous studies have stated that the observed difference in the peak height ratio between spectra is evidence of differential expression of the same protein by multiple strains of the same species (23). Along with this, we found that there were certain unique signals which were absent in Mycobacterium tuberculosis Ly_67 but present in Mycobacterium tuberculosis H37RV and vice-versa. Therefore, such potential typical 'strain specific biomarkers' can be employed to distinguish between strains of a given species (Fig.2)
Through the course of our study, MALDI-TOF MS has performed well in terms of identification of Mycobacterium tuberculosis and has displayed numerous advantages in comparison to conventional methods of diagnosis. It was found to be an accurate and highly versatile system. The system could analyse samples rapidly within minutes and thereby facilitating high throughput outcome. In addition to this, the simple extraction procedure, low running cost and the non requirement of high technical expertise provides MALDI-TOF MS an edge over other methods for identification. Although there are several advantages of MALDI-TOF MS as a diagnostic tool, there are certain limitations to it like the application of excessive supernatant while charging the plate leads to degraded quality of results and special care must be taken to avoid cross contamination induced by liquid smears between neighbouring spots.
In conclusion, 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, thereby saving considerable time for therapeutic intervention and better patient care. Also, more data needs to be generated to objectively evaluate its clinical usefulness.