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Keywords:

  • multiplex polymerase chain reaction;
  • rapid detection;
  • mycobacteria;
  • Mycobacterium tuberculosis;
  • India

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

For early detection and species differentiation of mycobacteria, polymerase chain reaction (PCR) techniques are currently in wide use. However, individual techniques using amplification of different targets with appropriate primers still have some limitations, which have to be overcome. The ideal technique would use DNA sequences which should be present in all mycobacteria and absent in others and would be able to discriminate one species from the other, as non-tuberculous mycobacteria (NTM) are on rise in terms of frequency of detection. We developed a multiplex PCR based on amplification of 165, 365 and 541 bp target fragments of unrelated genes, hsp 65 coding for 65 kDa antigen, dnaJ gene of mycobacteria and insertion element IS 6110 of Mycobacterium tuberculosis, respectively. This multiplex PCR was tested over 5 years from 1996 to 2001 with 411 clinical specimens from suspected cases of tuberculosis and mycobacterioses and compared with standard laboratory techniques. The multiplex PCR was positive for 379 cases compared with 280 cases by standard techniques (P < 0.0001). It could distinguish between strains of the M. tuberculosis complex and NTM; the results are comparable with standard techniques. Thus the multiplex PCR can be useful in early detection, species differentiation and epidemiology.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

Tuberculosis is a major public health problem both in developed and developing countries. Currently, more than one-third of the world's population is infected with Mycobacterium tuberculosis; eight million new cases and approximately two million deaths are reported each year (Dye et al. 1999). The situation was complicated with the advent of HIV infection which resulted in resurgence of tuberculosis even in industrialized countries between the mid-1980s and early 1990s (Small & Fujiwara 2001), and is further exacerbated by the emergence of multidrug-resistant (MDR) strains (Reichman 1991). Diagnosis is based on clinical pictures, radiological examination, the tuberculin test, and acid fast staining of a direct smear obtained from clinical specimen and culture (Bates 1979). There are various methods of culture in practice. Culture is the gold standard, but it requires a lot of time, whereas clinical urgency necessitates immediate laboratory back up. Moreover, in some paucibacillary cases such as genital tract tuberculosis, culture is often negative (Baum et al. 2001). Recently, the BACTEC system (Johnson Laboratories, BBI, Microbiological, Towson, MD, USA) came into use, which gives a very quick result compared with culture, but cannot yet do away with the standard culture technique for speciation of the isolate. It is also expensive and involves handling of radioisotopes. Serodiagnosis of tuberculosis also has many limitations (Daniel 1990). As an alternative to these classical methods, radiolabelled or non-radiolabelled nucleic acid-based technologies show promise as a more rapid, sensitive and specific means of identification of mycobacteria with some limitations.

An inexpensive, definitive and quick technique for detection of mycobacteria is urgently needed in developing countries like India. Again the therapeutic aspect of tuberculosis is complicated due to lack of proper laboratory facilities for species level identification and rapid emergence of MDR mycobacteria. The choice of therapy is dependent on the infecting species (Bass et al. 1990; Wallace et al. 1990) and identification of the organism(s) at an early phase of disease is required for optimal therapy and medical care decisions, e.g. whether isolation of the patient is necessary.

Polymerase chain reaction (PCR) is a sensitive method for detecting mycobacterial DNA or RNA directly in clinical specimens such as sputum, bronchial lavage, cerebrospinal fluid (CSF), ascitic fluid, pus, biopsy material, etc. Numerous PCR assays, which use conserved DNA or RNA sequences as targets for amplification, have been described for diagnosis of tuberculosis by detecting M. tuberculosis complex and mycobacterioses. This technology has shortened test periods from several weeks to 1–2 days or even less. But none of these methods are universal due to region-specific variations in the genome of mycobacteria (Yuen et al. 1993; Weil et al. 1996). Considering that so far no single target sequence exploited has yielded 100% sensitivity and a total absence of false positive results when used alone, there is a need to develop a PCR-based method as an efficient, single step and sensitive multiplex PCR from three different well established DNA target amplifications; and to use this newly developed multiplex PCR system successfully for differentiation of M. tuberculosis and non-tuberculous mycobacteria (NTM).

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

The following mycobacterial (tuberculous as well as NTM) reference strains were obtained from Central JALMA Institute for Leprosy, Agra (Indian Council of Medical Research) and were grown in Löwenstein–Jensen slants according to standard method: M. tuberculosis (H37Rv), M. bovis, M. smegmatis, M. intracellulare and M. avium. Clinical materials used comprised sputum, pleural fluid, CSF, ascitic fluid, blood, joint space fluid, endometrial tissue, urine, lymph gland biopsy material and pus from cold abscesses. Samples were collected from cases of suspected pulmonary tuberculosis, pleural effusion, meningitis, ascitis, pyrexia of unknown origin, arthritis, suspected female genital tract tuberculosis, renal tuberculosis, lymphadenitis and cold abscess, respectively (Table 1) and screened with conventional microbiological tests such as Ziehl–Neelsen acid fast staining for recording smear-positivity, identification by cultural isolation and biochemical tests. Löwenstein–Jensen medium was used for primary isolation. Tests to identify M. tuberculosis complex and NTM were: (i) niacin test, (ii) catalase test, (iii) heat stable catalase test, (iv) pigment production test, (v) Growth at 42–44 °C, (vi) aryl sulphatase test and (vii) state-of-the-art nucleic acid amplification tools like a multiplex PCR technique.

Table 1.  Distribution of samples
S. no.Type of sample*Number of samples
  • *

     All samples were collected from individual patients.

 1Sputum179
 2Pleural fluid29
 3CSF38
 4Ascitic fluid27
 5Blood38
 6Joint space fluid and material31
 7Endometrial tissue19
 8Urine17
 9Lymph gland biopsy material21
10Pus from cold abscess12
Total 411

After pre-treatment (Kolk et al. 1992) and necessary decontamination (Kubica et al. 1963), clinical specimens of urine, peripheral blood mononuclear cells (PBMC) containing traces of blood were prepared for mycobacterial DNA extraction by standard techniques (Boyum 1968). Other samples used were CSF (Kiyotoshi et al. 1992), blood, ascitic fluid, pleural fluid (Brisson-Noel et al. 1989), joint space fluid, endometrial tissues, lymph gland biopsy material and pus (Herrera et al. 1996).

Extraction of mycobacterial genomic DNA

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

One loop-full of the cultured mycobacteria (reference as well as isolated mycobacteria) was suspended in 500 μl of 1X TE [10 mm Tris–HCl, 1 mm ethylenediaminetetraacetic acid (EDTA), pH 7.5]. Then the suspension was lysed with 0.5% sodium dodecyl sulphate (SDS) and proteins were digested with 0.5 mg/ml of proteinase-K for 15 min at 37 °C.

Deoxyribonucleic acid was extracted from all mycobacterial strains and processed clinical materials by addition of an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v). The aqueous phase was transferred to another tube; DNA was precipitated with 3 m sodium acetate (pH 5.5) and 2.5 volumes of ethanol for 30 min at −80 °C. After washing, the pellets were dissolved in 100 μl of 1X TE and the concentration of nucleic acid was determined.

Extraction of mycobacterial DNA from sputum

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

Sputum samples were decontaminated by incubation with NaOH-sodium citrate-N-acetyl-l-cysteine for 20 min and centrifuged (Kubica et al. 1963). The pellet was washed once, resuspended in Tris–HCl buffer (pH 8.3) and digested in a proteinase-K detergent solution followed by DNA extraction as above.

Primers and development of multiplex PCR

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

Three separate PCR reactions, under different conditions and using three different sets of primers aimed to amplify three DNA target fragments of two conserved regions and an insertion element of DNA targets, have been positively incorporated into a single multiplex PCR system. The individual PCR reactions with respective sets of primers are as follows:

  • (i) 
    Two oligonucleotide primers derived from the sequence of gene that codes for the 65 kDa antigen of M. tuberculosis; a pair of 24 base synthetic oligonucleotide (primers) bracketing a 165-bp region of a gene codes for a 65 kDa antigen (Shinnick 1987). Briefly, according to Pao et al. (1990), 50 ng of purified total cellular DNA was amplified with thermostable Taq DNA polymerase in a Thermal Cycler (Perkin Elmer, Cetus, USA) GeneAmp PCR System 2400, Norwalle, CT 06859). To establish positivity, 50 μl of DNA amplification reaction mixture contained 10 mm Tris–HCl (pH 8.3); 50 mm KCl; 1.5 mm MgCl2; 0.01% (w/v) gelatin; 20 pmol of each of the two primers (from 5′ to 3′ ends CTA GGT CGG GAC GGT GAG GCC AGG and CAT TGC GAA GTG ATT CCT CCG GAT); 2.5 nmol of each of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP); 1 U of Taq DNA polymerase (Perkin Elmer) and appropriate amounts of specimen DNA, positive and negative control DNAs. The temperature of the reaction mixture was first raised to 94 °C for 20 s to denature the DNA, then cooled to 63 °C for 20 s. The temperature of the reaction mixture was then raised to 72 °C for 1 min, to extend DNA chain growth. This process was repeated 32 times, with 10-min incubation at 72 °C at the end.
  • (ii) 
    In this reaction two genus-specific oligonucleotide primers (forward, 5′-AAG AGG AAG GAG AGA GGG G-3′ and reverse, 5′-GTC GTT GAG GTT GAA CTC-3′) were used based on nucleotide sequence of dnaJ gene of M. tuberculosis (Syun-Ichi et al. 1993). The amplification of the 365-bp (region between sequence position 1377–1741 of M. tuberculosis) band has been observed in M. tuberculosis and M. avium, but not in other species of mycobacteria. This amplification is used for the identification of tuberculosis and NTM. Amplification was performed in a volume of 50 μl containing 1 U of Taq polymerase (Perkin Elmer), 50 mm KCl, 10 mm Tris–HCl (pH 8.3), 1.5 mm MgCl2, 0.01% gelatin, 50 pmol of each of the two primers and 200 μm of deoxynucleoside triphosphate mixtures (dATP, dCTP, dGTP and dTTP). Fifty nanograms of sample DNA was added lastly with positive and negative control DNA. Amplification was repeated with 38 cycles of denaturation at 94 °C for 30 s, primer annealing at 65 °C for 1 min and extension at 72 °C for 2 min with final extension of 10 min at 72 °C (Syun-Ichi et al. 1993).
  • (iii) 
    Two oligonucleotide primers within IS 6110 insertion element, designated primers Pt-8 (5′-GTG CGG ATG GTC GCA GAG AT-3′) and Pt-9 (5′-CTC GAT GCC CTC ACG GTT CA-3′), were used for PCR, resulting in amplification of a 541-bp DNA fragment (Kox et al. 1994). This IS 6110 insertion element is almost specific for M. tuberculosis complex. The composition of the PCR mix was 10 mm Tris–HCl (pH 8.3), 50 mm NaCl, 2.0 mm MgCl2, 0.01% (w/v) gelatin, 0.2 mm (each) deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP), 0.2 μm (each) primers Pt-8 and Pt-9, 1 U of Taq polymerase (Perkin Elmer) per 50 μl reaction volume. Approximately, 50–70 ng of DNA samples were added finally with positive and negative control DNAs. The reaction was performed by 40 cycles of 1.5 min of denaturation at 94 °C, 2 min of annealing at 65 °C and 3 min of primer extension at 72 °C, followed by final extension for 10 min at 72 °C (Kox et al. 1994).

Multiplex PCR

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

A new multiplex PCR was developed in our laboratory using primers and amplifying fragments of DNA targets as described above in PCR (i), (ii) and (iii) with the following proposed PCR mixture composition and condition: 10X PCR buffer-II [(GeneAmp, Perkin Elmer), 10 mm Tris–HCl (pH 8.3), 50 mm KCl, 0.01% gelatin (w/v), 1.5 mm MgCl2], 0.2 μm of each of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP), 0.2, 1 and 0.2 μm of primers, respectively, used in the PCR 1, 11 and 111, 1 U Taq DNA polymerase (Perkin Elmer). The reaction mixture was aliquoted in a volume of 45 μl in a PCR tube (500 μl) and 20–40 ng of sample DNA adjusted 1–5 μl, was added to each reaction tube to make a final volume of 50 μl. Positive and negative control DNAs were run in each experiment. The GeneAmp 2400 model (Perkin Elmer) of thermal cycler was used. The reaction mixture was first pre-incubated at 85 °C for 5 min, followed by initial denaturation for 10 min at 94 °C. The amplification was repeated with 40 cycles of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min and extension at 72 °C for 2 min with final extension of 10 min at 72 °C. Ten to 15 μl of the amplified reaction mixtures were fractionated electrophoretically in a 2% agarose gel, stained with ethidium bromide and visually inspected under UV light for bands of DNA of appropriate sizes (Figure 1). Next, DNAs extracted from all the reference strains including H37Rv as mentioned earlier, grown in Löwenstein–Jensen medium and other DNAs extracted from different clinical materials, as described earlier in the text, were screened with our newly developed multiplex PCR system.

image

Figure 1. Multiplex PCR using primers for heat-shock protein (165 and 365 bp) and insertion element IS 6110 (541 bp) and individual PCR showing 165, 365 and 541 bp of M. tuberculosis. Lane 1 – multiplex PCR of reference M. tuberculosis (H37Rv). Lane 2 – 541 bp amplicon of the same strain. Lane 3 – 365 bp amplicon of the same strain. Lane 4 – 165 bp amplicon of the same strain. M –φX174 marker.

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Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

McNemar's χ2-test was used for testing the differences between the proportions (Kirkwood 1990) and 95% confidence intervals (CI) of these differences were calculated.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

The multiplex PCR using the three sets of primers for three unrelated individual DNA fragment targets (165, 365 and 541 bp) was successfully developed with a minimum amount of DNA sample. At first multiplex PCR was applied to DNA extracted from M. tuberculosis reference strain (H37Rv strain) along with individual primer sets as proposed PCR conditions (Figure 1). From Figure 1 it is clear that the multiplex PCR system can simultaneously amplify three targets sequences, as described, of H37Rv strain.

The newly developed multiplex PCR system was tested with reference strains of M. smegmatis, M. bovis, M. intracellulare and M. avium along with M. tuberculosis H37Rv strain. In case of M. bovis only the 165 bp target was amplified whereas in M. smegmatis and M. intracellulare amplicons were 20–40 bp shorter than that of M. tuberculosis or M. bovis. But in these three cases 365 bp and 541 bp DNA fragment targets were not amplified. Mycobacterium avium can amplify both 165 bp (20–40 bp shorter) and 365 bp DNA fragment targets but not 541 bp DNA targets (Figure 2).

image

Figure 2. Multiplex PCR of different reference mycobacterial strains including non-tuberculous mycobacteria (NTM) and clinical samples using primers for heat-shock protein (165 and 365 bp targets) and insertion element IS 6110 (541 bp target). Lane 1 – pGEM marker. Lane 2 – reference M. tuberculosis strain (H37Rv). Lane 3 –M. tuberculosis (obtained from patient's cervical lymph node aspirate). Lane 4 –M. avium. Lane 5 –M. intracellulare. Lane 6 –M. bovis. Lane 7 –M. smegmatis. Lanes 8 and 9 –M. tuberculosis (obtained from patients' sputa). Lane 10 – negative control.

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To determine the sensitivity of the multiplex PCR, five series of 1000, 100, 10 and 1 fg of M. tuberculosis reference strain DNA were amplified with three sets of primers. In all five reference strains, 100 fg of DNA could be detected by agarose gel electrophoresis. PCR with 10 fg of DNA were positive in three of the five reference strains (i.e. amplifying the three targets).

Of the 411 samples studied, 280 (68.1%) were positive by smear and 241 (58%) were positive by culture. Of the 241 positive by culture, 143 (59%) were M. tuberculosis, 23 (9.5%), belonged to M. tuberculosis complex other than M. tuberculosis and 75 (31%) were NTM (Table 2). Compared with this, 379 (92%) samples were positive by multiplex PCR; 205 (54%) of these were positive for M. tuberculosis, 64 (16.8%) belonged to M. tuberculosis complex other than M. tuberculosis and 110 (29%) were positive for NTM (Table 2).

Table 2.  Comparison of the multiplex PCR results of clinical samples from patients suspected of mycobacteriosis with results of Z–N staining, culture and histopathology
 Acid-fast stainingCultureBiochemical testHistopathologyPCR
Type of sample(+) (–)(+) (–)M. tbM. tb complex (except M. tb)NTMTotal(+) (–)Total samples testedM. tbM. tb complex except M. tb)NTM
Sputum (179)1314810871591732173814250
Pleural fluid (29)25419101126241545
CSF (38)2711211714163622311
Ascitic fluid (27)1982251309251519
Blood (38)18201325706342338
Endometrial tissue (19)1361189021971218927
Joint space fluid and  material (31)1912211013173113182714310
Urine (17)7105124011751213823
Lymph node biopsy  material (21)147129624211110181035
Pus from cold abscess (12)759370211812

The positivity rate in PCR for diagnosis of tuberculosis was 92.2% (95% CI: 89–95%) compared with 68.1% of the smear. This is found to be statistically significant (P < 0.0001). The 95% CI for this difference between the proportions was 19%, 29% which indicates a positivity rate between 19% and 29% higher than smear in detecting tuberculosis cases (Table 3).

Table 3.  Comparison of PCR and smear technique for detection of tuberculosis cases among 411 specimens
 PCR (%) 
 (+)(–)Total (%)
  • *

     Difference in proportions was statistically significant (P < 0.0001).

Smear (+)273 (66.4)7 (1.7)280 (68.1)*
Smear (–)106 (25.8)25 (6.1)131 (31.9)
Total379 (92.2)32 (7.8)411

Compared with 241 culture positive samples for detection of M. tuberculosis complex, the PCR had a significantly higher rate of ability to detect tuberculosis caused by M. tuberculosis complex (P < 0.015). The M. tuberculosis complex positivity rate in PCR and culture methods among culture positive cases was 14.9% (95% CI: 10.0–19.0%) and 9.5%, respectively, indicating that the PCR technique is significantly better (P < 0.015) than culture. The difference in proportions (5.4%) was statistically significant (95% CI 2–9%) (Table 4). Of the 379 PCR positive samples, in all but three it was possible to amplify either all three or any two target fragments. The 541 bp fragment target of IS 6110 was not amplified in these three cases.

Table 4.  Comparison of culture method and PCR method for detection of M. tuberculosis complex other than M. tuberculosis among 241 culture positive samples
 PCR (%) 
(+)(–)Total (%)
  • Difference in proportions was statistically significant (P < 0.015).

Culture (+)17 (7.1)6 (2.5)23 (9.5)*
Culture (–)19 (7.9)199 (82.6)218 (90.5)
Total36 (14.9)205 (85.1)241

Compared with culture for detection of tuberculosis and mycobacterioses, the multiplex PCR system's sensitivity, specificity, positive predictive value and negative predictive value are 100.00%, 18.82%, 63.58% and 100.00%, respectively.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References

Definitive diagnosis of tuberculosis and mycobacterioses (NTM infections) has always been a serious problem. Traditional techniques such as smear positivity require presence of a large number of organisms in the clinical materials, whereas the culture technique for isolation is time consuming and requires even more time for species identification.

Deoxyribonucleic acid amplification is a useful tool in the diagnosis of mycobacterial diseases with high degree of sensitivity and specificity for detection of mycobacterial target DNA sequences. PCR is the method of choice for the diagnosis of tuberculosis in cases where the suspicion is high but Ziehl–Neelsen staining is negative. When the sample is positive in acid fast staining, PCR permits distinction between M. tuberculosis complex and other mycobacterial infections. Some of the clinical specimens that were positive by PCR but negative in culture were obtained from patients who were being treated for suspected tuberculosis. This is consistent with the fact that treated patients can still harbour mycobacteria long after culture for mycobacteria has become negative (Brisson-Noel et al. 1989). This may suggest that the DNA amplification method could detect mycobacteria that are unable to grow in vitro. The detection limit of PCR was much better than that of any other technique available in detecting mycobacteria including DNA probe method (Shoemaker et al. 1985; Pao et al. 1988).

Preliminary results indicate that our system can detect the mycobacterial DNAs in clinical specimens and identify species within 24 h. Successful use of DNA sequence(s) for the detection and identification of mycobacteria crucially depends on the right and logical choice of the target sequence(s), which ideally should be present in all mycobacterial species and absent from all other bacteria, and on the possible presence of particular sequence(s) specific to (a) M. tuberculosis, (b) M. bovis and (c) NTM.

So far no single target sequence has provided 100% sensitivity and a total absence of false positive when used alone. As discussed by Pao et al. (1990), a 165-bp DNA fragment of gene coding for 65 kDa antigen protein of M. tuberculosis is exploited for identification and differentiation of M. tuberculosis and M. bovisBCG (Bacillus Calmette-Guérin) from many other species of mycobacteria. The size of DNA fragments produced were 165 bp when M. tuberculosis and BCG DNAs were amplified. DNAs from other species of mycobacteria (M. avium, M. chelonae, M. fortuitum, M. gordonae, M. kansasi, M. paratuberculosis, M. phelei, M. smegmatis and M. xenopi) produced amplified DNA that was approximately 20–40 bp shorter than that from M. tuberculosis (Pao et al. 1990). Primers used by Syun-Ichi et al. (1993) are genus specific oligonucleotides, based on the nucleotide sequence of dnaJ gene of M. tuberculosis directed towards amplification of region between sequence positions 1377–1741 of M. tuberculosis (365 bp DNA fragment of M. tuberculosis and M. avium). This PCR is used for recognition of mycobacterial species with a broader spectrum. Again two oligonucleotide primers within IS 6110 insertion element, designated primers Pt-8 and Pt-9, were used for PCR product resulting in synthesis of 541 bp fragment. This IS 6110 insertion element is almost specific for M. tuberculosis complex (Kox et al. 1994).

Considering the increase in mycobacterial infections resulting from the emergence of NTM over the past decades, there is a need for a method that can detect virtually any deep-seated mycobacterial organism present in small numbers in suspected clinical samples irrespective of the mycobacterial species affiliation. For this purpose, we developed a multiplex PCR assay with three sets of primers that could sensitively and specifically identify clinically important mycobacterial species such as those of the M. tuberculosis, M. tuberculosis complex other than M. tuberculosis (specially M. bovis) and different major pathogenic NTM such as M. avium, M. intracellulare and non-pathogenic mycobacteria like M. smegmatis. The newly developed multiplex PCR was found to be highly effective in detecting mycobacterial infection from 411 suspected clinical specimens (positivity 92%) compared with standard technique (positivity 68%, P < 0.0001).

As more mycobacteria other than M. tuberculosis are increasingly found in clinical specimen, we evaluated our multiplex PCR to discriminate among the different species. The results obtained are comparable with standard techniques, although the method is more efficient in detecting M. tuberculosis complex other than M. tuberculosis (16.8%) compared with standard techniques (9.5%, P < 0.015). Thus this multiplex PCR is suitable for early detection of (i) human mycobacterial infection, (ii) M. tuberculosis infection at species level and (iii) other NTM causing human mycobacterioses at species level.

Although previous studies have reported that all members of M. tuberculosis complex have at least one copy, if not six to 15 copies, of IS 6110 insertion element in their genome (Van Soolingen et al. 1991; Otal et al. 1991), Yuen et al. (1993) first reported the existence of a group of M. tuberculosis strains that appear to have either one or no copy of IS 6110 insertion element. Most of these strains appeared to be isolated from patients of Vietnamese origin. This finding is consistent with our findings with some samples of suspected M. tuberculosis from north-eastern India. Thus, PCR diagnosis based exclusively on the IS 6110 sequence could not discriminate between M. tuberculosis and other mycobacteria from the M. tuberculosis complex, and the use of an IS 6110-based PCR method for diagnosis may, in some cases, lead to false-negative results. Therefore, the usefulness of IS 6110 for the epidemiological study may be limited in certain populations. In this situation, our multiplex system is still capable of identifying the mycobacteria through the 165 and 365 bp amplicon products. There is no report of M. africanum infection in humans in India. Therefore, the other two species of importance within M. tuberculosis complex, i.e. M. tuberculosis and M. bovis, can be specifically identified by this multiplex PCR.

In conclusion, the results presented here demonstrate that our multiplex PCR can be applied to a wide variety of clinical samples, making this system a useful tool in the diagnosis of mycobacterial diseases. This is the method of choice for the diagnosis of mycobacterial infections in cases where the suspicion is high but Ziehl–Neelsen staining or culture is negative. When the sample is positive by any one of the conventional tests, the multiplex PCR permits distinction among M. tuberculosis, M. bovis and other NTM.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Extraction of mycobacterial genomic DNA
  6. Extraction of mycobacterial DNA from sputum
  7. Primers and development of multiplex PCR
  8. Multiplex PCR
  9. Statistical analysis
  10. Results
  11. Discussion
  12. References
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