Respiratory Medicine CME
Volume 2, Issue 4 , Pages 151-154, 2009

Newer diagnostic techniques for tuberculosis

Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh 202002, U.P, India

Article Outline

Abstract 

Because of the low sensitivity of sputum smears and the delay in cultures (2–4 weeks for a result), a variety of new technologies have been developed for the more rapid and sensitive detection of PTB. This article address the new diagnostic techniques for Tuberculosis. These techniques help in early daignosis of tuberculosis.

Keywords: Tuberculosis, Bactec, PCR

 

Back to Article Outline

Educational aims 


•To outline new diagnostic techniques for diagnosis of TB infection

•To outline new diagnostic techniques TB Disease.

Back to Article Outline

1. Introduction 

Because of the low sensitivity of sputum smears and the delay in cultures (2–4 weeks for a result), a variety of new technologies have been developed for the more rapid and sensitive detection of PTB. At present, the use of these techniques is the subject of much debate.

New diagnostic techniques in can be broadly divided into two parts

1)Techniques for diagnosis of TB infection.

2)Techniques for the diagnosis of TB disease.

Back to Article Outline

2. Techniques for diagnosis of TB infection 

2.1. Interferon-assays(IFN-γ) 

This test measures the amount of interferon-γ; in the blood. On stimulation by mycobacterial peptides the amount of IFN-gamma increases in the blood. This test is different from conventional BCG test as it uses proteins known as ESAT-6 and CFP-10, which are present in Mycobacterium tuberculosis but absent from all BCG strains and from most non-tuberculosis mycobacteria. It is intended for use as a diagnostic aid for M. tuberculosis infection, including both tuberculosis disease and latent tuberculosis infection (LTBI). The advantages of, Interferon-assays compared with the tuberculin skin test, are that results can be obtained after a single patient visit, and that, because it is a blood test which is performed in a qualified laboratory, the variability associated with skin test reading can be eliminated.

An additional advantage of this newer generation blood test is that the test is not affected by past BCG vaccination, and will eliminate the unnecessary treatment of patients with BCG-related false positive results. Furthermore, the Interferon-assays does not affect the result of future Interferon-assays tests (i.e. no “boosting,” as with the TST).

Various interferon- assays available are-

•Enzyme linked immunosorbent assay (ELISA) e.g QuantiFERON TB and QuantiFERON TB Gold assay.

•Enzyme linked immunospot – ELISPOT (e.g T SPOT TB assay).

Back to Article Outline

3. Techniques for the diagnosis of TB disease 

3.1. Direct methods for detection of Mycobacteria or its products 


(a)w?>Microscopy – Advancement had been made to increase the sensitivity of sputum microscopy. A new method to improve culture and acid-fast staining of mycobacteria is introduced. This new method utilized N,N-dimethyl-N-(n-octadecyl)-N-(3-carboxypropyl) ammonium inner salt, also known as C18-carboxypropylbetaine (Zwitterionic detergent).1

(b)Culture – The major drawback in culturing mycobacteria on conventional media is that it takes at least 4 weeks. Various rapid methods are now available, these include
(i)Microcolony detection on solid media

(ii)Septi-check AFB method

(iii)Microscopic observation of in broth culture (MODS)

(iv)BACTEC 460 radiometric culture system

(v)BACTEC MGIT 960 system

(vi)MB/Bact system

(vii)ESP II culture system


(i) Microcolony detection on solid media
This is a better technique for culturing tubercle bacilli.2 This technique is less expensive and requires about half the time needed for conventional culture, however the recovery of mycobacteria is less efficient.

(ii) Septi-check AFB method
It is biphasic culture system made up of a modified Middlebrook 7H9 broth with a three-sided paddle containing chocolate, egg based, and modified 7H11 solid agar, the bottle is inverted regularly to inoculate the solid media. The growth is observed by observing the three-sided paddle.

(iii) Microscopic observation of in broth culture (MODS)
This technique is appropriate for disease endemic high burden countries. It requires 7H9 broth, oleic acid dextrose catalase (OADC) and antimicrobial supplement.3

(iv) BACTEC 460 radiometric culture system
Culture media contains 14C-labeled palmitic acid. If present in the broth, mycobacteria metabolize the 14C-labeled substrates and release radioactively labeled 14CO2 in the atmosphere, which collects above the broth in the bottle. The instrument withdraws this CO2-containing atmosphere and measures the amount of radioactivity present. Bottles that yield a radioactive index, called a growth index, greater than or equal to 10 are considered positive.

(v) BACTEC MGIT 960 system
Culture tube contains Middlebrook 7H9 broth and a fluorescent compound embedded in a silicone sensor. Growth is detected visually using an ultraviolet light. Oxygen (O2) diminishes the fluorescent output of the sensor; therefore, O2 consumption by organism present in the medium are detected as an increase in fluorescence.

(vi) MB/Bact system
This system is based on the colorimetric detection of CO2. It had slightly longer time for detection of growth as compared to BACTEC 460 system.4

(vii) ESP II culture system
Organism are cultured in a modified Middlebrooks 7H9 broth with enrichment and a cellulose sponge to increase the culture's surface area. The instrument detects growth by monitoring pressure changes that occurs as a result of O2 consumption or gas production by the organism as they grow. It should be used in combination with solid media.5

3.2. Identification of Mycobacterial species 

Earlier biochemical's were used for identification of mycobacterial species,6 however these were time consuming. To over come this various methods have come up, these are –

a)Lipid profiles – Since mycobacteria have characteristic lipid profile, these can be analyzed by high-pressure liquid chromatography.7

b)SDS-PAGE – The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of an easily and rapidly prepared soluble protein fraction were used in conjunction with conventional techniques to identify different strains of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium. bovis BCG, Mycobacterium africanum, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium gastri, Mycobacterium simiae and Mycobacterium malmoense. With the SDS-PAGE technique, all strains of the M. tuberculosis complex were recognized as belonging to one species. By visual analysis of the SDS-PAGE polypeptide profiles, only minor differences between strains of the same species were seen and each species show a characteristic polypeptide profile.8

c)Sherlock Mycobacterial Identification System – The Sherlock Mycobacterial Identification System (SMIS; MIDI, Inc.) has been developed for the rapid, computer-assisted identification of mycobacterial species based on the separation and quantification of mycolic acids using HPLC technology.9

d)GenoType assay – The GenoType assay allows for the identification of 16 different mycobacterial species that are most frequently isolated in the clinical laboratory.10 Isolation is commonly done by PCR amplification of the 16S–23S ribosomal DNA spacer region followed by hybridization of the biotinylated amplified DNA products with 16 specific oligonucleotide probes.10 The specific probes are immobilized as parallel lines on a membrane strip.10

e)PCR restriction enzyme analysis – It identifies 65kDa hsp gene and 16s rRNA gene of mycobacteria.

f)DNA chips – Also called DNA-microarrays. These consists of very large number of evenly spaced spots of DNA fixed to microscopic slide. Each spot is a unique DNA fragment transferred by a gridding robot from 96-well plates on to the slides. DNA extracts from ‘unknown’ isolates yield distinctive patterns of hybridization on the slide.

Back to Article Outline

4. Molecular methods 


(a)Polymerase Chain Reaction – A technique of DNA amplification that uses specific DNA sequences to serve as marker for the presence of microorganism, it can even detect a single bacteria. The commercial PCR assay is based on amplification of 584bp region of a gene encoding 16sRNA.

Nested PCR – The two sets of primers used for amplification were derived from the gene sequence encoding the insertion sequence IS6110 as follows: external primers were derived from position 367 to 392 (5′-CCGGCCAGCACGCTAATTAACGGTTC-3′) and position 769 to 746 (5′-TGTGGCCGGATCAGCGATCGTGGT-3′); and internal primers were derived from position 455 to 472 (5′-CTGCACACAGCTGACCGA-3′) and position 670 to 652 (5′-CGTTCGACGGTGCATCTG-3′). The reaction mixture consisted of 10mM Tris/HCl (pH 8.3), 50mM KCl, 2mM MgCl2, 0.15mM dATP, dGTP, dCTP, 0.45mM dUTP, 2pmol external primers, 75pmol internal primers, 2 U AmpliTaq Gold polymerase, and 0.5 U heat labile uracil-N-glycosylase in a total reaction volume of 100μl. The mixture was first incubated at 37°C for 10min with uracil-N-glycosylase to destroy any contaminating amplicons, and then at 94°C for 12min to activate the AmpliTaq polymerase, followed by subsequent temperature cycling at 94°C for 45s and 72°C for 1.5min for the first 15 cycles, followed by 94°C for 45s, 55°C for 45s, and 72°C for onemin for 45 cycles. Positive and negative controls were included in each run and all precautions to prevent cross contamination were observed. Amplified products were electrophoresed through a 2% agarose gel in Tris borate buffer. Target bands of 21bp were visualised by staining with ethidium bromide.

Other modification of PCR include – the strand displacement amplification(SDA)


-transcription based amplification(TAS)

-self - sustaining sequence replication

Real time PCR – a technique which can detect the target within 30–120min using PCR. It combines rapid thermocycling with the ability to detect target by fluorescently labeled probes as the hybrids are formed, i.e in real time. This technique allows multiplexing reactions, quantitation of targets, and online monitoring.

(c)Ligase chain reaction – Amplification of probe nucleic acid rather than target nucelic acid provides a strategy for enhancing the sensitivity of molecular diagnostic techniques. By this approach an amplified probe is the final reaction product to be detected, while the target sequence is neither amplified nor incorporated into this product.

(d)Amplification of the probe signal – Rather than amplifying either the target or probe, the signal used to detect hybridization between the amplicon and probe can be amplified by increasing the number of reporter molecules per probe

(e)Sequencing and enzymatic digestion of nucleic acid – This method is used independently or in association with hybridization or amplification procedures.

5. Serological diagnosis of TB.11 


i)Immunochromatographic tests:
-ICT Tuberculosis and

-RAPID TEST TB


ii)Enzyme linked immunosorbent assays:
-TUBERCULOSIS IgA EIA

-PATHOZYME-TB complex,

-PATHOZYME-MYCO IgG,

-PATHOZYME-MYCO IgA, and

-PATHOZYME-MYCO IgM,


i) Immunochromatographic tests
Five highly purified antigens (including one of 38kDa) secreted by M. tuberculosis during active infection are immobilized in four lines on the test strip. The test detects the presence of immunoglobulin G (IgG) antibodies to these antigens. A total of 30μl of serum is added to a blue pad and then diffuses along the test strip. When the test card is closed, anti-human IgG attached to colloidal gold particles binds to any bound human IgG antibodies, producing one or more pink lines. The presence of one or more pink lines in the strip's test area is considered a positive test result.

The RAPID TEST TB – a one-step colored immunochromatographic assay. It detects antibodies to the recombinant 38-kDa antigen from M. tuberculosis expressed in and purified from Escherichia coli. A total of 100μl of serum is added to the reaction tube, and the test strip is placed into the tube, which is then capped. After 15min of incubation, the presence of two colored bands is considered a positive test result.ii) Enzyme linked immunosorbent assays

TUBERCULOSIS IgA EIA detects IgA antibody to a mycobacterial Kp90 immuno-cross-reactive antigenic compound (ImCRAC). 100μl of diluted (1:400) serum was distributed in microtiter wells, and incubated at 37°C in a dark humid environment for 60min. The wells were subsequently incubated with 100μl of peroxidase-labeled anti-human IgA conjugate at 37°C in a dark humid environment for 60min. After another wash cycle, 100μl of peroxidase substrate, tetramethylbenzidine containing hydrogen peroxide, was added to the wells. The colorimetric reaction proceeded in the dark for 30min at room temperature (20 to 25°C) until 100μl of stop agent was added. The absorbance values at 450nm were recorded with an automatic reader system. The ratio of the OD for the unknown serum sample to the cutoff OD was used to interpret the results.

The PATHOZYME-TB complex test detects serum IgG antibody to a recombinant 38-kDa antigen from M. tuberculosis expressed in and purified from E. coli. This kit is specific for the diagnosis of disease due to M. tuberculosis complex. The procedures were similar to those described for TUBERCULOSIS IgA EIA, except that the sera were diluted 1:50, the second incubation was for 30min rather than 60min, the first and second incubations were not in a dark humid environment, and the third incubation was in the dark at 37°C for 15min. Three standards (with 2, 4, and 16serounits/ml) were provided for the generation of a semilogarithmic reference curve. Because the sera were diluted 1:50, the units extrapolated from the reference curve were multiplied by 50 to obtain serounits for result interpretation.

The individual PATHOZYME-MYCO lgG, lgA, and lgM assay. The three assays measure the levels in serum of IgG, IgA, and IgM antibodies, respectively, to two antigens; lipoarabinomannan (LAM) and recombinant 38-kDa antigen. These kits detect infection due to mycobacterium species. The procedures were identical to those described for PATHOZYME-TB complex except that the sera were diluted 1:100 rather than 1:50 and all three incubations were at room temperature. For the IgG and IgA assays three standards (with 2, 4, and 16serounits/ml) were provided for generation of a semilogarithmic reference curve. Because the sera were diluted 1:100, the units extrapolated from the reference curve were multiplied by 100 to obtain serounits for result interpretation. For the IgM assay, low- and high-positive control sera were provided. The OD of the low-positive control was used for the interpretation of the results.

Back to Article Outline

6. Conclusion 

New diagnostic techniques currently available are no more sensitive (and a great deal more expensive) than properly performed sputum smear and culture examination. Their usefulness in high prevalence countries, especially where there is a high prevalence of HIV co-infection, has not yet been established.

Back to Article Outline

6.1. Clinical focus 


i)Interferon γ assay test is not affected by past BCG vaccination, and will eliminate the unnecessary treatment of patients with BCG-related false positive results.

ii)The Interferon-γ assays does not affect the result of future Interferon-assays tests (i.e. no “boosting,” as with the TST).

iii.)Molecular methods have sensitivity greater than smear examination, but less than culture. False positives and false negatives occur, Cannot differentiate between live and dead bacilli. Very expensive test.

iv)Serological tests have not been adequately evaluated in high prevalence TB settings, false negative results are common in HIV infected persons, and antibody tests cannot distinguish between active TB and latent infection.

Back to Article Outline

Conflict of interest statement 

The authors have no conflict of interest.

Back to Article Outline

CME section 

This article has been accredited for CME learning by the European Board of Accreditation in Pneumology (EBAP). You can receive one CME credit by successfully answering these questions online.

(a)Visit the journal CME site at http://www.resmedcme.com

(b)Complete the answers online, and receive you final score upon completion of the test.

(c)Should you successfully complete the test, you may download your accreditation certificate (subject to an administrative charge).

Back to Article Outline

Educational questions 

Answer the following questions: true/false

1.The peptides used in the interferon –gamma assay test mimic proteins is ESAT-6, CFP-10.

2.The interferon – gamma assay test is affected by past BCG vaccination.

3.Culture media for BACTEC – 460 contains Palmitic acid.

4.In ESP II culture system, the instrument detects pressure changes that occurs a result of O2 consumption.

5.PCR can differentiate between live and dead bacilli.

Back to Article Outline

References 

  1. Watterson Simon A, Wilson Stuart M, Yates Malcolm D, Drobniewski Francis A. Comparison of three molecular assays for rapid detection of rifampin resistance in M. tuberculosis. J Clin Microbiol. 1998;36:1969–1973
  2. Mejia GI, Castrillon L, Robledo JA. Microcolony detection on 7H11 thin layer culture as an alternative for rapid diagnosis of M. tuberculosis. Int J Tuberc Lung Dis. 1999;3:138
  3. Caviedes Luz, Lee Tien-Shun, Gilman Robert H, Sheen Patricia, Spellman Emily, Lee Ellen H, et al. Rapid, efficient detection and drug susceptibility. Testing of M. tuberculosis in sputum by microscopic observation of broth cultures. J Clin Microbiol. 2000;38:1203–1208
  4. Rohner P, Ninet B, Metral C, Emler S, Auckenthaler R. Evaluation of the MB/BacT system and comparison to the BACTEC 460 system and solid media for isolation of mycobacteria from clinical specimens. J Clin Microbiol. 1997;35:3127–3131
  5. Woods GL, Fish G, Plaunt M, Murphy T. Clinical evaluation of difco ESP culture system II for growth and detection of mycobacteria. J Clin Microbiol. 1997;35:121–124
  6. Vestal AL. Procedures for isolation and identification of mycobacteria. NO. CDC 77-8230 CDC Atlanta: US Department of Health, Education and Welfare Publication; 1977;
  7. Duffey PS, Guthertz LS, Evans GC. Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures. J Clin Microbiol. 1996;34:1939–1943
  8. De Jong A, Hoentjen AH, Van Der Zanden AG. A rapid method for identification of Mycobacterium species by polyacrylamide gel electrophoresis of soluble cell proteins. J Med Microbiol. Jan 1991;34(1):1–5
  9. Kellogg James A, Bankert David A, Withers Gisela S, Sweimler William, Kiehn Timothy E, Pfyffer Gaby E. Application of the Sherlock Mycobacteria identification system using high-performance liquid chromatography in a clinical laboratory. J Clin Microbiol. March 2001;39(3):964–970
  10. Boden D, Weizenegger M, Benz K, Ponstingl W, Hengstler M, Rusch-Gerdes S, et al. Reverse hybridization assay for rapid identification of Mycobacteria from culture samples. Clin Lab. 1998;44:687–692
  11. Pottumarthy Sudha, Wells Virginia C, Morris Arthur J. A comparison of seven tests for serological diagnosis of Tuberculosis. J Clin Microbiol. June 2000;38(6):2227–2231

PII: S1755-0017(09)00084-0

doi:10.1016/j.rmedc.2009.09.015

Respiratory Medicine CME
Volume 2, Issue 4 , Pages 151-154, 2009

Article Tools