PDF version

E. Kalafati-Tzimaka,¹ I. Georgitzikis,¹ D. Delikatzi,¹ D. Chatzidimitriou,¹ M. Tzimaka² and D. Patakas³

ABSTRACT A comparative analysis was made of 3 conventional tests for tuberculosis (TB) versus a DNA probe technique among suspected TB patients at a reference centre in Greece. During 2004, we tested 2961 biological specimens from 2234 patients with the following methods: Ziehl–Neelsen staining, Löwenstein−Jensen culture, BACTEC mycobacteria growth indicator tubes (MGIT) and the Gen-Probe AMPLIFIED™ Mycobacterium tuberculosis direct test (MTD). Of a total of 136 TB patients diagnosed and under anti-TB treatment, 133 of them (98%) were positive by amplified MTD. There were 112 TB (82%) detected by the MGIT method, 102 (75%) by Löwenstein–Jensen culture and 75 (55%) by Ziehl–Neelsen staining. Using MTD the positive result is ready within hours compared with days or weeks.

Utilisation du test AMPLIFIED™ Mycobacterium tuberculosis Direct Test dans le diagnostic de la tuberculose

RÉSUMÉ Une analyse comparative entre trois tests classiques de dépistage de la tuberculose et une technique utilisant une sonde à ADN a été réalisée sur des cas suspects de tuberculose dans un centre de référence en Grèce. Pendant l’année 2004, nous avons effectué des tests sur 2961 échantillons biologiques provenant de 2234 patients à l’aide des méthodes suivantes : la coloration de Ziehl-Neelsen, la culture sur milieu de Löwenstein-Jensen, les tubes MGIT (Mycobacteria Growth Indicator Tubes) de BACTEC et le test AMPLIFIED™ Mycobacterium tuberculosis direct test (AMTD) de Gen-Probe. Sur un total de 136 patients tuberculeux diagnostiqués et sous traitement antituberculeux, 133 (98 %) étaient positifs au test AMTD. Cent douze cas de tuberculose (82 %) ont été détectés grâce à la méthode MGIT, 102 (75 %) à la culture sur milieu de Löwenstein-Jensen et 75 (55 %) à la coloration Ziehl-Neelsen. Avec le test AMTD, les résultats positifs sont prêts en quelques heures et non plus en plusieurs jours ou semaines.

¹Department of Mycobacteria Tuberculosis of Northern Greece, Pneumonological Clinic Laboratory; ²Department of Mass Media Communication; ³Pneumonological Clinic, Aristotle University of Thessaloniki, Thessaloniki, Greece (Correspondence to E. Kalafati-Tzimaka: This e-mail address is being protected from spambots. You need JavaScript enabled to view it ).
Received: 20/12/05; accepted: 13/06/06
EMHJ, 2008, 14(5): 1119-1125 

---

Introduction

Tuberculosis (TB) like no other disease has taken its toll of human life over the millennia and has spread worldwide [1]. It is estimated that 2 million people each year die from TB and nearly one-third of the world’s population is infected with the bacterium that causes TB. Left untreated, a person who develops active TB will infect an average of 10 to 15 other people every year. TB is curable only if it is diagnosed quickly and appropriately with medication. The laboratory diagnostic tests used for de- tecting Mycobacterium tuberculosis disease are summarized in Table 1.

Conventional culture methodologies can detect TB growth as early as 1 week, but may take up to 8 weeks [2–8]. Compara- tively, the M. tuberculosis direct (MTD) test provides detection of M. tuberculosis complex rRNA within 2.5 to 3.5 hours after beginning the test procedure. The need for a quick diagnosis and a quick start of anti-TB drug therapy lead our laboratory to the use of the amplified MTD genetic technique (San Diego, California, Gen-Probe) for the diag- nosis and laboratory follow up of suspected TB patients [9]. The Gen-Probe amplified MTD test utilizes transcription-mediated amplification (TMA) and the hybridization protection assay (HPA) to qualitatively detect M. tuberculosis complex ribosomal ribonucleic acid (rRNA). The MTD test will detect rRNA from both cultivable and noncultivable organisms [10].

This paper reports a comparative analy- sis of 3 conventional tests for TB versus am- plified MTD among suspected TB patients at a TB reference centre in Greece where the Gen-Probe method has been improved to achieve better specificity and sensitivity by titration of the method with positive and negative controls every day and where there is follow-up of patients before and after anti-TB therapy for more than 6 months.

Methods

Background to the study

The study was conducted during 2004 at the TB reference centre of northern Greece— the pneumonological clinic laboratory of G. Papanikolaou Hospital, Aristotle Univer- sity of Thessaloniki. Our reference centre covers mostly the population of northern Greece (approximately 2 500 000 people). Samples from both Greek nationals and immigrant adults are tested. During 2004 a total of 7023 suspected TB patients were examined at the centre, two-thirds of whom were hospitalized in the pneumonologi- cal and the medical pathology department while one-third were referred to our labo- ratory as outpatients from pneumonolo- gists. The majority of the patients suffered from fever, night sweating, weakening, body weight loss, cough (dry or mucopu- rulent expectoration) and haemoptysis with bloody sputum or large haemoptysis. Some of these patients were asymptomatic but had chest X-ray findings compatible with TB infection (nodules or patchy shadows).

Data collection

We tested 2961 biological specimens from 2234 patients with the Gen-Probe amplified MTD and with 3 conventional methods: Ziehl–Neelsen staining, Löwenstein–Jensen culture and BACTEC mycobacteria growth indicator tubes (MGIT) (Becton, Dickin- son and Company, Franklin Lakes, New Jersey). Table 2 shows the site of the 2961 biological specimens. Samples from the re- maining 4789 patients were examined with the 3 conventional methods only.

The criteria for the clinical diagnosis of pulmonary TB were as follows [11]. Smear- positive cases: at least 2 positive smears, or 1 positive smear and radiographic abnor- malities compatible with pulmonary TB, or 1 positive smear and 1 positive culture. Smear-negative cases: at least 3 negative smears and 1 or more positive cultures, or at least 2 series of negative smears from samples taken at least 2 weeks apart, with persisting radiographic abnormalities com- patible with active TB, not improved with treatment using broad-spectrum antibiotics for at least 1 week.

Laboratory methods

Standard methods were used for Ziehl– Neelsen staining [12], Löwenstein–Jensen culture [13] and MGIT [14].

Ziehl–Neelsen staining

The smear was stained with carbol fuchsin, destained with sulfuric acid and alcohol then restained with methylene blue. The stained smear was examined for acid-fast bacilli (AFB) using a binocular microscope with an immersion lens (× 100). The criteria for 1+ positive were 10–99 AFB per 100 immersion fields; for 3+ positive were > 10 AFB per field.

Löwenstein–Jensen culture

After decontamination, specimens were centrifuged and the sediment neutralized using a mild acid before adding Löwenstein– Jensen medium. The inoculated tubes were incubated at 37 °C for 4–12 weeks. The culture medium was inspected for large, rounded, buff-coloured, “cauliflower-like” colonies visible to the naked eye which indicate a positive result. The criteria for 1+ positive were < 10 colonies; for 3+ positive were > 100 colonies 

BACTEC MGIT

A fluorescent compound was embedded in silicone on the bottom of round-bottom tubes. Tubes entered into the BACTEC MGIT 960 system were continuously incu- bated at 37 ºC and monitored every 60 min for increasing fluorescence. The fluorescent compound is sensitive to the presence of oxygen dissolved in the broth from ac- tively respiring microorganisms. A positive tube contains approximately 105–106 colony forming units per millilitre (CFU/mL). Cul- ture vials which remained negative for a minimum of 42 days (up to 56 days) were removed from the instrument as negatives.

Gen-Probe amplified MTD test

The Gen-Probe MTD test is a 2-part test in which amplification and detection take place in a single tube. Initially, nucleic acids are released from mycobacterial cells by sonication. Heat is used to denature the nucleic acids and disrupt the second- ary structure of the rRNA. The Gen-Probe TMA method, using a constant 42 °C tem- perature, then amplifies a specific myco- bacterial rRNA target by transcription of DNA intermediates, resulting in multiple copies of mycobacterial RNA amplicon.

M. tuberculosis complex-specific sequenc- es are then detected in the RNA amplicon using the Gen-Probe ΗΡΑ method [15]. The M. tuberculosis hybridization reagent contains a single-stranded DNA probe with a chemiluminescent label. This probe is complementary to M. tuberculosis complex- specific sequences. When stable RNA:DNA hybrids are formed between the probe and the specific sequences, a hybridized probe is selected and measured in a luminometer. Our methods have been described before [16]. We observed that the manufacturer’s amplification cell negative control was not steady, which encouraged us to modify the interpretation of the results using a control that was calculated on a daily basis and was relevant to the biological specimens that were to be examined, under the same conditions daily.

Specimens were collected in sterile plas- tic containers, and stored at 2 °C to 8 °C for no more than 4 days (generally less than 24 hours) prior to processing. Specimens that are grossly bloody should not be tested with the MTD test. Sediments were decon- tamination using NALC-NaOH and then centrifuged at > 3000 × g. Specimens and controls were pipetted into lysing tubes, buffer was added and after sonication for 15 minutes the lysates were ready for am- plification.

Amplification tubes were prepared and 50 μL reconstituted M. tuberculosis ampli- fication reagent and 200 μL mycobacterium oil reagent were added and 25 μL of lysate was transferred to the bottom of the am- plification tubes. Tubes were incubated at 95 °C for 15 minutes in a dry heat bath. The enzyme reagent was reconstituted and 25 μL enzyme mix added to each amplification tube and incubated at 42 °C for 30 minutes. Hybridization was done at room tem- perature with reconstitute lyophilized my- cobacterium hybridization buffer, vortexed until clear, followed by 100 μL of recon- stituted hybridization reagent, vortexed 3 times until the reaction mixture was uni- formly yellow. This was incubated at 60 °C for 15 minutes in a dry heat bath.

Selection was done by adding 300 μL mycobacterium selection reagent to the tubes and vortexing until uniformly pink, incubating at 60 °C for 15 minutes in a dry heat bath, then cooling at room temperature for at least 5 minutes but not more than 1 hour.

The tubes were read in a luminometer (Gen-Probe Leader) with a 2 s read time. The results are expressed in relative light units (RLU). The cut-off value was set by the manufacturer: samples with values ≥ 30 000 RLU were considered positive to M. tuberculosis complex rRNA, and values of < 30 000 RLU were interpreted as nega- tive.

Results

Of a total of 136 TB patients clinically diagnosed and under anti-TB treatment, 133 of them (98%) were positive by ampli- fied MTD (130 were detected with RLU ≥ 30 000 and 3 were detected with RLU < 30 000). The 3 TB patients detected with RLU < 30 000 had values of 24 354, 27 977 and 29 351.

There were 112 TB patients (82%) de- tected by the MGIT method, 102 TB pa- tients (75%) by Löwenstein–Jensen culture and 75 TB patients (55%) by Ziehl–Neelsen staining (Table 3).

Discussion

While the conventional methods for cultiva- tion of Mycobacteria on solid media such as Löwenstein–Jensen and Middlebrook agars are relatively straightforward, they do require several weeks of incubation for the detection of organisms, and may lack sensitivity when used alone. For ex- ample, using the classic culture method (Löwenstein–Jensen) the positive result is ready in 3 weeks or more and the negative results ready after 2 months. The sensitiv- ity of Löwenstein cultivation is 75%–80% [17]. The positive result is ready by Ziehl– Neelsen staining in 4 days, but the sensitiv- ity of Ziehl–Neelsen is only 55%–65% [17]. MGIT supports and will detect the growth of a wide variety of Mycobacterium species, but requires a large number of tubes and requires 8–14 days.

Using amplified MTD the positive re- sult is ready in 3.5 hours, the sensitivity is 96%–100% and the specificity 95%–100% [17]. The amplified MTD test is specific for, but does not differentiate among, mem- bers of the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti and M. canetti [18]. However, M. microti infects only animals, M. bovis is uncommonly transmit- ted from infected animals to humans, and M. africanum causes pulmonary disease in humans in tropical Africa. M. tuberculosis is by far the most common member of the complex responsible for the human disease worldwide.

The DNA probe detection methods have been practised for years in research labora- tories, but their use has recently been broad- ened to clinical laboratories. Advantages of the rapid diagnosis include the reduced costs of medical treatment. The medical treatment of a TB patient, according to the bibliography, costs €235 per day. Previ- ously, suspected patients were hospital- ized for at least 6 days at a cost of around €1408. Besides the economic side, there are social and psychological advantages. Rapid diagnosis means that the patient is not marked as having an infectious disease, a stigma that, even if the result is negative, may be indelible. Families, who naturally try to protect the other family members, may not accept having the suspected patient at home. Psychological stress that can arise in the patient while waiting for the result is another problem.

For optimum results, however, close cooperation between the clinician and the laboratory is needed to define those speci- mens which urgently have to be screened by amplified MTD, i.e. those cases in which the clinical usefulness of amplified MTD is justified.

References

1. Global tuberculosis control: surveillance, planning, financing. WHO report 2005. Geneva, World Health Organization, 2005 (WHO/HTM/TB/2005.349).
2. Fujiki A. TB microscopy. Tokyo, Japan, Research Institute of Tuberculosis, 2000.
3. External quality assessment for AFB smear microscopy. Silver Spring, Mary- land, Association of Public Health Labo- ratories, 2003.
4. Grosset J. Bacteriology of tuberculo- sis. In: Reichmann LB, Hershfield AS, eds. Tuberculosis: a comprehensive in- ternational approach. New York, Marcel Dekker, 1993:49–74.
5. Roberts GD, Koneman EW, Kim YK. Mycobacterium. In: Balows et al., eds. Manual of clinical microbiology, 5th ed. Washington, DC, American Society for Microbiology, 1991:304–39.
6. Zanetti S et al. Evaluation of a nonradio- metric system (BACTEC 9000 MB) for de- tection of mycobacteria in human clinical samples. Journal of clinical microbiology, 1997, 35(8):2072–5.
7. Reischl U et al. Clinical evaluation of the automated COBAS AMPLICOR MTB assay for testing respiratory and nonres- piratory specimens. Journal of clinical microbiology, 1998, 36(10):2853–60.
8. Kalafati-Tzimaka E et al. Direct diag- nosis of MbTb complex by the genetic technique TMA of the AMTD Gen Probe system. In: Abstracts of the 24th Annual Congress of the European Society for My- cobacteriology. Tartu, Estonia, 30 June–3 July 2003.
9. Coll P et al. Routine use of E-MTD (Gen- Probe) for direct detection of mycobacte- rium tuberculosis in clinical samples. In: Proceedings of the 23rd Annual Congress of the European Society for Mycobacteri- ology. Dubrovnik, Croatia, 23–26 June 2002.
10. Wayne LG. Microbiology of the tubercle bacilli. American review of respiratory disease, 1982, 125(3 Pt2):31–41.
11. Maher D et al., eds Treatment of tubercu- losis: guidelines for national programmes, 2nd ed. Geneva, World Health Organiza- tion, 1997 (WHO/TB/97.220).
12. Ait-Khaded N, Enarson DA. Tuberculosis: a manual for medical students. Geneva, World Health Organization, 2003:15 (WHO/CDS/TB/99.272).
13. Ait-Khaded N, Enarson DA. Tuberculosis: a manual for medical students. Geneva, World Health Organization, 2003:18 (WHO/CDS/TB/99.272).
14. Hanna BA et al. Detection of Mycobac- terium tuberculosis directly from patients specimens with mycobacteria growth indi- cator tube; a new rapid method. Abstract C 112. In: Abstracts of the 94th General Meeting of the American Society of Mi- crobiology. Washington DC, American Society for Microbiology, 1994.
15. Arnold LJ et al. Assay formats involving acridinium-ester-labeled DNA probes. Clinical chemistry, 1989, 35:1588–94.
16. Kalafati-Tzimaka E et al. Patients’ control, during the year 2003, at the MbTb refer- ence center of the Aristotle University of Thessaloniki of pneumological clinic in the general hospital “G. Papanikolaou”. Sci- entific annals of the Faculty of Medicine, Aristotelian University, 2004, 31(2):72–6.
17. Kalafati-Tzimaka E et al. MGIT, Löwen- stein Jensen, AMTD (Gen-Probe) and Ziehl-Neelsen. Evaluation of the sensi- tivity and specificity of the four methods during 2004. In: Abstracts of the 26th Annual Congress of the European Society for Mycobacteriology. Istanbul, Turkey, 26 June–1 July, 2005.
18. Van Soolingen D et al. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Interna- tional journal of systematic bacteriology, 1997, 47:1236–45.