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Early Diagnosis of Mycobacteriosis

The diagnosis of mycobacterioses is carried out in compliance with particular regulations. It is recommended to analyze pulmonary clinical specimens of three different days. To detect extra-pulmonary TB the appropriate specimen, for example urine or cerebrospinal fluid has to be chosen.
The primary samples are decontaminated in order to remove any interfering accompanying flora. N-Acetyl-L-Cystein-NaOH (sodium hydroxide) or 4% NaOH according to Petroff are widely-used decontamination substances. Subsequently, a microscopic preparation is analyzed and two solid media and one liquid medium are inoculated. In high-burden countries normally the microscopy is done directly from specimen without decontamination.For economic reasons culture is only been done for follow-up patients, microscopic acid-fast bacilli (AFB) positive specimens and relapse or failure cases.
The microscopic analysis of smears on acid-fast bacilli is indeed a quick but also an insensitive and not very specific method: as it is impossible to distinguish between either living or dead bacteria or between tuberculosis bacteria and NTM, a culture report guarantees an improvement of sensitivity and specificity.  A positive result using solid culture can be expected after about 3-4 weeks, a negative assessment however is only available after incubation of 8 weeks. The sensitivity limit of the solid culture is approx. 102 bacteria/ml. By using liquid culture systems, in addition to increasing sensitivity the detection time can also be reduced. Therefore a positive result is already available after 1-2 weeks, and safe exclusion is possible after an incubation time of 6 weeks.

However, since a morphological evaluation is not possible from liquid culture, only molecular genetic systems offer quick identification and differentiation.

Direct detection
With DNA- or RNA-based techniques it is possible to detect specific fragments of the mycobacterial genome. This involves multiplication of short nucleic acid sequences of the mycobacterial genome using enzymatic methods and the subsequent detection of the amplified fragments. Therefore in the event of severe symptoms and also in the case of persons at risk, rapid detection directly from patient specimens is possible with the aid of NAT (Nucleic Acid Amplification Technology) within one day.
The Mycobacteria Product Series of Hain Lifescience enables the detection of mycobacteria directly from pulmonary patient specimen either if it is microscopic AFB negative or positive.

  • Microscopy AFB-negative:
    GenoType® Mycobacteria Direct, simultaneous detection of the M. tuberculosis complex and 4 clinically relevant NTM(M. avium, M.intracellulare, M. kansasii, M. malmoense)
    GenoQuick® MTB, rapid direct detection of the M. tuberculosis complex from patient specimens
     
  • Microscopy AFB-positive:
    GenoType® MTBDRplus, identification of the M. tuberculosis complex and its resistance to isoniazid (INH) and rifampicin (RMP)
    GenoType® MTBDRsl, identification of the M. tuberculosis complex and its resistance to fluoroquinolones, injectable drugs and ethambutol

Differentiation
In addition to the determination of TB pathogens requiring obligate treatment from ubiquitous mycobacteria, species differentiation is also of crucial importance for the selection of adequate antibiotics, as drug resistance differs considerably between species. Conventional methods are time-consuming and require trained staff. They normally use biochemical method for detection of species specific enzymes as growth rate and producing of pigment. This may take several weeks. To reduce the detection time molecular genetic testing is therefore the method of choice. It is fast and easy to learn.
 

Hain Lifescience opens different ways to identify mycobacteria from cultivated samples within 4-5 hours only!

Resistance testing
Treatment of TB involves an uninterrupted administration of antibiotics for several months. If the treatment plan is not observed consistently or the treatment is discontinued prematurely, drug resistance will be developed. Nearly half a million new cases a year are estimated as multidrug-resistant (MDR) TB. These strains are resistant to the most important tuberculostatics, rifampicin and isoniazid. A rapid detection of MDR-TB is essential to adapt quickly to effective antibiotic treatment. Meanwhile it is also possible to determine a resistance to drugs like ofloxacin (fluoroquinolone), amikacin/kanamycin (aminoglycosides) and capreomycin (cyclic peptide) with the help of molecular genetic technology. A MDR-TB in addition to a resistance to any fluoroquinolone and at least to one of the aminoglycosides/cyclic peptides is defined as XDR-TB, extensive (or extreme) drug-resistant TB.
The traditional drug susceptibility testing (DST) on solid Löwenstein-Jensen media according to Canetti (gold standard) and DST in liquid media like Middlebrook 7H9 can only be done from cultivated samples. The time of determination add up to 42 days on solid media and 12 days in liquid media. In case of MDR/XDR TB it is absolutely essential to reduce the detection time.

GenoType® MTBDRplus and GenoType® MTBDRsl  can be performed directly from AFB-positive pulmonary patient specimen and from cultivated samples. Results are obtained in 4-5h.

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