This invention relates to a method of distinguishing between different pathogens in a biological sample.

Tuberculosis (TB) is a disease mainly caused by Mycobacterium tuberculosis (M. tuberculosis) in humans and infects approximately one-third of the world's population. A relatively high prevalence of TB causing mycobacteria, other than M. tuberculosis, such as M. bovis and non-tuberculous mycobacteria (NTM) such as M. avium and M. kansasii, are frequently encountered in clinical specimens, such as sputum.

In 2008, there were an estimated 9.4 million newly identified TB incidences and 1.8 million deaths globally attributable to TB. Different treatment regimens are followed for different mycobacterial infections and it is becoming more important for diagnostic techniques to differentiate between various mycobacterial species.

Detection limits of diagnostic techniques further play an important role in the early diagnostic phase of TB when a sputum sample is used, as only a small amount of bacteria are present, which are often undetected and consequently cause the misdiagnosis of TB in patients. Additionally, TB patients who are co-infected with HIV and childhood infections are frequently left undiagnosed due to lower sputum bacterial loads and lack of TB diagnostic methods with improved detection limits.

TB diagnostic methods and their disadvantages;

  1. Smear microscopy - regarded as the TB diagnostic method most commonly used worldwide, due to its simplicity and affordability.
  • Has a detection limit of 5 000 to 10 000 bacteria ml"1 sputum, consequently detecting only 60 to 70% of all TB incidences in adults, and thus requires a bacteriological culture method to confirm the diagnosis.
  • Number of identified cases can be as low as 20 to 30%, when performed under sub-optimal conditions, attributable to inexperienced personnel, for example.
  • Unable to differentiate between various mycobacterial species in the sputum, hence, it can only detect the presence of mycobacteria in general, but cannot establish which mycobacterial species is responsible for infection.
  1. Bacteriological culture method
  • More sensitive than the smear microscopy method, and has a detection limit of 10 to 100 bacteria ml"1 using solid or liquid media
  • It requires two to six weeks before bacterial colonies can visually be detected, hence leading to a long waiting period prior to the initial treatment of the patient.
  • 15 to 20% of all adult TB incidences are reported to have negative sputum cultures, consequently leading to a false-negative result.
  • 1 to 4% of all incidences present a false positive, due to poorly executed anti-contamination procedures.
  • 1 to 4% of all incidences present a false positive, due to poorly executed anti-contamination procedures.


  1. Nucleic acid amplification
  • Has sensitivity of only 60 to 70% in smear-negative, culture-positive samples.
  • High incidence of false-positive results due to cross-contamination.
  • Due to the residual genetic material in the patients, positive results may still be obtained after effective treatment.
  • Cannot be used in patients with previous mycobacterial infections and for monitoring of a patient's response to therapy.
  • In the clinical practice environment, the method requires two days before a diagnostic result can be obtained, and in most cases, additionally requires prior culturing of the organism, in order to overcome the previously mentioned disadvantages. As a result, an additional two to six weeks diagnostic time is required.

According to the WHO, no serology TB diagnostic assays have yet been approved for clinical use due to a lack of adequate sensitivity and specificity of these methods.

WO 2004/059280 discloses a method for measuring a plurality of different organisms in a sample (mucus, nasal, pharyngeal or genital discharge sample). The measurement includes detecting the presence or absence of a proteinaceous, lipid, carbohydrate and/or nucleic acid marker indicative of an organism using specific antibodies.

Furthermore, WO 2004/059280 discloses an extraction method for extracting one or more markers from the matrixes of one or more samples and/or rendering the samples more suitable for analysis.

WO 2006/079846 discloses a method of detecting and identifying bacteria comprising the steps of collecting volatile bacterial products, subjecting the volatile products to a gas chromatography system employing a surface acoustic wave detector, establishing chromatographic profiles for different bacteria, and comparing the chromatographic profile of the biological sample (including sputum, breath and blood) with profiles in the library. The system is said to be suitable for use in the detection of TB and indicates that mycolic acids could be potential biomarkers. However, no specific markers for TB are described.

WO 2009/045116 discloses a method for detecting a mycobacterium by analysing a gas mixture for one or more identified biomarkers selected from a group consisting of methyl phenyl acetate, methyl p-anisate, methyl micotinate, and o-phenylanisole.

WO 2009/091375 relates to a method for identifying the presence of M. tuberculosis in a sample by detecting at least one volatile organic compound indicative of the presence of M. tuberculosis in the sample.

A disadvantage associated with the abovementioned methods is that they do not differentiate between various disease-causing species, including those belonging to the genus Mycobacterium.

It is accordingly an object of the present invention to provide a method for distinguishing between different pathogens in a biological sample with which the aforesaid disadvantages could be overcome or at least minimised.


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