World Tuberculosis Day. PCR diagnosis of Mycobacterium tuberculosis with GeneProof

Link to the source - https://wise-mag.com/index.php/2025/06/30/tuberculosis-how-do-we-get-it/

 

World Tuberculosis Day is observed every year on 24 March. This date was not chosen at random, as it was on this day in 1882 when Robert Koch announced the discovery of the bacterium that causes the disease. Since then, the path to the diagnosis and treatment of tuberculosis has been opened.

 

Tuberculosis is still one of the most deadly infectious diseases in the world. It is estimated that a quarter of the world’s population is infected with tuberculosis bacteria; most of these people are not ill or infectious, but they are at risk of developing the disease in the future. This depends, first and foremost, on several factors, the most important of which is a person’s immune status [8].

 

Tuberculosis (TB) remains the leading cause of death by a single infectious agent globally. The World Health Organization (WHO) reported that there were an estimated 10.6 million new cases of disease and 1.6 million deaths from TB in 2022 (World Health Organization, 2023). Most of the cases were contributed by low- and middle-income countries, which accounted for two-thirds of the global total cases [1].

 

According to statistics from the Public Health Centre of the Ministry of Health of Ukraine, 1,281 people with tuberculosis were registered in October 2025:

• 945 new cases (meaning a case of tuberculosis in a patient who has never previously been treated for the disease or has taken anti-mycobacterial drugs for less than 1 month).
• 269 patients with relapse.
• 67 — others (including: people who resumed treatment; patients following an unsuccessful course of treatment; patients with an unknown history of previous treatment).

 

268 of the total number had drug-resistant tuberculosis. Among the 1,225 new cases (meaning individuals with a new case of the disease, a relapse, or an unknown history of previous treatment), 1,132 people with tuberculosis were diagnosed with the pulmonary form of the disease, and 93 with the extrapulmonary form; 891 were male and 334 were female. 211 patients were HIV-positive. In October 2025, 894 people were successfully treated for tuberculosis. Information as of 12 November 2025 [5].

 

Link to the source - https://phc.org.ua/news/statistika-tuberkulozu-v-ukraini-zhovten-2025-roku

 

The direct and indirect costs of TB, and the social consequences, are often catastrophic for the individual patient and for the community. Therefore, the rapid and precise detection of Mycobacterium tuberculosis is of primary importance for the administration of empirical antibiotic therapy and for the appropriate implementation of public health measures to effectively cut transmission and prevent the disease from spreading. In fact, an intensive effort is needed to develop new medical technologies for prevention, diagnosis, and treatment of TB [2]. 

 

Diagnosis

 

Methods for diagnosing tuberculosis infection include the traditional tuberculin skin test (the Mantoux test), an interferon-gamma release assay (IGRA), and new skin tests which are similar in specificity to IGRA tests but resemble the Mantoux test in their method of administration.

 

The Mantoux test. The tuberculin test, known as the Mantoux test, is a traditional, well-known and widely available method for detecting tuberculosis infection. However, it can produce false-positive results due to prior BCG vaccination, as well as in people with weakened immune systems or in cases of delayed immune response.

 

An Interferon-Gamma Release Assay (IGRA). The gamma-interferon release assay is a modern laboratory blood test that allows the presence of tuberculosis infection in a person to be determined with a high degree of accuracy. The patient’s venous blood is used for the test. This method of testing does not produce false-positive results associated with a history of BCG vaccination or the presence of a non-tuberculous mycobacterial infection.

 

New skin tests — a combination of methods. Skin tests combine the simpler Mantoux test method with the specificity of the IGRA test. Based on the available evidence, these tests help to avoid the negative consequences of false-positive diagnostic results, as their diagnostic accuracy is close to that of gamma-interferon release assays and higher than that of the Mantoux test. Another advantage of these tests is their lower cost compared to IGRA tests [6].

 

Tuberculosis is treatable if detected in time, a full course of treatment is completed, and all medical recommendations are followed. In early 2023, Ukraine updated its standards of care for tuberculosis to align with the latest WHO recommendations. The priority and primary diagnostic method are the microbiological test, as it allows for an accurate diagnosis and the prompt prescription of an effective treatment regimen. To perform this test, the patient provides a sputum sample.

 

Initially, an automated molecular genetic test using semi-quantitative real-time PCR for in vitro diagnosis is usually employed, which within a few hours not only confirms the presence of the disease but also determines sensitivity to rifampicin — a drug frequently used to treat tuberculosis. This method can be used to test not only sputum but also other bodily fluids, including stool samples from children who are not yet able to cough up sputum.

 

For the diagnosis of tuberculosis, examinations such as X-rays or computed tomography (CT) scans of the chest are also used. In several countries, so-called computer-aided detection is used to interpret X-ray results. Essentially, this involves artificial intelligence that has been trained to analyse numerous chest X-rays and identify tuberculosis specifically and accurately. In Ukraine, the first three regions to adopt the use of AI in X-ray analysis were Lviv, Sumy and Ivano-Frankivsk.

 

Tuberculosis can affect not only the lungs but also other organs. In cases of extrapulmonary tuberculosis, in addition to these methods, ultrasound scans, bronchoscopy and other techniques may be used. If tuberculosis cannot be confirmed using the abovementioned diagnostic methods, a biopsy is performed to obtain tissue samples for subsequent microbiological and morphological examination [7].

 

The currently accepted ‘‘gold standard’’ laboratory method for detecting and identifying Mycobacterium tuberculosis is a combination of acid-fast bacillus (AFB) smear for initial screening and culture for bacterial isolation and identification. Microscopic examination of AFB smears can yield a result within 24 h. Unfortunately, smear is neither specific for M. tuberculosis nor very sensitive, requiring > 103 to 104 organisms per ml of sputum. Mycobacteria can also be detected and identified directly from sputum specimens by analyzing the mycobacterial mycolic acid by high-performance liquid chromatography. However, the sensitivity of this method is low, and direct detection from sputum is possible only in smear-positive specimens. Bacterial culture is superior to AFB smear and mycolic acid analysis, both in terms of sensitivity and specificity. However, mycobacteria are fastidious organisms with very strict growth requirements. As a result, culture-based diagnostic methods are slow. 

 

The need to perform biochemical testing to identify the mycobacterial species adds to the time required to obtain a diagnosis. Even when nucleic acid probes are used for ‘‘rapid’’ species identification from cultures, results are often not available for 2 to 8 weeks. Clearly, a more rapid test for the detection of pulmonary TB and other nontuberculous mycobacterial infections would greatly benefit patient management.

 

The PCR has proven to be a very useful tool in the rapid diagnosis of infectious diseases and specifically mycobacterial infections. Many of the mycobacterial PCR assays employ species-specific primers that allow for the detection of a single or a limited number of mycobacterial species. Species-specific amplification assays are useful in the detection and identification of mycobacterial species. However, only a limited number of mycobacterial species can be detected. This limits the usefulness of these assays, since additional amplifications are needed to detect other species. There are other limitations associated with these assays. The most commonly used of these assays is one targeting the insertion element IS6110 found only in species within the M. tuberculosis complex. However, there are isolates of M. tuberculosis that do not harbor IS6110, although they are uncommon [3].

 

Among the commercially available PCR kits that utilise the IS6110 multi-copy insertion sequence and the MPB64 immunogenic protein gene sequence, the PCR kit for the detection of Mycobacterium tuberculosis from the Czech manufacturer GeneProof for in vitro diagnostic purposes stands out. The advantages of this kit include:

 

• dual target detection:
  • Sual targeting prevents detection failures caused by occurrence of mutations.
  • Secured by targeting the multi-copy sequence IS6110 and MPB64.

 

• high specificity — detection of all species from Mycobacterium tuberculosis complex (M. tuberculosis, M. africanum, M. bovis, M. microti, M. canetti, M. pinnipedi, M. caprae, M. orygis, M. mungi and vaccination strain Bacillus Calmmette-Guérin, BCG).

 

• wide range of validated clinical materials:
  
  • Enables detection from a broad spectrum of respiratory and gastrointestinal samples.
  • Sputum, BAL, swab, urine, CSF, plasma, stool.

 

• easy-to-use concept:
  
  • Single tube Ready-to-Use Master Mix contains all components for PCR amplification.
  • No additional PCR reagents pipetting necessary.
  • Simplifies laboratory workflow.
• contamination prevention — Master Mix contains Uracil-DNA glycosylase (UNG) and dUTPs eliminating carryover contamination [4].

 

Link to the source — https://www.geneproof.com/geneprooftm-mycobacterium-tuberculosis-pcr-kit/p1077

 

CONCLUSIONS

 

Consequently, tuberculosis is the leading infectious disease in developing countries. It remains a significant global public health problem, as a substantial number of new cases are recorded every year, including in Ukraine.
Polymerase chain reaction (PCR) has been an increasingly used tool for a more sensitive and rapid diagnostic of many infectious diseases, including TB. Over the years, a significant improvement in PCR technologies has been achieved with the development of real-time PCR (rtPCR). The main advantages of rtPCR are a shortened turnaround times; automation of the procedure, which reduces hands-on time; and a decrease in the risk of cross-contamination [2].

 

REFERENCES

 

1. Parwati I., Chaidir L., Yunus M., Montain M., Budhiarko D., Selasih S., Ristandi R., Rachman R., Nurhayati R., Pambudi I and Budiyati A. (2024). Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia. Front. Microbiol. 15:1372647. doi: 10.3389/fmicb.2024.1372647.
2. Rozales F., Machado ABMP, De Paris F., Zavascki A., Barth A. PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data. Epidemiology and Infection. 2014;142(7):1517-1523. doi:10.1017/S0950268813002598.
3. Tevere V., Hewitt P., Dare A., Hocknell P., Keen A., Spadoro J., Young K. Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe. J. Clin. Microbiol. 1996; 34(4): 918-23. doi: 10.1128/jcm.34.4.918-923.1996. PMID: 8815108; PMCID: PMC228917.
4. Набір для ПЛР GeneProof Mycobacterium tuberculosis. 2026 GeneProof. — Режим доступу: https://www.geneproof.com/geneprooftm-mycobacterium-tuberculosis-pcr-kit/p1077.
5. Статистика туберкульозу в Україні: жовтень 2025 року. — Державна установа «Центр громадського здоров’я Міністерства охорони здоров’я України». — Режим доступу: https://phc.org.ua/news/statistika-tuberkulozu-v-ukraini-zhovten-2025-roku.
6. Сучасні методи діагностики ТБ-інфекції та туберкульозу. — Державна установа «Центр громадського здоров’я Міністерства охорони здоров’я України». — Режим доступу: https://phc.org.ua/news/suchasni-metodi-diagnostiki-tb-infekcii-ta-tuberkulozu.
7. Як діагностують туберкульоз в Україні. — Міністерство охорони здоров'я України. Офіційний сайт. — Режим доступу: https://moz.gov.ua/uk/jak-diagnostujut-tuberkuloz-v-ukraini.
8. Як не втратити час? Всесвітній день боротьби з туберкульозом. — Державна установа «Центр громадського здоров’я Міністерства охорони здоров’я України». — Режим доступу: https://phc.org.ua/news/yak-ne-vtratiti-chas-vsesvitniy-den-borotbi-z-tuberkulozom.