March 24^th is World Tuberculosis Day which aims at raising public awareness of a disease that is still widespread all over the world and figures among the top ten causes of death worldwide.

On this occasion, we interviewed Dr. Daniela Maria Cirillo, Deputy Director of the Division of Immunology, Transplants and Infectious Diseases and Group Leader of the Emerging Bacterial Pathogens Unit at the IRCCS Ospedale San Raffaele, who has been working in clinical microbiology, molecular mechanisms of drug resistance, and research of new diagnostic approaches for tuberculosis for about thirty years.

What is tuberculosis and how is it diagnosed?

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. As its name suggests, it is a mycobacterium, a particular genus of bacteria that has a lipid-rich cell wall that makes it resistant to the environment and antibiotics. “It is often thought that tuberculosis only affects the respiratory system, which is certainly the most affected target, but actually the disease can interest (involve) every organ and system,” says Dr. Cirillo, who continues: “It is a contagious disease, which is transmitted by contact through respiratory droplets that the person expels by coughing or simply talking.”

Although more widespread in countries in Southeast Asia and Africa, tuberculosis is present all over the world. Every year, 10 million new infections are counted, and approximately 5-10% of them lead to the disease, which can cause death if not well-treated: data from the end of October 2024 from the World Health Organization (WHO) indicate that in 2023 approximately 1.25 million people worldwide died of tuberculosis, which is the disease that provokes the most deaths from a single pathogen, i.e. the agent responsible for its onset. The risk of progression from infection to disease is much higher in exposed subjects who are immunosuppressed, i.e., who have poor immune defenses.

A critical aspect of the disease concerns its symptoms. Infected subjects may show some symptoms, such as cough, fever, or chest pain, but they can also remain asymptomatic. “We have always thought that this disease first remained in the “infection” phase, and then progressed to become symptomatic, and therefore the subject who begins to cough is the one who can actually infect” says Cirillo, who adds: “instead, there are cases of potentially infectious tuberculosis in subjects who are “healthy” from a symptomatic point of view but in whom there are lesions that can be detected with radiological tools”. Since these subjects do not show any symptoms that can be associated with a disease, they are not diagnosed even though they can continue to infect: “it is therefore essential to pay great attention to identifying cases of “asymptomatic tuberculosis”, which are easy to treat but represent a risk for the community”.

Today, the diagnosis of tuberculosis infections are usually made using the Mantoux test. This test is done by injecting tuberculin, a protein derivative obtained from the tuberculosis bacterium, into the deep layer of the skin, which can lead to a localized immune response in people who have previously been infected by mycobacteria. The diagnosis of tuberculosis infection can also be made through the IGRA test, an acronym for Interferon Gamma Release Assay. The test detects the presence of interferon gamma in the blood, a protein produced by lymphocytes, cells of the immune system, in response to the presence of M. tuberculosis.

However, these tests detect individuals who have actually been exposed to the bacterium, providing little information about the disease progression.

“Through the tests described above we can only understand who among those who have been exposed has mounted an acquired immune response against the bacterium,” says Dr. Cirillo, who adds: “however, in the majority of infected people, the immune system is able to eradicate the mycobacteria and therefore prevent the development of the disease.” We thus have no way to detect who, among the infected, is at risk for progression of the disease and who will instead remain healthy, having already eradicated the mycobacteria.

The diagnosis of tuberculosis, on the other hand, is based on the identification of the mycobacteria through culture (a method that enables us to selectively grow a specific type of microorganism) or through some molecular techniques, such as the sequencing of nucleic acids (DNA, RNA) which allows us to determine the exact order of the nucleotides, their fundamental units.

Therapeutic perspectives and multidrug-resistant tuberculosis

Given the lack of a test able to detect infected subjects who will not develop the disease, therapeutic approaches for tuberculosis infection are based on the so-called preventive therapy, which recommends the administration of a therapeutic regimen with the drugs isoniazid and rifampicin for a duration of three months. This treatment is directed at people who have been exposed to the tuberculosis bacterium and who are at greater risk of developing the disease, such as immunosuppressed patients.

“This is a preventive approach, so we treat ten patients to prevent just one case. Of course, this approach is not possible in countries where the incidence of the disease is very high, because we would have to extend this treatment to many subjects, but without sufficient resources available” says Dr. Cirillo. This is why it is necessary to have dedicated tests to predict who will develop the disease, which in some subjects can progress very quickly and is difficult to diagnose, such as in children. In countries with a high incidence and always for preventive purposes newborn children undergo the BCG vaccine administration, whose name derives from the Bacillus of Calmétte and Guerin, a mycobacterium derived from the strain (a group of microorganisms that share similar genetic characteristics) of bovine tuberculosis.

The gold standard treatment regimen for tuberculosis disease, in its classic pulmonary form, is to administer 4 different drugs over the course of 4-6 months. However, a critical aspect in the therapeutic field concerns drug resistance. Following some genetic mutations in their genome, bacteria, including tuberculosis bacteria, can become resistant and therefore survive in the presence of an antibacterial agent. “This is the reason why no one would treat tuberculosis with a single drug; [it’s]because the bacterial population capable of surviving the antibiotic action would be selected. Indeed, four drugs are used in the first intensive phase of treatment, which lasts two months, and two in the second phase, which instead lasts four months.” The problem becomes more complicated when mutations arise that make the strains resistant to rifampicin, a key drug for treating both the infection and the disease.

“In this case, we talk about tuberculosis resistant to rifampicin; if there is also resistance to isoniazid, then we talk about multidrug-resistant tuberculosis or MDR (Multidrug-Resistant Tuberculosis)” says Cirillo.

The therapeutic approach for patients with MDR tuberculosis has recently changed: “until a couple of years ago, patients with multidrug-resistant tuberculosis were treated for two years with extremely toxic drugs, and with a fairly low success rate, around 50-60%”. In the last two years, new drugs have been developed that allow the so-called BPaLM treatment (bedaquiline, pretomanid, linezolid and moxifloxacin). This therapeutic regimen lasts only 6 months and requires that the drugs are taken orally, thus increasing the chance that patients will adhere to therapeutic prescriptions.

However, genetic mutations of bacteria occur much more quickly than the time needed to develop a new drug. For this reason, “while our diagnostics have been based on the identification of mutations for resistance to certain drugs, such as rifampicin and isoniazid, today unfortunately we do not have suitable diagnostic tests to detect resistance to bedaquiline, which in some countries involves up to 30% of subjects with multidrug-resistant tuberculosis.”

The importance of molecular diagnostics

The problem of antimicrobial resistance raises the need for diagnostics able to reveal the genetic mutations responsible for antimicrobial resistance, in order to prescribe the most effective drug.

This need can be satisfied with a molecular diagnostic approach that uses next-generation sequencing techniques to identify the specific mutation responsible for a certain type of resistance of the bacterium that causes the infection. “In this way, if the specific type of resistance of the bacterial strain that caused the ongoing disease is known, it is possible for the infectious disease specialist to specifically adapt the antibiotic therapy, which eventually will be tailored to the pathogen and the patient”, explains Dr. Cirillo.

New therapeutic options

Given the severity of the disease and the problem of antimicrobial resistance, research is trying to find new options for the treatment of tuberculosis.

From this perspective, a scientific article published in The New England Journal of Medicine in January of this year reports the results of an international clinical study that evaluated the efficacy of five different antibiotic regimens for the treatment of tuberculosis resistant to rifampicin but sensitive to fluoroquinolones, a class of antibiotics including moxifloxacin, which is used in BPaLM for the treatment of multidrug-resistant tuberculosis.

The study involved 754 patients, aged 15 years and older: 130 of them were treated with standard therapy, while the remaining 624 were subjected to one of the five experimental regimens. The regimens, each lasting 9 months, included different combinations of some drugs, in particular bedaquiline, delamanid, clofazimine, linezolid, levofloxacin, moxifloxacin and pyrazinamide.

The results indicate that these regimens have the same efficacy as the standard therapy. In addition, side effects were more frequent in a fraction of patients undergoing conventional therapy, rather than in patients treated with one of the five experimental regimens.

"The aim of the study is to provide alternatives to sick people who cannot undergo the BPaLM therapeutic regimen. Having few data on the use of pretomanid in children and pregnant women, BPaLM, which also uses this drug, is not indicated for these people" comments Cirillo, who concludes: "the study published in the New England Journal of Medicine therefore shows promising alternatives for patients like children and pregnant women, because BPaLM uses delamanid, a drug similar but alternative to pretomanid, which can be used to treat the disease also in these people".

To encourage the development of new treatment regimens against tuberculosis, the Unite4TB consortium is also underway. Unite4TB is a public-private partnership that brings together academic institutions, small and medium-sized businesses, pharmaceutical companies, and public organizations and has the aim of testing new regimens against tuberculosis disease in phase 2 clinical trials in which the efficacy of a drug (or combination of drugs) is tested on a limited number of patients. The therapeutic regimens that demonstrate the highest probability of success during these phase 2 trials can then undergo phase 3 trials, during which the efficacy and dosage of the treatment can be studied in depth.

Simplifying the treatment of tuberculosis is essential to achieving the fundamental objective of eliminating the disease. However, to be successful we must not only identify and streamline new treatment regimens but also prioritize the ongoing development of more sensitive and accessible diagnostic tools, enabling earlier detection and preventing disease transmission, which are equally vital to the ultimate eradication of the disease.