10/​07/​2021

Tuberculosis – A New Generation of Rapid TB Diagnostics is Urgently Needed

Tuber­cu­losis (TB) is one of the most important infec­tious diseases and thus a topic of global impor­tance. A signif­icant reduction of the TB incidence or complete elimi­nation of TB is not unreal­istic. Therefore, the article provides important infor­mation on the topic of TB and, at the same, important background infor­mation on the fasci­nating biology of the causal agent of TB, Mycobac­terium tuber­cu­losis (Mtb). In order to be able to fight TB effec­tively, innov­ative diagnostic approaches are needed – especially in the field of  Point-​​of-​​Care (PoC) appli­ca­tions. For this reason the article focuses on the high requirement profile of compre­hensive TB diagnostics and what impact the universal Lateral Flow Devel­opment Platform Milenia® HybriDetect can have.

Mycobac­terium tuber­cu­losis – the causative agent of TB

Mycobac­terium tuber­cu­losis (Mtb) is a rod-​​shaped, gram-​​positive, non-​​motile bacterium that belongs to the actinobac­teria, one of the most diverse divisions of the bacterial domain. Many people know Mtb as the etiological agent of one of the most important infec­tious diseases – tuber­cu­losis (TB). TB is an airborne disease that belongs to the Top 10 causes of death worldwide and it is present in every country and age group. According to WHO data, around 1.4 Mio. people died from TB in 2019. Mycobac­terial drug resis­tance in particular is one of the most important issues in the context of TB diagnosis, medication and prevention.

Mtb has extremely inter­esting charac­ter­istics that under­score its patho­genic potential and explain why TB is a serious global threat. In contrast to many other patho­genic bacteria, Mtb is able to remain in the host organism for a long time without causing signif­icant damage. In a first host reaction, Mtb cells can be enclosed in special struc­tures, the so-​​called granuloma. But Mtb proves to be very adaptable and can change its metab­olism and thus survive in the host for a long time. In this latent phase, when the host is not conta­gious, Mtb proves to be a “master of manip­u­lation”. For example, proteins that modify or imitate the host’s signal pathways are secreted in a very targeted manner.

Figure 1. Latent Infection of TB (Source of this image: Madhukar Pai et al., 2016)

But if the host’s immune system is weakened, e.g. due to medication or virus infection, the mycobac­teria quickly adapt and begin to divide. At a certain point, Mtb can no longer be retained in the granuloma struc­tures and the bacteria are released. From this point on mycobac­teria cause TB-​​active disease. The host develops charac­ter­istic symptoms and thus is able to infect others.

Figure 2. Active Disease (Source of this image: Madhukar Pai et al., 2016)

Beside common symptoms (cough with sputum and blood at times, chest pains, weakness, weight loss, fever and night sweats) the role of Mtb as major risk factor for other lung diseases (lung cancer, COPD), autoimmune diseases or metabolic syndromes (obesity, diabetes, ather­o­scle­rosis) is inten­sively discussed in science.

Learn more – Madhukar Pai et al., 2016 – Excellent Review on Tuber­cu­losis

Drug Resistant Mycobac­terium tuber­cu­losis

Mycobac­teria are without a doubt fasci­nating. However, some charac­ter­istics of these pathogens make the airborne disease TB a very serious health security threat. Basically, TB is a disease that can be cured efficiently with medication. But this is only the case for drug-​​susceptible Mtb strains. Unfor­tu­nately, it is not uncommon for Mtb to develop resistance(s) to overcome potent drugs. This is very often due to the misuse or mishan­dling of anti-​​TB drugs, but also due to the biology of the mycobac­teria themselves. A basic distinction is made between monore­sistant TB, multi-​​resistant TB and exten­sively drug resistant TB. The following defin­i­tions are cited from the CDC-​​homepage.

  • Monodrug-​​resistant TB is caused by an Mtb strain that is resistant to one of the first line anti-​​TB drugs.
  • Multidrug-​​resistant TB (MDR TB) is caused by an Mtb strain that is resistant to at least isoniazid and rifampin, the two most potent TB drugs. These drugs are used to treat all persons with TB disease.
  • Exten­sively drug-​​resistant TB (XDR TB) is a rare type of MDR TB that is resistant to isoniazid and rifampin, plus any fluoro­quinolone and at least one of three injectable second-​​line drugs (i.e., amikacin, kanamycin, or capre­omycin). Because XDR TB is resistant to the most potent TB drugs, patients are left with treatment options that are much less effective.

A global total of 206.030 people with multidrug– or rifampicin-​​resistant TB (MDR/​RR-​​TB) were detected and notified in 2019, a 10% increase from 186.883 in 2018. About half of the global burden of MDR-​​TB is in 3 countries – India, China and the Russian Feder­ation.
(World Health Organi­zation)

Intrinsic & Acquired Resis­tance

In the case of mycobac­terial resis­tance, a funda­mental distinction can be made between intrinsic resis­tance and acquired resis­tance. The intrinsic resis­tance can be traced back to species-​​specific properties that do not require additional mutations. This includes for example the lipid rich compo­sition of the mycobac­terial cell wall, which is often described as waxy. Furthermore, transport systems (efflux pumps) can efficiently remove active substances from the cell and thus lead to drug tolerance or resis­tance. However, enzymatic drug modifi­cation or inacti­vation are also important mecha­nisms of mycobac­terial intrinsic resis­tance. On the other hand, there is the so-​​called acquired resis­tance. In the presence of an anti-​​TB drug, bacterial cells are subject to selective pressure. If a point mutation occurs that reduces the effec­tiveness of the anti-​​TB drug, a selective advantage arises. In extreme cases, the accumu­lation of such mutations can lead to multidrug– or exten­sively drug resistant Mtb strains.

More Infor­mation about Drug Resis­tance in Mycobac­teria:

The Need for Innov­ative, Rapid & Field-​​Applicable TB Diagnostics

Although TB ​​can be found in all regions of the world, an overwhelming majority of active TB cases can be traced back to struc­turally weak regions such as Africa or Southeast Asia. In addition to the devel­opment of alter­native anti-​​TB drugs and vaccines, innova­tions in TB diagnostics are one of the major challenges of the near future in order to actually overcome TB. In particular, the focus must be on test formats that allow simple, rapid and robust analysis. The challenge is to achieve sensi­tiv­ities and speci­ficities that come close to current laboratory methods. The combi­nation of innov­ative molecular biology & super simple and robust visual­ization of the results can be the basis of a new gener­ation of diagnostic tools that could have a signif­icant impact in the field of TB-​​diagnostics.

Aside from improving the quality of public health systems in resource-​​limited settings, novel, cost-​​effective point-​​of-​​care diagnostic tools, drugs and an effective vaccine are urgently needed

(S.M. Gygli et al., 2017; Swiss Tropical and Public Health Institute; FEMS Micro­bi­ology Reviews, fux011, 41, 2017, 354 – 373)

Through research and innovation, WHO works to accel­erate devel­opment of rapid diagnostics and treat­ments for drug-​​resistant TB

(World Health Organi­zation)

Research work must continue towards devel­oping new molecular and advanced techniques for rapid and accurate diagnosis of TB, with better perfor­mance charac­ter­istics, that can be easily imple­mented for routine TB diagnosis in low-​​resource countries.”

(Eddabra, R., Ait Benhassou, H. Rapid molecular assays for detection of tuber­cu­losis. Pneumonia 10, 4 (2018).)

Milenia® HybriDetect – A Universal Tool for Field-​​Applicable, Molecular TB Diagnostics

The universal lateral flow test strips of the Milenia® HybriDetect platform are ideal for the visual­ization of DNA-​​or RNA-​​amplification products, but also for the rapid assessment of highly sensitive CRISPR-​​Cas based detection methods. The demands on modern and compre­hensive TB diagnostics are high. It is about safe methods for species identi­fi­cation or the detection of drug resis­tances. But it is also about techniques for deter­mining host-​​specific risk factors and associated virus diagnostics (e.g. HIV). In the following list, repre­sen­tative publi­ca­tions are given that deal with TB-​​associated issues or in which relevant techniques are described.

The universal Lateral Flow Devel­opment Platform Milenia ® HybriDetect enables equipment free analysis of molecular biological products, even in a multiplex setup. (Isothermal) ampli­fi­cation techniques or innov­ative CRISPR-​​Cas-​​based diagnostics are used frequently. In this „type of appli­cation“, the strip is cited again and again in scien­tific publi­ca­tions, but it is also used as a kit component in commercial POC test systems. But our universal test strips can do a lot more …

Learn more about general detection principles using Milenia® HybriDetect.

Milenia® HybriDetect – A Platform for the Detection of Proteins & Nucleic Acids 

Ultimately, the functional principle is based on the recog­nition of an analyte that is labeled with biotin and FITC /​ FAM /​ Fluorescein. In internal exper­i­ments, we want to test how well the Milenia® HybriDetect is suitable as a devel­opment platform for antibody-​​based rapid tests. Our aim was to provide an extremely simple and robust test system for the Point-​​of-​​Care identi­fi­cation of Mtb from an enrichment culture without DNA-​​/​RNA-​​amplification. A pair of monoclonal anti-​​MPT64 antibodies were biotiny­lated and fluorescein-​​labeled, respec­tively. MPT64 is a Mtb–charac­ter­istic protein that is secreted in large quantities by the bacterium into the culture medium. Thus, culture super­natant can be used as sample matrix for the analysis, in order to confirm the presence of Mtb. After co-​​incubation of the sample with the anti-​​MPT64-​​antibody cocktail, the Milenia® HybriDetect test strip is directly placed in the reaction tube. Mycobac­terial growth is confirmed, if a clearly recog­nizable test line appears within 5 to 10 minutes. Once the antibodies were available, the test system was success­fully estab­lished within a single lab day. We will try to provide a detailed summary of these exper­i­ments soon. The following figure shows the basic function­ality.

Rapid Detection of Mycobacterium tuberculosis using universal Lateral Flow Assay
Figure 3. MPT64-​​Detection using Milenia® HybriDetect (Assay Procedure — Detection Principle – Results Inter­pre­tation — Initial Result)

Outlook

TB is still an important topic that threatens the whole world. A signif­icant reduction of the TB incidence or complete elimi­nation of TB is not unreal­istic. But it requires investing in science in order to find new vaccines and alter­native drugs. The third important pillar beside drugs and vaccines is the devel­opment of future-​​oriented diagnostics. TB in particular illus­trates how complex the demands of compre­hensive future orien­tated Point-​​of-​​Care compatible diagnostics are – from simple and rapid identi­fi­cation of charac­ter­istic biomarkers such as the MPT64 to very precise and sensitive analyzes for the detection of resis­tances or even genetic predis­po­sition of the host.

Milenia-HybriDetect-as tool for multilayered TB Diagnostics
Figure: Milenia® HybriDetect – A Valuable Tool for Multi­layered, Field-​​Applicaple TB Diagnostics

The use of universal lateral flow devices such as the Milenia® HybriDetect can make an important contri­bution to multi-​​layered point-​​of-​​need diagnostics in the future.

Refer­ences
  1. Pai, M., Behr, M. A., Dowdy, D., Dheda, K., Divangahi, M., Boehme, C. C., Ginsberg, A., Swami­nathan, S., Spigelman, M., Getahun, H., Menzies, D., & Raviglione, M.: Tuber­cu­losis. Nature reviews. Disease primers, 2, 16076. https://​doi​.org/​10​.​1038​/​n​r​d​p​.​2016.76 (2016)
  2. Iacobino, A.; Fattorini, L.; Giannoni, F.: Drug-​​Resistant Tuber­cu­losis 2020: Where We Stand. Appl. Sci. 2020, 10, 2153. https://​doi​.org/​10​.​3390​/​a​p​p​10062153 (2020)
  3. Navisha Dookie, Santhuri Rambaran, Nesri Paday­atchi, Sharana Mahomed, Kogieleum Naidoo: Evolution of drug resis­tance in Mycobac­terium tuber­cu­losis: a review on the molecular deter­mi­nants of resis­tance and impli­ca­tions for person­alized care, Journal of Antimi­crobial Chemotherapy, Volume 73, Issue 5, May 2018, Pages 1138 – 1151, https://​doi​.org/​10​.​1093​/​j​a​c​/​d​kx506 (2018)
  4. Sebastian M. Gygli, Sonia Borrell, Andrej Trauner, Sebastien Gagneux: Antimi­crobial resis­tance in Mycobac­terium tuber­cu­losis: mecha­nistic and evolu­tionary perspec­tives, FEMS Micro­bi­ology Reviews, Volume 41, Issue 3, May 2017, Pages 354 – 373, https://​doi​.org/​10​.​1093​/​f​e​m​s​r​e​/​f​ux011 (2017)
  5. Eddabra, R., Ait Benhassou, H.: Rapid molecular assays for detection of tuber­cu­losis. Pneumonia 10, 4 (2018). https://doi.org/10.1186/s41479-0180049-2 (2018)
  6.  Joon, D., Nimesh, M., Gupta, S., Kumar, C., Varma-​​Basil, M., & Saluja, D.: Devel­opment and evalu­ation of rapid and specific sdaA LAMP-​​LFD assay with Xpert MTB/​RIF assay for diagnosis of tuber­cu­losis. Journal of micro­bi­o­logical methods, 159, 161 – 166. https://​doi​.org/​10​.​1016​/​j​.​m​i​m​e​t​.​2019​.​03.002 (2019)
  7.  Jaroenram, W., Kampeera, J., Arunrut, N., Siritham­majak, S., Jaitrong, S., Boonnak, K., Khumwan, P., Prammananan, T., Chaiprasert, A., & Kiatpath­omchai, W. : Ultra­sen­sitive detection of Mycobac­terium tuber­cu­losis by a rapid and specific probe-​​triggered one-​​step, simul­ta­neous DNA hybridization and isothermal ampli­fi­cation combined with a lateral flow dipstick. Scien­tific reports, 10(1), 16976. https://​doi​.org/​10​.​1038​/​s​41598-020 – 73981-​​6 (2020)
  8.  Ma, Q., Liu, H., Ye, F., Xiang, G., Shan, W., & Xing, W.: Rapid and visual detection of Mycobac­terium tuber­cu­losis complex using recom­binase polymerase ampli­fi­cation combined with lateral flow strips. Molecular and cellular probes, 36, 43 – 49. https://​doi​.org/​10​.​1016​/​j​.​m​c​p​.​2017​.​08.004 (2017)
  9.  Myhrvold, C., Freije, C. A., Gootenberg, J. S., Abudayyeh, O. O., Metsky, H. C., Durbin, A. F., Kellner, M. J., Tan, A. L., Paul, L. M., Parham, L. A., Garcia, K. F., Barnes, K. G., Chak, B., Mondini, A., Nogueira, M. L., Isern, S., Michael, S. F., Lorenzana, I., Yozwiak, N. L., MacInnis, B. L., … Sabeti, P. C. : Field-​​deployable viral diagnostics using CRISPR-​​Cas13. Science (New York, N.Y.), 360(6387), 444 – 448. https://​doi​.org/​10​.​1126​/​s​c​i​e​n​c​e​.​a​a​s8836 (2018)
  10.  Osborn, M. J., Bhardwaj, A., Bingea, S. P., Knipping, F., Feser, C. J., Lees, C. J., Collins, D. P., Steer, C. J., Blazar, B. R., & Tolar, J. : CRISPR/​Cas9-​​Based Lateral Flow and Fluores­cence Diagnostics. Bioengi­neering (Basel, Switzerland), 8(2), 23. https://​doi​.org/​10​.​3390​/​b​i​o​e​n​g​i​n​e​e​r​i​n​g​8020023 (2021)
  11.  Crannell, Z. A., Rohrman, B., & Richards-​​Kortum, R. : Equipment-​​free incubation of recom­binase polymerase ampli­fi­cation reactions using body heat. PloS one, 9(11), e112146. https://​doi​.org/​10​.​1371​/​j​o​u​r​n​a​l​.​p​o​n​e​.​0112146 (2014)
  12.  Rohrman, B. A., & Richards-​​Kortum, R. R. : A paper and plastic device for performing recom­binase polymerase ampli­fi­cation of HIV DNA. Lab on a chip, 12(17), 3082 – 3088. https://​doi​.org/​10​.​1039​/​c​2​l​c​40423k (2012)
  13. : Centers for Disease Control and Prevention (CDC), General Infor­mation on Tuber­cu­losis (TB)  Website – CDC-​​TB ()
  14. : World Health Organi­zation (WHO), Key Facts about Tuber­cu­losis (TB), Website-​​WHO-​​TB ()