Autor : González, Claudio D.1*, Fruhwald, Gladys E.2, Duré, Roberto M.3, Armitano, Rita I.4, Amiano, Nicolás O.5, García, Verónica E.6, Cerqueiro, María Cristina7, Grupo de Estudio Diagnóstico de TB: Amiano, Nicolás O.5; Armitano, Rita I.4; Bisero, Elsa D.8; Cerqueiro, María Cristina7; Duré, Roberto M.3; Fruhwald, Gladys E.2; García, Verónica E.5; González, Claudio D.1; González, Norma E.9; Lombardero, Lorena A.7; Luque, Graciela F.7; Melillo, Karina C.73; Símboli, Norberto F.9
1 Pneumophthisiology Unit, Hospital General de Agudos José M. Ramos Mejía, Autonomous City of Buenos Aires, Argentina. 2 Pulmonology Service of OSPERYH (Health Insurance for Rental and Horizontal Property Workers). 3 Bronchoscopy Unit, Hospital de Infecciosas Francisco J. Muñiz, Autonomous City of Buenos Aires, Argentina. 4 Laboratory for Mycobacteria. Hospital General de Agudos Parmenio Piñero, Autonomous City of Buenos Aires, Argentina. 5 Researcher at CONICET (National Scientific and Technical Research Council). Laboratory of Immunity and Tuberculosis of the IQUIBICEN (Institute of Biological Chemistry, Faculty of Exact and Natural Sciences), University of Buenos Aires (UBA), Autonomous City of Buenos Aires. Argentina. 6 Consulting Physician in the Department of Phthisiology. Hospital de Niños Dr. Ricardo Gutiérrez, Autonomous City of Buenos Aires, Argentina. 7 Pediatric Service. Pediatric Pulmonology Department, Hospital Nacional Prof. Dr. Alejandro Posadas, El Palomar, Province of Buenos Aires, Argentina. 8 Pneumophthisiology Unit, Hospital General de Niños Pedro de Elizalde, Autonomous City of Buenos Aires, Argentina. 9 Mycobacteria Service, National Institute of Infectious Diseases - ANLIS Dr. Carlos G. Malbrán, City of Buenos Aires, Argentina.
https://doi.org./10.56538/ramr./FQAI7141
Correspondencia :Claudio Daniel González E-mail: (claudiodgonzalez57@gmail.com)
Recibido : 17/11/2021
Aceptado: 17/03/2022
1. DIAGNOSIS BASED ON CLINICAL CRITERIA
Claudio D. González
The system of symptoms and signs
most widely used for the screening for suspected tuberculosis (TB) was provided
by the WHO (World Health Organization). The presence of fever, night sweats,
weight loss, and cough correlates with 77% of sensitivity and 68% of diagnostic
specificity in HIV reactive patients.1
Furthermore, in HIV non-reactive patients, a comparison has been
made between the diagnostic capacity of the symptoms (cough, hemoptysis,
fever, night sweats or weight loss), of the radiology, and the rapid diagnostic
molecular tests, such as Xpert, LAMP and Truenat. Any of the said TB symptoms
reached 71% sensitivity and 64% specificity; radiologic anomalies had 85%
sensitivity and 98% specificity, and rapid tests in adults at risk reached 69%
and 99%.1
2. DIAGNOSIS BASED ON CHEST IMAGING
The use of simple chest X-ray has
been recommended a long time ago for the screening of the population with
suspected TB. The sensitivity of the radiology in the diagnosis of pulmonary
TB oscillates between 87% and 98%, and the specificity is approximately 75%.2 As additional information, it is observed that radiological
findings can appear even before the four mentioned symptoms. The validity of
simple chest radiology is explained by the fact that at least 90% of patients
had evident radiological manifestations which are then confirmed in their
bacteriology.2,
3
Regardless of the accesibility of
the radiological resource, in certain groups it may be necessary to perform
large-scale population screening. For that purpose, the Stop TB Partnership
initiative recommends for the first time the use of three specialized
software products of detection computerized assistance (CAD, computer aided
detection) based on artificial intelligence that provide an automated and
standardized interpretation of chest digital X-rays for radiologists or
teleradiographs. Results are expressed as abnormality scores; the software may
be used for detection or screening and is limited to simple X-rays for
pulmonary TB in individuals of 15 years or older.4, 5
The section about TB diagnosis in
children describes the usefulness and limitations of radiology in the
diagnosis of TB.
3. DIAGNOSIS OF TB IN CLINICAL POINTS OF CARE (POC)
Gladys Esther Fruhwald
With the need to facilitate the
access of patients to TB diagnostic methods, this system of decentralized care
(POC) has been suggested.6 It requires minimum staff training and simple diagnostic
equipment with rapid results.
For the purpose of obtaining
early detection of the disease, specially in
vulnerable patients such as HIV carriers or children, the WHO proposes getting
a new system that allows for a diagnosis without sputum samples or with the
use of biomarkers. It also recommends a screening test that allows for the
identification of those who need more tests, possibly sputum, blood or urine samples. In any case, the method replacing
the sputum sample should have a sensitivity that can be comparable to the XPERT
system; it has to be simple and, if possible, it shouldn’t require any power or
temperature control.7
Screening tools to identify who
needs more tests can be used by taking into account the symptoms (cough, night
sweats, weight loss, fever and hemoptysis) or the chest X-rays.8
In the screening through symptoms, risk populations should be
categorized by community (impoverished neighbourhoods, immigrants and people
with direct contact with cases of TB), hospital departments and primary care
centers; in the screening of people with history of HIV disease, undernourished
individuals, diabetic patients or other immunosuppressed groups, place of
residence (detention centers, shelters, immigration centers), and workplace
(activities with high risk of having TB such as mining, health workers, etc.).9
Screening through portable chest
X-ray is a simple, highly sensitive method, as shown by the experience in
Kenya, where 92% sensitivity was obtained in patients with HIV and 100% in
patients without that disease, with a specificity of 73%.10
In this case, the X-ray is more sensitive than the screening
through symptoms, specially if any of those symptoms is taken into account.
Finally, it is necessary to
highlight the fact that in the diagnostic algorithms designed with the use of
these simple resources, the X-ray is most useful when placed at the beginning
of the algorithm.11 The
combination of screening through symptoms with imaging screening would allow
compensating the lack of information of the latter, which can be missing or get
lost, and seems to be more common precisely among the most vulnerable
populations. The addition of a confirmation molecular test seems to be the
ideal strategy for obtaining early TB diagnosis in POC units.12
The use of a digital radiology system of
automated reading could help reduce the bias in the interpretation of the
technician in charge.13
4. DIAGNOSIS THROUGH INVASIVE METHODS
Roberto Miguel Duré
4.1 Role of bronchoscopy in the diagnosis of TB
Below is a description of four
groups with risk of showing pulmonary TB, with indication of further evaluation
through bronchoscopy.14
i. Patients with primary forms,
HIV-negative, usually children.
ii. Patients with primary or
post-primary forms, HIV-positive, generally with atypical radiological
presentation.
iii. Patients with typical,
post-primary forms, HIV-negative.
iv. Patients with treatment
failure and non-conclusive sputum samples, with suspicion of resistance to
treatment drugs.
Patients from group 2 have a
higher risk of disseminating their condition and also the possibility of other
disease markers, that is why the use of this resource
is more urgent in these patients. In adult patients from group 3, the risk of
transmission will necessarily be low and the possibility of beginning an
empiric treatment, according to the previously exposed criterion, should be
evaluated. Finally, in children, gastric lavage should be performed as a first
option before an endoscopy, and the decision to begin treatment shouldn’t be
delayed in this risk group.
This analysis would allow for the
indication and rational use of the endoscopy in negative pulmonary forms,
without disregarding the risk of bad evolution of each group.15
Indication of bronchoscopy according to clinical presentation
The clinical forms in which the
endoscopy has proven effective are:
i. Miliary TB,
generally detected in group 2 and, to a lesser extent, in patients from group
3.
ii. Forms of intrathoracic
tuberculous lymphadenopathy, commonly observed in groups 1 and 2.
iii. Other typical or atypical
radiological forms, generally related to group 3.
iv. Asymptomatic patients with
history of contact with TB focus and CT compatible with a tree-in-bud image.
This is a new indication related to topographic image analysis. Although there
isn’t any evidence to conduct an invasive study, isolated cases being found
will probably justify the use of that kind of study in the future.15, 16
For these forms of presentation,
the available procedures are: the bronchial brush (BB), bronchial biopsy
(BBy), transbronchial needle aspiration (TBNA), transbronchial biopsy (TBB)
and bronchoalveolar lavage (BAL).17-24
Bronchial brushing
(BB)
The use of the BAL has been
extended more than the BB for the diagnosis of TB. However, the performance of
the swab plus the culture studies is between 43% and 57%.15, 16
Bronchial biopsy
(BBy)
The performance of the bronchial
biopsy with some of the cited endoscopic images is 53%.42 Although the prevalence of endobronchial TB
is low (2.5% of the cases), we must mention that the most common forms, the
caseous, the hyperemic-edematous and granular forms may evolve to the
fibroestenotic form, or resolve completely within the first three months of
treatment.18,
19
Transbronchial needle
aspiration (TBNA
It is recommended for mediastinal
ganglionar forms, especially in right paratracheal, right and subcarinal
bronchial and hiliar groups, in that order, because those are the most
accessible for transbronchial needle aspiration.19
At present, histology needles No 19 such as
the Wang or Schiepatti are suggested. In a study of 84 HIV-negative patients,
Bilacerogu reaches 75% of the diagnosis with that method, adding the
histological aspect with the biopsy culture (histological examination, 57% of
efficacy per se).21 With
predominantly right nodes, the sensitivity is 83%, specificity 100%, NPV
(negative predictive value) 38%, PPV (positive predictive value) 100% and
precision of 85%21.
The usefulness of endobronchial ultrasound-guided transbronchial needle aspiration
(EBUS - TBNA) isn’t well-known yet as a diagnostic method of TB, but it could
increase the profitability of needle aspirations.22
Transbronchial biopsy
(TBB)
The miliary forms or segmental
infiltrates have high specificity in the diagnosis of TB, and a sample shall
always be sent for histological examination (5 samples) and culture. The
performance of the TBB in TB is 73%, mainly based on the histological result.21
Bronchoalveolar
lavage (BAL)
BAL is the most used endoscopic
procedure for the diagnosis of pulmonary TB with negative sputum.
In comparative studies, the BAL
contributed to the TB diagnosis in 30%, compared to 21% for gastric lavage and
16% for post-bronchoscopy sputum in a sample of 215 patients.23
The BAL had 89.7% sensitivity,
100% specificity, 100% positive predictive value, 94.6% negative predictive
value and 96.3% test precision in suspected cases of pulmonary TB with negative
sputum/swab and culture.24
4.2. Diagnosis through measurement of adenosine deaminase enzyme
Rita Armitano
Pleural TB is the most common
extrapulmonary manifestation of the infection caused by bacteria of the M.
tuberculosis complex, and occurs with variable frequency according to each
country in up to 30% of patients, regardless of coinfection by HIV.
The anatomopathological study and
culture of the pleural biopsy are the diagnostic methods of choice. The
histopathology shows a sensitivity of 56%-78% and a specificity of 95%, whereas
the culture has a sensitivity that oscillates between 69% and 97% and a
specificity of 96%.25
The adenosine deaminase (ADA)
test in pleural fluid is a useful diagnostic examination, especially in
patients who come from an environment with high prevalence of TB. There are
numerous studies supporting its determination as a supplementary test for the
diagnosis of tuberculous pleurisy.
The ADA enzyme participates in
the catabolism of purine bases, the proliferation and differentiation of
lymphoid cells and the maturation of macrophages. That enzyme is produced by
monocytes and macrophages that catalyze the conversion of adenosine and
deoxyadenosine to inosine and deoxyinosine, respectively. The level of ADA in
pleural fluid reflects the presence of cells in the pleural cavity, mainly
activated T-lymphocytes.26
At present, the reference method
is the one described by Giusti, based on the detection of ammonia released in
the enzymatic reaction and its subsequent quantification from a coloured
compound.26
The determination of the
parameters for the ADA test, like in any other diagnostic test, is in direct
relation to the prevalence of the disease in question and other diseases that
could influence the population being studied, to the study design and the
methodology. Consequently, there are different
discrimination values (cut-off points) for this test which according to the
data published in the international literature vary from 30 U/L to 80 U/L. In
accordance with the national recommendations based on a study conducted by the
National Network of TB Bacteriology, where 152 patients with tuberculous
pleural effussion were investigated through the manual colorimetric method of
Giusti-Galanti, an ADA value of ≥ 60 U/L would have a sensitivity of 84%
and a specificity of 94% for the diagnosis of tuberculous pleurisy.27 In agreement
with this work, the Mycobacteria Service INEI-ANLIS Carlos G. Malbrán,
has shown that said cut-off level groups 80% of patients with pleural TB,
therefore an inferior result wouldn’t discard the diagnosis. One disadvantage
of this method is the presence of false positives, for example in the case of
non-tuberculous empyemas, the malignant proliferation of T-cells, systemic
lupus erythematosus and rheumatoid arthritis pleurisy. In any case, the result
will have to be analyzed according to the reference clinical situation, always
considering the fact that diagnostic certainty requires microscopic examination
and culture that confirm the presence of the M. tuberculosis complex.27
A study conducted in a reference
laboratory of the Tuberculosis Care Network of CABA during 2016 evaluated the
performance of an automated method for determining the presence of ADA in
pleural fluid.28 A total of 26
samples from patients with suspicion of tuberculous pleurisy were processed.
The samples were divided in two aliquots. One of those aliquots was referred to
the Mycobacteria Service INEI-ANLIS Carlos G. Malbrán for determination
through the Galanti-Giusti manual colorimetric method, and the remaining
aliquot was processed by the automated method DIAZIME with the COBAS 6000 -
Module C50 analyzer, according to the manufacturer’s instructions, at the
Central Laboratory of the Hospital General de Agudos Parmenio Piñero.28 The cut-off
values were those of the Galanti and Giusti method: 60 UI/L 37°C, and DIAZIME
method: 30 UI/L 37°C. In order to calculate the Kappa index, the mean values of
each sample were classified in the following agreed categories: negative
Galanti and Giusti method: ≤50 UI/L 37°C; borderline value: 50-70 UI/L
37°C; positive value: ≥70 UI/L 37°C. DIAZIME method: negative value:
<29.4 UI/L 37°C; borderline value: 29.4-30.4 UI/L 37°C; positive value:
>30.4 UI/L 37°C.
18 (69.2%) of the 26 processed
samples were negative and 8 (30.8%) were positive, with both methods. No
borderline results were obtained through any of the methods. The concordance
strength in the classification per categories between the two used methods was
excellent (k = 1.000).
These results suggest the
usefulness of the automated method for the determination of ADA in pleural
fluid, because apart from its high concordance with the reference method, no
false positives or negatives were detected in this work.28
Another advantage of ADA
determination is the fact that it is a simple method, easy to use and fast,
with a mean result time of 2 hours, a value that can be reduced even further
with the use of automated methods such as the one previously mentioned, with
the possibility to increase the number of analyzed samples during a working
day. Apart from the cases of false positives and negatives mentioned before, we
must consider the alterations in the results caused by difficulties in the
conservation and transport of the sample, the presence of hemolysis and
exposure to high temperatures as other potential disadvantages. There is one
limitation of the ADA research that we must emphasize: the method doesn’t have
an acceptable precision regarding the cerebrospinal fluid and other serous
collections, due to the narrow range between normality and the cut-off points
suggested for these extrapleural samples.28
4.3. Diagnosis of infection through the interferon gamma assay (IGRA)
Nicolás Amiano and
Verónica García
The latent infection caused by M.
tuberculosis (LTBI) is a subclinical infection defined on the basis of the
cellular immune response against mycobacteria antigens. The identification of
the LTBI is important for the implementation of public health policies related
to the control of the disease through the identification of individuals with
high risk of developing active TB. Currently there isn’t any assay that could
be used as reference method (gold standard) for the identification of LTBI. The
low bacterial load in tissue associated with LTBI prevents any diagnosis
focused on the identification of the bacteria or its components. So, the
diagnosis of LTBI consists in showing the cellular immune response of the
individual against microbacterial antigens. In Argentina, the test to diagnose
LTBI uses the purified protein derivative (PPD), but in the last years,
developed countries implemented the interferon gamma release assays (IGRAs).
These tests came from the search of antigens in exclusive regions of the M.
tuberculosis genome (not present in M. bovis, BCG or any other
mycobacteria species) as the main tools for developing new diagnostic methods.
The basis of these assays lies in the fact that T cells from individuals
previously sensitized with M. tuberculosis release interferon gamma
(IFN-γ when being
re-stimulated with pathogen-specific antigens; the most used ones are CFP-10
and ESAT-6.29
Since patients with active TB are
infected with M. tuberculosis, they have been used as standard for the
IGRA and PPD in order to determine the sensitivity of these assays; generally,
the IGRAs are more sensitive than the PPDs. Therefore, in patients with active
TB the skin test is usually positive, almost in 70% of the cases, whereas the
IGRAs are positive in 80%-85% of the cases. Is this the real sensitivity of
these diagnostic tests of latent infection? Maybe not, because the state of
the immune system of individuals who progressed to active disease is different
from that of subjects with latent infection, and this could affect the results
of the assays that are based on the performance of cell-mediated immunity to
detect previous exposure to M. tuberculosis.30
The IGRAs that are available in
the market are the following:
I. T-SPOT.TB (Oxford Immunotech,
UK). With this method, the mononuclear cells of peripheral blood obtained by
centrifugation are stimulated by CFP-10 and ESAT-6 for 16-24 h, and the ELISPOT
technique analyzes the number of points (spots) indicating activated T cells
that are producers of IFN-γ against the antigens.
II. QuantiFERON-TB Gold
(QFT-QIAGEN, U.S.A, Germany) and QuantiFERON-TB Gold-Plus (QIAGEN, U.S.A,
Germany). With this method, a sample of peripheral blood from the individual is
stimulated with specific M. tuberculosis antigens for 16-24 h. Then,
centrifugation is performed and plasma IFN-γ levels are determined through ELISA (enzyme-linked
immunosorbent assay).
III. LIOFeron®TB/LTBI (LIONEX GmbH, Germany). The
performance of this test is similar to that of QFT-QUIAGEN.
IV. VIDAS® TB-IGRA
(BIOMÉRIEUX, France)
However, neither the PPD nor the
available IGRAs allow the discrimination between active and latent TB. Also,
these tests can’t be used to predict if an individual with LTBI will be developing
active TB or if the treatment for LTBI is effective in reducing the risk of
developing active TB. An analysis carried out with 167 individuals (patients
with TB and persons cohabiting with patients) from hospitals in the city of
Buenos Aires showed 78% concordance between QFT and PPD and 22% discordance
(Kappa = 0.530 SE of kappa = 0.067), indicating a moderate strength of
concordance.31
IGRA for the identification of
infected contacts
The follow-up of the contacts of
TB patients and the identification of subjects with LTBI after exposure to
individuals with active TB are important elements of TB control. Several
studies have provided different estimations of the rate of progression to active
disease two years after the conversion of PPD/IGRA, but the general lifetime
risk that is normally described accounts for 10%-15%.32
Even though certain studies have suggested a higher risk of
progression to active TB after a positive IGRA result, this difference wasn’t
significant in comparative meta-analyses. Therefore, the PPD or IGRA could be
used to investigate contacts of active TB. Nevertheless, in populations whose
contacts have a history of vaccination against BCG (bacille Calmette-Guerin),
the highest specificity of the IGRA could allow a better orientation of
preventive therapy. Still, it is important to highlight the fact that the
specificity of the PPD is minimally affected by immunization with BCG if the
vaccine is administered before one year of age.33
For that reason, the IGRAs could be used in adults exposed to
patients with active TB (for example, for the follow-up of the contacts) and
the results would be more reliable in contacts vaccinated with BCG after one
year of age.
IGRA to identify latent infection
in immunocompromised patients
HIV infection increases the risk
of LTBI progressing to a clinical disease in a significant way. Several
studies confirm that the sensitivity of the IGRAs is reduced in subjects
infected with HIV, with similar findings for PPD.34
Low counts of CD4 T lymphocytes (<200 cells/μL) are associated with negative or indeterminate IGRA
results. But some meta-analyses suggest that the T-SPOT.TB test has more
sensitivity than the QFT in HIV-positive subjects for the diagnosis of active
TB. On the contrary, other studies show that none of the existing IGRAs has
proven to be more sensitive than PPD for the detection of LTBI in HIV-positive
patients, and that IGRAs generally work in a way similar to PPD tests.35
Patients with immune-mediated
inflammatory diseases (IMIDs), such as rheumatoid arthritis, ulcerative colitis
and Crohn’s disease, inter alia, have an increased risk of developing active TB
due to the immunosuppressive therapy they receive. Several studies have shown
that IGRAs do not seem to be better than the PPD tests for the diagnosis of
LTBI in patients with IMIDs.36 However, more
formal meta-analyses or longitudinal studies about the risk of active TB in
these patients with negative and positive IGRAs should be conducted.
In our country, a diagnostic
method of the IGRA type has been developed at the Laboratory of Immunity and
Tuberculosis (IQUIBICEN, CONICET - UBA) (Diagnos-TB) that is similar to
QuantiFERON but better, thanks to the addition of an extra tube that allows
differentiating individuals with LTBI from healthy individuals, patients with
TB and individuals recently exposed to M. tuberculosis, with 79%
sensitivity and 83% specificity.31 This method
will be offered to the community in the future as High-Level Technological
Services (STAN [Servicio Tecnológico de Alto Nivel, for its acronym in
Spanish] - IQUIBICEN: www.iquibicen.fcen.uba.ar) at a cost clearly lower than
imported IGRAs. At the same time, the paperwork for the ANMAT (National Administration
of Drugs, Foods and Medical Devices) approval will be prepared.
5. THE FUTURE OF TB DIAGNOSIS. RESEARCH METHODS
María Cristina Cerqueiro
The translational medicine of
this century has brought a major breakthrough in the diagnosis of TB, thanks to
the movement of discoveries from basic research to medical practice. This is a
slow process that usually poses a whole lot of challenges. In so far as new
tests arise, it is not enough to identify variants of statistical
significance; the clinical relevance must be supported in terms of validity and
usefulness by profitability, noninvasiveness, efficacy
and risk reduction. For that purpose, the WHO uses the criteria of the Target
Product Profile (TPP).7 For example,
in order to identify disease progression, the method or element is required to
reach minimum sensitivity precision ≥ 75% and specificity ≥ 90%;
for a new diagnostic test, 65% sensitivity and 98% specificity are expected;
for the screening, a minimum sensitivity ≥ 95% and specificity ≥
80%, with a cost ≤ USD 2 for a test not based on sputum that can be applied
in the POC.
In order to improve the quick TB
diagnosis, new tools are of crucial importance: noninvasive tests not based on
sputum, and portable and affordable devices to apply those tests in a simple
way, apart from the improvement in already existing tests. Translational
research reinforces progress preventing and managing the whole TB spectrum;
there is an urgent need for investment and support to strengthen the research capacity
and its application in the healthcare field.
The TB diagnosis could be
improved, specially in places with higher incidence of this disease, with
easily accessible clinical samples, such as urine, stool, oral swabs, exhaled
air or aerosols and the use of POC tests that do not require a source of
energy, and are cheap and easy to use.37
Also, such diagnostic tests must be widely applicable to every
type of population, including children and immunocompromised individuals who
frequently show negative sputum or tend to have atypical presentations,
compromising the performance of the quickest NAATs (nucleic acid amplification
tests).
THE “OMICS” PLATFORMS AND THEIR APPLICATION TECHNIQUES
The TB diagnosis requires the
knowledge of the complex group of host-pathogen interactions, a process that
has not yet been completely elucidated. Scientific breakthough related to such
knowledge and the application of technologies has facilitated the development
of tools and platforms of the whole biological system. These “omics” approaches
are used in search of diagnostic tests for the tuberculous infection, its
progression to disease, to monitor treatment efficacy and results, and to
improve the understanding of the pathogenesis of the disease and its virulence in
the application of vaccines and new treatments.38
Since 2015, breakthroughs in genomics
have allowed the use of the whole genome sequencing (WGS) and the discovery
of new biological mechanisms in TB. Unlike the serological techniques, this
diagnostic tool can discriminate between TB reinfection and relapse, confirm
the presence of current infection, and provide information for the
epidemiological characterization and tracing of transmission.39
Epigenomics studies changes in the function of the genes without changing the
sequence, for example, the non-specific effects of the Calmette- Guérin
bacillus or the Mendelian susceptibility to mycobacteria.40
Proteomics researches the dynamics of protein products coded by the genome
(proteome), ultimately facilitated with protein mass spectrometry (MS).41
The transcriptomics approaches
research the patterns of genic expression to derive molecular signatures from
the host and the physiology of the pathogen. They use RNA quantification methods,
such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
and RNA sequencing (RNA-Seq). They analyze ribosomal RNA (rRNA) to detect the
viability of the bacillus, the gene regulation of micro RNA (miRNA) with the
next-generation sequencing (NGS) and also the cellular signatures obtained from
databases of gene ontology (GO) and flow cytometry.42
The infection of the immune cells
of the host caused by M. tuberculosis causes several changes in metabolism;
the metabolism of glucose and lipids is fundamental to defining the fate of
the host cell function within the context of mycobacteria survival inside the
granuloma.39 Metabolomics
focuses on understanding the interactions that occur in the disease environment.
It allows the identification of metabolites with active and passive effects on
phenotypes of interest, characterizes the metabolites of small molecules in the
biological systems and their participation in various biological processes:
cell differentiation and maturation, insulin signaling, T cell survival, energy
transfer, immune responses of macrophages and cell-to-cell communication.43 The MS and
nuclear magnetic resonance (NMR) are the chosen techniques.
Fluxomics studies the dynamics of molecules, measures the metabolic phenotype of
the biological system and provides an identification of carbon and nitrogen
flow in the host.38
BIOMARKERS
The discovery of biomarkers can
be based on different methodologies, such as the imaging techniques that show
the fluctuation of a substance or the alteration in the structure and function
(CAD, biochemical techniques or omics techniques, to cite a few examples). A
biomarker could be a cell or molecule that can be detected in a biological
sample collected from the body that expresses M. tuberculosis exclusively
or differentially or host molecules that express differentially in response to
infection by M. tuberculosis.39 One example
of what is used currently in the field of TB would be the IFN-gquantified by
the IGRA, the LAM (lymphangioleiomyomatosis) antigen in urine or the Koch
bacillus in the bacilloscopy.
In the last 20 years, only a few
of the thousands of biomarkers reported in the bibliography have offered
promising tools for clinical decision-making in TB. The omics approach is a
high-performance method that allows obtaining biomarkers of multiple
dimensions in only one step.
Bacterial biomarkers can derive
from the analysis of their genes (urinary cell-free DNA), transcriptomic
profiling or proteomics signatures. ESAT, CFP-10, Rv3615c, Rv3798c, MPT64, lipoproteins,
mycolic acids or antigens in volatile compounds are detectable in sputum,
plasma, expired air or urine.39,
40
Exosomes derived from Mycobacterium
tuberculosis (Mtbexo) are a type of bioactive vesicle produced by the
internal budding of endosomes, and are present in biological fluids.
Preliminary evidence suggests that exosomes play a role in cell-to-cell
communication and modulate immune and inflammatory responses of the host.39, 44
Host biomarkers study cellular
immune signatures with high diagnostic sensitivity and specificity for active
TB and distinguish the active from the latent infection, antigen expression in
T cells, activation, memory, or proliferation markers with the participation of
interleukins, various cytokines, the HLA-DR level or enzymatic molecules that
intervene in signaling routes.39,
40
The genes and signatures or
transcriptional records of RNA, of various materials, could distinguish the
latent infection from active TB and predict disease progression.42
For example, the combination of monocytes and macrophages and
their relative expression of miRNA provide a more recent vision of the
mechanism that generates the survival of the bacillus, the manipulation of the
host’s defense and the origin of latent infection and disease resistance.
The multi-omic integration, which
represents multiple levels of biological organization, allows a more precise
reconstruction of dynamic molecular networks that sustain healthy and sick
states, using artificial intelligence applications with a variety of
statistical and automatic learning approaches (machine learning).42
From the molecular diagnosis
systems, lateral flow chromatographic assays, plasmid-based technology, and
the volatile gas analysis (Aenose) to artificial intelligence processing, the
search for future TB biomarkers shall be based on the principle of
“patient-centered medicine” proposed by translational medicine.
DEVICES
In the last years, the
development of research in POC diagnostic platforms has shown many advantages
in its down-scaling process. The micro-nanodevices based on LOC platforms
(Lab-on-a-chip) with microfluidic techniques also show high sensitivity, high-performance
and accurate results, as well as low cost and portability in a compact format.44 They are
still in their first stages, like the lateral flow chromatographic immunoassay
(LFA), that uses porous membrane and other portable nanotechnologies (Oxford
Nanopore Technologies-ONT).
Paper-based analytical devices (μPAD electrochemicals) represent the largest part of POC devices because paper is a low-cost,
biocompatible substrate with high feasibility to integrate different function
modules, thus favouring their use in the diagnosis of biological samples and
the identification of biomarkers. Other devices include the development of bioassays,
flow cytometry, Luminex bead-based assays and multiple electro-chemioluminiscent
immunoassays (Meso Scale Discovery, MSD), which have achieved great success
in the detection of multiple cytokines in serum and plasma samples.44
Aptamers, in turn, are short, single-stranded nucleic acids that fold into specific three-dimensional structures, and this allows them to bind to target molecules through different processes, for example, IFN
ꙋdetetion with graphene-field effect transistors
(GFETs) or with LFA.45
The clustered Regularly
Interspaced Short Palindromic Repeats assay (CRISPR) that uses the
trans-excision activity CAS (SHERLOCK and DETECTR) is a genomic edition
method that functions as molecular scissors, cutting and modifying
CRISPR-MTB detects M. tuberculosis directly in clinical samples, including
sputum, BAL, CSF, pleural fluid, ascites and pus with improved sensitivity
(that is to say, with an almost unique copy); it requires lower sample volume
and provides faster results. CRISPR/Cas9 and CRISPR/Cas12a detect DNA or RNA
and are viewed with blue luminescence signal. Also, cytokines have been
detected which are viewed with LFA.46
Large-scale use of new diagnostic
tests in clinical applications and public health is yet limited by a series of
factors: lack of standardized solutions, technical requirements of the
laboratories, high costs associated with the use and maintenance of these
molecular technologies, Internet infrastructure and cloud computing.47
We hope that addressing these
barriers improves patients’ results, but we still need a real POC test to meet
the remaining challenges, such as sample preparation and human resources
demands.
Conflicts of
interest.
The authors of this work declare
that there is no conflict of interest.
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