Autor : Muñoz Luis1 *, Gallego Claudio1, Joza Karla1, Marchetti Eliana2, Cordoma Natalia2, Poropat Alejandra1, Armitano Rita Ines2, Salomone César1*
1Department of Respiratory Medicine, Hospital General de Agudos Parmenio P. Piñero CABA 2Central Laboratory, Bacteriology Section, Hospital General de Agudos Parmenio P. Piñero CABA
Correspondencia :Luis Muñoz. E-mail: luismcneumo@gmail.com
Abstract
Tuberculosis (TB) is one of the ten leading causes of death
worldwide, and the main cause from a single infectious agent. Early detection
of the Mycobacterium tuberculosis complex (MTC) and of mutations conferring
resistance to the main drugs used in antituberculous treatment contributes to
reducing the transmission of the infection, and consequently the spread of
resistant TB. The GeneXpert MTB/ RIF test identifies the MTC and simultaneously
detects mutations most frequently associated with rifampicin resistance,
through real-time PCR testing.
The purpose of this study was to compare the performance of the
GeneXpert MTB/RIF method in bronchoalveolar lavage (BAL) with bronchial lavage
(LB) in immunocompetent patients with clinical suspicion of pulmonary TB
without any previous microbiological documentation.
Materials and Methods: We prospectively enrolled patients
with radiologic pulmonary infiltrates compatible with active or residual TB
without previous treatment, with negative direct bacilloscopy or nonproductive
cough, for the assessment of active disease. We identified the most affected
segment through computed axial tomography and bronchoscopy with BAL in said
segment, followed by BL of the affected lobe. A BAL recovery > 40% was
considered significant. The samples obtained were processed for bacilloscopy,
culture and GeneXpert MTB/RIF. We analyzed sensitivity (S), specificity (SP),
positive predictive value (PPV) and negative predictive value (NPV), taking the
solid culture medium as reference for the diagnosis of MTC.
Results: We included 20 patients; 3 were excluded
because they didn’t have a representative BAL sample. 17 patients were
evaluated (11 women, 65%), age 37.2 ± 16.3. The MTC was identified through
conventional methods in 10 patients: 10 with positive culture in BL and 9 in
BAL.
In comparison with the conventional methods, 6 out of 17 samples
obtained through BAL had a positive result for GeneXpert MTB/ RIF: S = 60.0% (CI
31%-83%), SP = 100% (CI 65%-100%), PPV = 100% (CI 61%-100%) and NPV = 64% (CI
35.4%-84.8%). With BL, 9 out of 17 had a positive result for Xpert MTB/RIF: S =
90.0% (CI 60%-98%), SP = 100% (CI 65%-100%), PPV = 100% (CI 70%-100%) and NPV =
88% (CI 53%-98%). All the cases identified with GeneXpert MTB/RIF were true
positives in relation to conventional cultures.
Conclusion: Considering the solid culture as reference
method, the BL was more sensitive than the BAL for the diagnosis of tubercuÂlous
infection through the GeneXpert MTB/RIF method in patients with suspected TB
without previous microbiological documentation.
Key words: Tuberculosis; Bronchoalveolar lavage;
Comparative study
Received: 12/15/2020
Accepted: 06/29/2021
Introduction
Tuberculosis (TB), a preventable and curable condition, is one of
the most widespread infectious disÂeases around the world and constitutes an
important threat to public health. There are approximately 2 billion people
around the world infected with the Mycobacterium tuberculosis complex
(MTC). Every year almost 9 million people develop the active disease and 2
million people die from it1.
The microbiological diagnosis of TB is complicated. The
bacilloscopy is a fast, simple, low-cost techÂnique but has low sensitivity2, 3. Although the culture
test is still the reference method or gold standard for the diagnosis of all
types of TB because it allows for gender and species identification, it
requires higher level laboratories with more demanding infrastructure and equipment
conditions, and the results are obtained after 15 days and even 8 weeks of
incubation of the clinical samples.
Early detection of the MTC and of mutations conferring resistance
to the main drugs used in antiÂtuberculous treatment has a great impact on the
management, prognosis and evolution of the disease apart from contributing to
reduce the transmission of the infection, and consequently the spread of
resistant TB1-4.
In this regard, during the last years different molecular biology
techniques have emerged noticeably accelerating the TB diagnosis in comparison
with the use of bacilloscopy and culture.
The GeneXpert MTB/RIF test is based on the detection of
MTC-specific nucleic acids through real-time PCR testing. The GeneXpert MTB/RIF
simultaneously identifies the MTC and detects the rpoB gene
mutations more frequently associated with resistance to rifampicin, the most
important drug against this disease. Itis a completely automated process that
allows the diagnosis within a period of two hours2.
The WHO (World Health Organization) recommends this test for the initial
diagnosis of TB1-4.
In patients with clinical suspicion of TB and nonproductive cough
or negative bacilloscopy, respiratory samples can be obtained for infectious
analysis through bronchoscopy, providing an early diagnostic confirmation5-9.
The flexible bronchoscopy is a diagnostic and therapeutic
procedure essential for respiratory mediÂcine6,7.
Bronchoalveolar lavage (BAL) is the reference bronchoscopic sample for the
study of infectious diseases that affect the pulmonary parenchyma8, 9, and bronchial lavage
(BL) is a complementary sample due to the possibility to be contaminated with
secretions from the upper respiratory tract.
When the BAL is not available or the samples obtained are not
significant for the analysis, the quesÂtion arises as to consider the BL as the
representative sample for the etiologic study in the situations previously
mentioned.
The purpose of this study was to compare the performance of the
GeneXpert MTB/RIF method in BAL with BL in immunocompetent patients with
clinical suspicion of pulmonary TB, without previous microbiological
documentation, in order to optimize the use of diagnostic resources.
Materials
and Methods
All the bronchoscopy procedures were performed by the staff of the
Respiratory Medicine Division. We used a videobronchoscope of the 70K series
with a diameter of 6.2 mm (model EB - 1970K, PENTAX). All visible bronchi and
pulmonary segments were examined through topical anesthesia; and samples were
collected from the pulmonary segment or subsegment which showed abnormal
lesions suggestive of active TB in the HRCT (High Resolution Computed
Tomography).
For the BAL, 100 to 120 ml of saline solution 0.9% were instilled
in five or six aliquots, the first being separated and attached to the BL. The
BL was done after the BAL. 20-40 ml of saline solution 0.9% were instilled in
the other pulmonary segments involved, and aspirated until 20 to 30 ml of
liquid were collected in the suction trap: then the first aliquot obtained with
the BAL was added.
We excluded patients in whom the BAL couldn’t be performed or
those who didn’t have significant samples. A BAL recovery > 40% was
considered significant. There weren’t any complications during the procedure.
Samples obtained were divided and referred for bacteriological and
cytological studies. At the BacteÂriology Service each sample was divided in
two: one part was used for identifying the MTC and detectÂing sensitivity to
rifampicin by GeneXpert MTB/RIF, whereas the remaining aliquot was used for the
bacilloscopy and culture in a liquid medium (MGIT 960, Becton Dickinson-BD) and
in a solid medium (Löwenstein Jensen and Stone Brink).
We determined sensitivity (S), specificity (SP), positive
predictive value (PPV) and negative predicÂtive value (NPV) taking the culture
in solid medium, Löwenstein-Jensen and Stone Brink as method of reference.
Results
17 patients were evaluated (11 women, 65%), with a mean age of
37.2 ±16.3 years. Distribution per nationality was: 10 (59%) Argentinians, 6
(35%) Bolivians and 1 (6%) Peruvian. Most patients didn’t show comorbidities (n
= 15, 88%) and didn’t have a history of TB (n = 16, 95%). The most frequently
found lesions were infiltrates or consolidations in the upper lobes (n = 17,
100%).
The combination of the results of the BAL and BL samples allowed
us to identify 10 patients with MTC through conventional methods (one with
positive bacilloscopy in BL, 10 with positive cultures in BL and 9 in BAL); the
remaining cases were clinically followed-up.
6 out of the 17 samples obtained through BAL were positive with
GeneXpert MTB/RIF: S = 60.0%, SP = 100%, PPV= 100% and NPV = 64%.
With BL, 9 out of 17 had a positive result for GeneXpert MTB/RIF:
S = 90.0%, SP = 100%, PPV = 100% and NPV = 88%. All the cases identified with
GeneXpert MTB/RIF were true positives in relation to conventional
cultures. The results obtained are shown schematically and with confidence
intervals in Table 1.
In this study we didn’t assess the importance of the GeneXpert MTB/RIF
test in detecting resistance to rifampicin in FBC (fibrobronchoscopy)
samples, though they weren’t detected in patients under evaluation.
Discussion
The bronchoscopic diagnosis of pulmonary tuberculosis is
traditionally based on bacilloscopy and MTC culture. The genomic analysis
through nucleic acid amplification techniques such as the GeneXpert MTB/RIF allows
for a rapid, high-sensitivity diagnosis.
Given the fact that WHO recommendations on GeneXpert MTB/RIF only
refer to sputum samples, more research is necessary on the use of this PCR in
bronchoscopic samples.
Le Palud et al, 2014 conducted a retrospective, observational
study about the diagnostic precision of the GeneXpert MTB/RIF test in
fibrobronchoscopic samples, where the type of sample was left to the discretion
of the specialist according to the patient’s tolerance to treatment10.
In our work, the diagnostic performance of the BL turned out to be
superior to that of the BAL for the identification of active pulmonary TB. With
regard to other studies that analyze the BAL11,
12 or the
BL13, 14 performance individually, we
compared the performance of GeneXpert MTB/RIF in BAL and BL together in
the same procedure in patients with clinical suspicion of TB and we considered
the culture as reference method. We observed that the BAL was less sensitive
than the BL for the diagnosis of TB (60% versus 90%, respectively), with the
same specificity (100%).
Bronchial lavage is referred to in research done in Japan (Kohno,
Kurashima and Takano), India (Arshad, Gupta15)
and South Korea (Yoo, Song) as the reference sample for the study of TB, using
BAL for other respiratory diseases. Those are centers with high incidence of
TB.
One limitation to the work is the small sample of individuals
included. Another observation regarding the procedure used for sample
collection (BL after BAL, including the same segment already assessed with the
latter) is that we could infer a better diagnostic performance of the BL when
we include the residual material from the BAL.
Conclusion
Considering the solid culture as reference method, the bronchial
lavage was more sensitive than bronÂchoalveolar lavage for the diagnosis of
tuberculous infection through the GeneXpert MTB/RIF method in patients
with suspected TB without previous microbiological documentation.
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