INTERNAL MEDECINE JOURNAL OF VIETNAM|NO 22/2021
42
CLINICAL RESEARCH
THE RELATIONSHIP BETWEEN THE 16S
rRNA REAL-TIME EXBP RT-PCR ASSAY RESULTS
WITH CLINICAL AND SUBCLINICAL SYMPTOMS
IN NEWLY DIAGNOSED TB PATIENTS
Nguyen Thi Han1, Do Quyet1, Mai Xuan Khan1, Nguyen Chi Tuan1
1Respiratory center, Military Hospital 103, Vietnam Military Medical University
ABSTRACT
Background: Globally, an estimated 10.0 million people fell ill with TB in 2018, a number that has been
relatively stable in recent years. The 16S rRNA real-time ExBP-RT-PCR test has been shown to be an
effective TB diagnostic test. Subjects and methods: The study was conducted on 43 newly diagnosed
pulmonary tuberculosis patients at the Department of Tuberculosis - Military Hospital 103 within 4 months
from February 2020 to June 2020. Aims: The current study assessed the relationship between the 16S rRNA
real-time ExBP RT-PCR assay results with clinical and subclinical symptoms in newly diagnosed TB patients.
Results: The 16S rRNA Real-time ExBP-RT-PCR in sputum specimens associated with hemoptysis (p
= 0.014), fever symptoms (p = 0.034). The 16S rRNA Real-time ExBP-RT-PCR cycle threshold in BAL
samples was inversely proportional to the positive level of AFB smear (r = -0.5215, p = 0.0153) and MGIT
time to positivity (r = -0.5867, p = 0.0041) Conclusions: The 16S rRNA real-time ExBP RT-PCR assay
was associated with hemoptysis and fever symptoms. The threshold cycle of 16S rRNA assay in BAL
specimens was inversely correlated with AFB positivity level and MGIT time to positivity.
* Keywords: 16S rRNA; New pulmonary tuberculosis; New PTB.
INTRODUCTION
The burden of tuberculosis varies enormously
among countries, from fewer than five to more
than 500 new cases per 100,000 population per
year, with the global average being around 130.
Vietnam ranks 11th out of 30 countries with a high
prevalence of tuberculosis in the world, according
to the global tuberculosis report 2019 [1]. Recently,
the 16S real-time ExBP-RT-PCR for tuberculosis
bacteria using the ultra-sensitive gene detection
technology has been proven as a highly sensitive
test in diagnosing pulmonary tuberculosis in
Vietnam [2]. To serve clinical practice in TB
diagnosis, we conducted a study: To evaluate
the relationship between the 16S rRNA assay
and clinical and subclinical characteristics in new
pulmonary tuberculosis patients.
SUBJECTS AND METHODS
1. Subjects
All patients diagnosed with new PTB, aged more
than 18 were recruited into this study. The main
inclusion criteria were: Abnormalities on CXR
compatible with PTB and a positive result on the
MGIT culture; principal exclusion criteria were
previous treatment for active TB disease.
Corresponding author: Nguyen Thi Han (nguyenhan.hvqy@gmail.com)
Date received: 04/5/2021
Date accepted: 02/6/2021
INTERNAL MEDECINE JOURNAL OF VIETNAM|NO 22/2021
43
CLINICAL RESEARCH
* Study location and time:
The current study was conducted at the Department
of Tuberculosis, Military Hospital 103 from February
2020 to June 2020.
* Sample size:
43 eligible patients with new pulmonary tuberculosis
were included in the study.
2. Methods
* Study design: A cross-sectional study.
43 PTB patients with MGIT cultures had sputum
and/or BAL specimens taken for the 16S rRNA
real-time ExBP RT-PCR and AFB smear. The
16S rRNA assay was performed according to
the Department of Genomics and cytogenetics,
Institute of Biomedicine and Pharmacy, Vietnam
Military Medical University. Specimens after
collection were transported to the laboratory within
30 minutes and stored immediately at a -80ºC
freezer. Before conducting the assay, the lab staff
examined the positive and negative controls to
rule out false-negative or false-positive results.
The positive control must be positive and had a Ct
value coinciding with the Ct value of the previously
titrated calibrator (positive control had a Ct value
of 27 ± 1.5). Negative controls must be negative.
- Cycle threshold value:
+ If Ct ≤ 27 cycles: CT high.
+ If 27< Ct ≤ 33 cycles: CT medium.
+ If 33< Ct ≤ 36 cycles: CT low.
+ If Ct ≥ 36 cycles: CT very low.
All patients were clinically examined for general
and functional symptoms and physical signs of
pulmonary tuberculosis.
* Data analysis and statistical method:
Continuous variables data were presented as
means ± SD [standard deviation], and categorical
data were presented as numbers and percentages.
For continuous variables with normal distribution,
test by Pearson’s correlation coefficient: If the value
of the correlation coefficient is negative (r < 0), the 2
variables are inversely correlated; If the correlation
coefficient value is positive (r > 0), the two variables
are positively correlated. Pearson (r) is significant
only when the observed significance level (sig) is less
than the significance level α = 5%. (sig < 0.05). For
categorical variables and continuous variables, use
the Spearman rank correlation test. If the correlation
coefficient r> 0, the 2 variables are positively
correlated. If r <0, the 2 variables are negatively
correlated. Spearman’s correlation coefficient is
significant when p <0.05. For categorical variables,
test the relationship by Chi-Square, if P <0.05, the
relationship is statistically significant.
The analysis was performed using STATA version
14 (College Station, Texas 77845 USA).
* Data collection:
Data collection procedures: All new PTB patients
were invited to the study. A consent form was
given to the participants before administering the
research. Personal information (e.g., name, phone
number…) was anonymized before the analysis.
RESULTS
1. Participants’ characteristics
Table 1: General characteristics of the participants.
N or mean ± SD %
Gender (n = 43)
- Male
- Female
30
13
69.77
30.23
Age, years
(n = 43)
- Mean ± SD
- Range
49.23 ± 20.12
20 - 90
INTERNAL MEDECINE JOURNAL OF VIETNAM|NO 22/2021
44
CLINICAL RESEARCH
N or mean ± SD %
Clinical manifestations
- Cough
- Hemoptysis
- Chest pain
- Night sweats
- Fever
- Weight loss
27
9
4
11
21
3
62.79
20.93
9.30
25.58
48.84
6.98
Sputum smear microscopy results (AFB
report*) (n = 36)
- Negative (0)
- Positive
- Scanty
- 1+
- 2+
- 3+
15
21
2
5
6
8
41.67
58.33
5.56
13.89
16.67
22.22
BALF smear microscopy results (AFB
report*) (n = 22)
- Negative (0)
- Positive
- Scanty
- 1+
- 2+
- 3+
16
6
3
2
0
1
72.73
27.27
50
33.33
0
16.67
Days to MGIT positivity (n=43)
- Mean ± SD
- Range
13.97 ± 8.35
3 - 37
*AFB report (WHO-IUATLD): No AFB = 0; 1 - 9 AFB
per 100 fields = Scanty (report number of AFB); 10
- 99 AFB per 100 fields = 1+; 1 - 10 AFB per field =
2+; More than 10 AFB per field = 3+.
MGIT= mycobacterial growth indicator tubes;
Table 1 showed that male patients were 30/43
(69.77%), female patients were 13/43 (30.23%), the
mean ± SD age was 49.23 ± 20.12 years. Cough was
the most common functional symptom, accounting for
27/43 PTs (62.79%), followed by fever, night sweats,
21/43 PTs (48.84%), and 11/ 43 PTs (25.58 %). The
least common symptoms were chest pain, weight
loss, and dyspnea, ranging from 0 - 9.30%.
2. The association between 16S rRNA results with some clinical features
Table 2: Association between the 16S rRNA real-time ExBP RT-PCR assay in respiratory
specimens and hemoptysis and fever.
16S rRNA Sputum BALF
(-) (+) (-) (+)
Hemoptysis
No 1 (3.70%) 26 (96.30%) 1 (5.88%) 16 (94.12%)
Yes 3 (33.33%) 6 (66.67%) 0 (0%) 5 (100%)
p* 0.014 0.579
Fever
No 4 (22.22%) 14 (77.78%) 1 (8.83%) 11 (91.67%)
Yes 0 (0%) 18 (100%) 0 (0%) 10 (100%)
p* 0.034 0.350
*Chi-Square test
INTERNAL MEDECINE JOURNAL OF VIETNAM|NO 22/2021
45
CLINICAL RESEARCH
Table 2 presents the association between
hemoptysis and fever versus 16S rRNA results
by the Chi-Square test. For patients who coughed
up blood with sputum samples, the p-value was
0.014 (< 0.05); with BAL samples, p was 0.579
(> 0.05). Therefore, 16S rRNA Real-time ExBP-
RT-PCR results in sputum specimens associated
with hemoptysis symptoms. For fever patients with
sputum specimens, p was 0.034 < 0.05, and with
BAL fluid samples, p was 0.350 > 0.05. Thus, 16S
rRNA cycle thresholds in sputum samples were
associated with fever symptoms.
As shown in Chart 1, patients with hemoptysis had a lower 16S rRNA cycle threshold (26.51 ± 3.58) than
patients who did not cough up blood (31.24 ± 5.76). Also shown in chart 2, patients with fever symptoms
had a lower 16S rRNA cycle threshold (median 27.75) than patients without fever (median 31.53).
3. The correlation between the 16S rRNA cycle threshold with some subclinical characteristics
Table 3: Correlation between the 16S rRNA cycle threshold and AFB smear results.
Correlation
AFB
Sputum BALF
r p r p
16S rRNA Ct -0.3286 0.0663 -0.5215 0.0153
Table 3 shows the correlation between the 16S rRNA cycle threshold and AFB smear results by Spearman
correlation coefficient. All r were < 0, the cycle threshold of 16S rRNA was inversely proportional to the
positive level of AFB. However, this relationship was only statistically significant with BAL specimens with
p = 0.0153 < 0.05.
Table 4: Correlation between the 16S rRNA positive level and MGIT time to positivity.
Correlation
MGIT time to positivity
Sputum BAL
r p r P
16S rRNA positivity level 0.3203 0.1570 -0.5867 0.0041
Test the correlation between 16S rRNA cycle threshold and MGIT time to positivity by Pearson correlation
coefficient, gave the following results: For sputum specimens: r = 0.3203 >0, p = 0.1570; so the MGIT time
Chart 1: Association between the 16S
rRNA cycle threshold and hemoptysis.
Chart 2: Association between the 16S
rRNA cycle threshold and fever.
INTERNAL MEDECINE JOURNAL OF VIETNAM|NO 22/2021
46
CLINICAL RESEARCH
to positive did not correlate with the positive level of 16S rRNA; For BAL specimens: r = -0.5867 > 0, MGIT
time to positive was inversely proportional to 16S rRNA positive level with p = 0.0041 < 0.05.
As shown in chart 3 and chart 4, 16S rRNA cycle
thresholds were inversely proportional to the
positive level of AFB on BAL specimens, and 16S
rRNA positive level on BAL specimens was inversely
proportional to MGIT time to positivity (p = 0.0041).
DISCUSSION
1. The association between 16S rRNA results
with some clinical features
In theory, the typical symptoms include fever, night
sweats, and weight loss, respectively, in about 75%,
45%, and 55% of patients, and the most common
symptom is a chronic cough in the proportion of
95% of patients [2]. Therefore, chronic cough is
considered a symptom that drives the diagnosis
of pulmonary tuberculosis in countries with a high
prevalence of endemic TB.
In the current study, the most common symptom
was cough (62.79%), followed by fever (48.84%)
and night sweats (25.58%). The weight loss was
seen only in 6.98% of patients.
According to Jinsoo Min et al., in a study of 390
pulmonary tuberculosis patients, the cough was
found in 61.3% of patients, equivalent to our study
at 62.79%. Hemoptysis was only found in 9.2% of
patients lower than our study (20.93%). Jinsoo’s
study also showed that 11.8% of patients had night
sweats, lower than our study at 25.58%. The fever
in Jinsoo’s study accounted for 29.7%, lower than
our study at 48.84% [3].
Testing the association between the 16S rRNA
assay with clinical symptoms showed that the 16S
rRNA results in sputum specimens associated with
hemoptysis were statistically significant. Accordingly,
for hemoptysis patients, the sensitivity of 16S rRNA
in sputum samples was 66.67% lower than in
patients without hemoptysis of 96.30% (p = 0.014).
Thus, the sensitivity of the 16S rRNA assay in the
group of patients with hemoptysis was lower than
in patients who did not cough up blood. Previous
studies with the Xpert MTB assay have also shown
that Xpert MTB decreases insensitivity in blood-
stained samples. In explaining the low sensitivity of
bloody samples, the scientists assumed that blood is
a DNA amplification inhibitor, which is also relevant
for all molecular assays used the technology of
gene amplification [4]. Recommendations from
WHO, the NTP, and the manufacturer of Xpert MTB
(Cepheid) also agree that the Xpert MTB test should
not be used on blood-stained samples because of
false-negative results. According to our study, the
sensitivity of 16S rRNA in patients with hemoptysis
was lower than in patients without hemoptysis, and
there was no specific study on bloody samples
so far. So if the study continues development,
Chart 3: Correlation between the 16S
rRNA cycle threshold and AFB smear on
BAL ecimens.
Chart 4: Correlation between the 16S
rRNA positive level and MGIT time to positivity
on BAL specimens.