| dc.description.abstract | Background
Tuberculin skin test (TST) and interferon gamma release assay (IGRA), currently used to diagnose latent
tuberculosis infection (LTBI) have low positive predictive values for progression to active tuberculosis
(TB). We explored the detection of Mycobacterium tuberculosis complex (MTBC) DNA in peripheral
blood mononuclear cells (PBMC) in the diagnosis of LTBI and monitoring response to isoniazid preventive
therapy (IPT).
Methodology
In a cross-sectional study (study 1), 121 close contacts of index pulmonary TB patients (59 HIV-negative
and 62 HIV-positive) were recruited to evaluate whether MTBC DNA was detectable in CD34-positive
versus CD34-negative PBMC of the close contacts. The close contacts each donated 100 milliliters (ml) of
whole blood for isolating PBMC using density gradient centrifugation and 4 ml for IGRA testing using the
QuantiFERON-TB Gold Plus assay (QFT-Plus). The PBMC were sorted into CD34-positive and CD34-
negative fractions using magnetic activated cell sorting. The hexadecyltrimethylammonium bromide
(CTAB) protocol was used to extract the PBMC DNA, from which MTBC DNA was then quantified using
droplet digital polymerase chain reaction (dPCR), targeting the MTBC-specific genes IS6110 and rpoB. In
a nested prospective study (study 2), the 62 HIV-infected participants in study one were given IPT for six
months to evaluate whether IPT reduced the proportion with detectable MTBC DNA and the number of
MTBC DNA copies in the CD34-positive and CD34-negative PBMC. The assays described above to detect
MTBC DNA were repeated at the end of six-month of IPT. In another cross sectional study (study 3), 289
close contacts of index pulmonary TB patients were recruited to determine their IGRA status and the
associated socio-demographic and clinical characteristics.
In descriptive analysis, the continuous predictor variables were reported as means with the standard
deviation and the categorical variables as proportions in terms of frequencies and percentages. For the
outcome variable (MTBC DNA copies), the median and interquartile range were used due presence of
outliers. In inferential analysis for studies one and two, the Fisher's exact test was used to compare the
proportions with detected MTBC DNA in the different categories of the predictor variables. The analysis
was performed with Stata/IC 15.0, StataCorp LLC Texas USA. The Wilcoxon signed rank test was used to
compare MTBC DNA copies for matched samples and the Mann Whitney U test for independent samples.
MacNemars test was used to determine the number of pairs with the outcome at baseline and follow-up.
The analysis was performed with GraphPad Prism 9.1. In study three, the random effect logistic regression
analysis was used to determine the factors associated with a positive IGRA test at both bivariate and
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multivariate logistic regression analysis. The analysis was performed with Stata/IC 15.0, StataCorp LLC
Texas USA. At all levels of comparison, the level of significance was a two tailed P-value < 0.05.
Study results In study one, MTBC DNA was detected in PBMC of 106/119 (89%, 95% CI 0.82-0.94) participants. There
were more participants in whom MTBC DNA was detected in CD34+ vs CD34-negative PBMC (103/119
[87%] vs 59/119 [50%]; p=0.007). More participants had IS6110 detected than rpoB in both CD34-negative
PBMC (57/119 [48%] vs 38/119 [32%]; p<0·0001) and CD34-positive PBMC (101/119 [85%] vs 80/119
[67%]; p<0·0001). The median IS6110 copy number in CD34-negtive PBMC was 0.0 (IQR: 0.0 - 666.7)
copies/hDNA and 825.0 (IQR: 166.7 - 56601.9) copies/hDNA in CD34-positive PBMC while the median
rpoB copy number in CD34-negative PBMC was 0.0 (IQR: 0.0 – 50.0) copies/hDNA and 120.7 (IQR: 0.0 - 4859.1) in the CD34-positive PBMC.
The proportion of participants in whom MTBC DNA was detected in PBMC did not differ between QFT
negative vs QFT-positive individuals (56/113 [50%] vs 57/113 [50%]; p=0·51). The IS6110 copies did not
also differ according to QFT status in either CD34-positive or CD34-negative PBMC. The CD34-negative
PBMC in the QFT-negative had a median IS6110 copy number of 0.0 (IQR: 0.0 - 664.4) vs. 0.0 (IQR: 0.0 - 310.0) copies/hDNA in QFT-positive. The CD34-positive PBMC in the QFT-negative had a median
IS6110 copy number of 1361.1 (IQR: 220.0 - 129315.1) vs. 537.6 (IQR: 115.0 - 3406.4) copies/hDNA in
the QFT-positive. However, more rpoB copies were found in QFT-negative compared to QFT-positive
individuals (P=0.03) in the CD34-positive PBMC [356.9 (IQR: 0.0 - 10609.3) vs. 53.0 (IQR: 0.0 - 400.0)
copies/hDNA] unlike in the CD34-negative PBMC [0.0 (IQR 0.0 - 38.2) copies/hDNA vs. 0.0 (IQR: 0.0 -
34.0) copies/hDNA]. On the other hand, the proportions with detectable MTBC DNA did not differ between HIV-infected vs.
HIV-uninfected participants (56/119 (47%) vs 50/119 (42%), p=0·44). Likewise, the IS6110 and the rpoB
DNA copies did not differ according to HIV status, in either the CD34-positive or CD34-negative PBMC.
The CD34-negative PBMC in HIV-negative had a median IS6110 copy number of 0.0 (IQR: 0.0 - 980.6)
vs. 0.0 (IQR: 0.0 - 270.0) copies/hDNA in the HIV-positive while the CD34-positive PBMC had a median
IS6110 copy number of 1050.0 (IQR: 166.7 - 130000.0) vs. 650.0 (IQR: 172.2 - 28500.0) copies/hDNA
respectively. Similarly, the CD34-negative PBMC in HIV-negative had a median rpoB copy number of 0.0
(IQR: 0.0 - 63.2) vs. 0.0 (IQR: 0.0 – 34.0) copies/hDNA in the HIV-positive while the CD34-positive
PBMC had a median rpoB copy numbers of 123.7 (IQR: 0.0 - 1903.0) vs. 117.6 (IQR: 0.0 - 3150.0)
copies/hDNA respectively. In study two, the proportion with detectable IS6110 copies did not differ between baseline and follow:
CD34-negative, 25/53 vs. 27/53 (p=0.448) and CD34-positive: 43/53 vs. 46/53(P=0.520). Likewise, the
proportions didn’t differ for rpoB: CD34-negative, 36/53 vs. 41/53 (P=0.214) and CD34-positive: 13/53 vs.
25/53 (P=0.345) respectively. Similarly, the baseline and post-IPT IS6110 and rpoB copies were not
different. The CD34-negative PBMC at baseline had a median IS6110 copy number of 0.0 (IQR: 0.0 -
270.0) vs. 21.4 (IQR: 0.0 - 245.1) copies/hDNA post-IPT while the CD34-positive PBMC had a median
IS6110 copy number of 650.0 (IQR: 172.2 - 28500.0) vs. 1179.0 (IQR: 225.0 - 6050.0) copies/hDNA
respectively. The CD34-negative PBMC at baseline had a median rpoB copy number of 0.0 (IQR: 0.0 -
34.0) vs. 0.0 (IQR: 0.0 - 97.8) copies/hDNA post-IPT while the CD34-positive PBMC had a median rpoB
copy number of 117.6 (IQR: 0.0 - 3150.0) vs. 181.3 (IQR: 17.4 - 521.7) copies/hDNA respectively.
In study three, overall, 105/192 (54%, 95% CI 0.48-0.62) participants had a positive QFT Plus result. The
risk of QFT-Plus positivity was independently associated with casual employment/unemployment vs. non
casual employment (adjusted odds ratio (aOR) 2.18, 95% CI 1.01-4.72), family vs. non-family relation to
the index patient (aOR 2.87, 95% CI 1.33-6.18), living in the same vs. a different house as the index (aOR
3.05, 95% CI 1.28-7.29), a higher body mass index (BMI) (aOR per additional kg/m2 1.09, 95% CI 1.00
1.18) and tobacco smoking vs. not smoking (aOR 2.94, 95% CI 1.00-8.60). HIV infection was not
associated with QFT-Plus positivity (aOR 0.91, 95% CI 0.42-1.96).
Conclusion
Mycobacterium tuberculosis complex DNA was detected in PBMC, and the copy number and frequency of
detection were higher in the CD34-postive vs. CD34-negative fraction, making PBMC a potential niche for
MTBC during latent TB. Isoniazid preventive therapy did not decrease the frequency of MTBC DNA
detection nor the copy numbers. IGRA positivity was associated with manual employment, closer contact with the index case, tobacco smoking and increment in BMI.
Recommendations 1. Before detection of M. tuberculosis DNA in PBMC can be adopted as a molecular test for latent TB
infection, prospective studies are required to determine whether a positive test associates with increased
risk of progression to active TB.
2. The above determinant of IGRA positivity may be utilized in identifying groups at risk of latent TB,
which need to be targeted for screening. | en_US |
