MYCOBACTERIOLOGY AND AEROBIC ACTINOMYCETES
Mycobactericidal Effects of Different Regimens Measured by
Molecular Bacterial Load Assay among People Treated for
Multidrug-Resistant Tuberculosis in Tanzania
Peter M. Mbelele,a,b Emmanuel A. Mpolya,b Elingarami Sauli,b Bariki Mtafya,c Nyanda E. Ntinginya,c Kennedy K. Addo,d
Katharina Kreppel,b Sayoki Mfinanga,e Patrick P. J. Phillips,f Stephen H. Gillespie,g Scott K. Heysell,h Wilber Sabiiti,g Stellah G.
Mpagamaa,b
aKibong’oto Infectious Diseases Hospital (KIDH), Siha, Kilimanjaro, Tanzania
bDepartment of Global Health and Biomedical Sciences, School of Life Sciences and Bioengineering, Nelson Mandela African Institution of Science and Technology
(NM-AIST), Arusha, Tanzania
cNational Institute for Medical Research, Mbeya Medical Research Centre, Mbeya, Tanzania
dDepartment of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
eNational Institute for Medical Research, Muhimbili Centre, Dar Es Salaam, Tanzania
fUCSF Center for Tuberculosis, University of San Francisco, San Francisco, California, USA
gSchool of Medicine, University of St Andrews, St. Andrews, Scotland, United Kingdom
hDivision of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA
ABSTRACT Rifampin or multidrug-resistant tuberculosis (RR/MDR-TB) treatment has
largely transitioned to regimens free of the injectable aminoglycoside component,
despite the drug class’ purported bactericidal activity early in treatment. We tested
whether Mycobacterium tuberculosis killing rates measured by tuberculosis molecular
bacterial load assay (TB-MBLA) in sputa correlate with composition of the RR/MDR-
TB regimen. Serial sputa were collected from patients with RR/MDR- and drug-sensi-
tive TB at days 0, 3, 7, and 14, and then monthly for 4months of anti-TB treatment.
TB-MBLA was used to quantify viable M. tuberculosis 16S rRNA in sputum for estima-
tion of colony forming units per ml (eCFU/ml). M. tuberculosis killing rates were com-
pared among regimens using nonlinear-mixed-effects modeling of repeated meas-
ures. Thirty-seven patients produced 296 serial sputa and received treatment as follows:
CitationMbelele PM, Mpolya EA, Sauli E,
13 patients received an injectable bedaquiline-free reference regimen, 9 received an Mtafya B, Ntinginya NE, Addo KK, Kreppel K,
injectable bedaquiline-containing regimen, 8 received an all-oral bedaquiline-based regi- Mfinanga S, Phillips PPJ, Gillespie SH, Heysell
men, and 7 patients were treated for drug-sensitive TB with conventional rifampin/isonia- SK, Sabiiti W, Mpagama SG. 2021.
Mycobactericidal effects of different regimens
zid/pyrazinamide/ethambutol (RHZE). Compared to the adjusted M. tuberculosis killing of measured by molecular bacterial load assay
20.17 (95% confidence interval [CI] 20.23 to 20.12) for the injectable bedaquiline-free among people treated for multidrug-resistant
tuberculosis in Tanzania. J Clin Microbiol
reference regimen, the killing rates were 20.62 (95% CI 21.05 to 20.20) log10 eCFU/ml 59:e02927-20. https://doi.org/10.1128/JCM
for the injectable bedaquiline-containing regimen (P=0.019), 20.35 (95% CI 20.65 to .02927-20.
20.13) log eCFU/ml for the all-oral bedaquiline-based regimen (P=0.054), and 20.29 Editor Christine Y. Turenne, St. Boniface10
Hospital
(95% CI 20.78 to 10.22) log10 eCFU/ml for the RHZE regimen (P=0.332). Thus, M. tuber-
Copyright © 2021 Mbelele et al. This is an
culosis killing rates from sputa were higher among patients who received bedaquiline but open-access article distributed under the terms
were further improved with the addition of an injectable aminoglycoside. of the Creative Commons Attribution 4.0
International license.
KEYWORDS Kibong'oto, Tanzania, MDR-TB treatment regimens, molecular bacterial Address correspondence to Peter M. Mbelele,
load assay, multidrug-resistant TB, mycobactericidal effects,Mycobacterium tuberculosis, mbelelepeter@yahoo.com.
all-oral bedaquiline regimen, injectable aminoglycoside regimen Received 19 November 2020
Returned for modification 12 January 2021
Accepted 28 January 2021
Accepted manuscript posted online
Measurement of pulmonary tuberculosis (PTB) treatment response in areas of en- 3 February 2021demicity largely depends on sputum smear microscopy (1). While the sputum Published 19 March 2021
smear microscopy detection threshold is at least 103 Mycobacterium tuberculosis colony
April 2021 Volume 59 Issue 4 e02927-20 Journal of Clinical Microbiology jcm.asm.org 1
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
forming units in 1ml (CFU/ml) of sputum sample, many patients with PTB, such as
those with human immunodeficiency virus and AIDS (HIV/AIDS), present with pauciba-
cillary disease and may be unable to produce a good quality sputum for detection of
acid-fast bacilli (AFB) (2, 3). Besides, microscopy does not distinguish viable from nonvi-
able M. tuberculosis and therefore does not inform how patients with rifampin- and/or
multidrug-resistant (RR/MDR)-TB respond to treatment (3). Patients with RR/MDR-TB
are typically monitored for cultured growth in Lowenstein-Jensen (LJ) solid medium or
the Mycobacteria Growth Indicator Tube (MGIT) liquid culture system. This culture sys-
tem is sensitive, with a detection limit of 10 to 100 CFU/ml of sputum, yet it is also
prone to contamination and can take up to 8weeks to determine a definitive positive
or negative result, thereby limiting the ability to take appropriate and timely clinical
action (4).
The DNA-based methods, such as Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA)
and line probe assays (LPA) like genotype MTBDRplus/sl (Hain Lifescience GmbH,
Nehren, Germany) are rapid in detecting M. tuberculosis compared to culture-based
methods (5, 6). Nevertheless, DNA of M. tuberculosis can persist in a patient’s sputum
for a prolonged time after successful treatment (7, 8). Even if the sputum sample is
pretreated with propidium monoazide (Biotium Inc., Hayward, CA, USA), a chemical
substance previously known to bind the DNA of dead bacilli, the Xpert MTB/RIF
(Cepheid, Sunnyvale, CA, USA) cannot distinguish viable from nonviable DNA of M.
tuberculosis (9, 10). Therefore, DNA-based assays are not suitable for monitoring TB
treatment response. Fortunately, there is growing evidence that RNA can serve as a
surrogate biomarker for microbial viability (11–13), and therefore can be used for
monitoring TB treatment response (14, 15). TB molecular bacterial load assay (TB-
MBLA) was first reported in 2011 as a biomarker for drug-sensitive TB treatment
response and proved a robust measure in different settings (14, 16). TB-MBLA is a
real-time quantitative PCR (RT-qPCR) assay that detects and quantifies killing of 16S
rRNA from both viable replicating and dormant M. tuberculosis in patient sputum dur-
ing treatment (14). It is rapid and results are available within 24 h, thereby allowing
informed clinical assessment of patient progress (14, 17). TB-MBLA was found to be
consistently read as positive for samples with as low as 10 CFU/ml of M. tuberculosis.
This bacterial load corresponds to a RT-qPCR quantification cycle (Cq) value of 30, the
limit of quantification for positive M. tuberculosis (14). Therefore, TB-MBLA has the
potential to replace or complement both smear microscopy and culture for monitor-
ing TB treatment response (14, 16, 18).
Because the treatment is difficult and complex, more that 40% of patients treated
for RR/MDR-TB in 2019 worldwide had an unfavorable treatment outcome (19).
However, the 10-year RR/MDR-TB treatment success, defined as the total number of
patients who achieve microbiological cure and those who complete the treatment
regimen, has been consistently above 75% in Tanzania (20). During this decade, the
injectable aminoglycoside class of antibiotics, such as kanamycin, has been one of
the backbones of RR/MDR-TB treatment regimens (21). Because administering inject-
able aminoglycoside is not only invasive to patients but is also associated with severe
adverse events, including ototoxicity and nephrotoxicity (22, 23), the WHO has
endorsed a transition from injectable to all-oral MDR-TB treatment regimens (24). To
align with this transition, countries where TB is endemic, including Tanzania, have
recently adopted new and repurposed TB medicines, such as bedaquiline, delamanid,
and linezolid, and constructed regimens with limited microbiological evidence of
effectiveness in patients with RR/MDR-TB. Hence, we deployed TB-MBLA to describe
killing of M. tuberculosis in patients receiving RR/MDR-TB and drug-susceptible TB
(DS-TB) treatment. We tested the hypothesis that M. tuberculosis killing rates from
the sputum, as measured by TB-MBLA, not only correlated with time-to-culture con-
version but were dependent upon the composition of the RR/MDR-TB antibiotic
regimen.
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 2
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Monitoring MDR-TB Treatment Response by TB-MBLA Journal of Clinical Microbiology
MATERIALS ANDMETHODS
Patients and ethical considerations. From August 2018 to December 2019, two populations of
patients with TB participated in this study. The primary target population was patients with RR/MDR-TB.
A small population of patients with DS-TB was included as a control to inform the reproducibility of pre-
vious TB-MBLA study findings (14, 16). Rifampin susceptibility in M. tuberculosis was confirmed using
Xpert MTB/RIF (25). Moreover, all patients harbored M. tuberculosis that was deemed susceptible to fluo-
roquinolones and aminoglycosides by line probe assay (Hain, LifeScience, Germany), the genotype
MTBDRsl version 2.0 (26). The study included all patients aged at least 18 years who were able to expec-
torate and provide quality early morning sputum. Quality sputum was defined by an adequate volume
of.5ml and absence of food particles. No sputum induction was done to patients who were unable to
provide quality sputum. Critically ill or moribund patients, as previously defined by Robertson et al. (27),
and pregnant women were excluded. Additionally, patients who interrupted treatment were excluded
from the final analysis. Prior to any study procedure, all patients signed a witnessed oral or written
informed consent. The study was approved by the National Institute for Medical Research (NIMR) in
Tanzania (NIMR/HQ/R.8a/Vol. IX/2662). Permission to conduct the study was granted by authorities of
the Kibong’oto Infectious Diseases Hospital (KIDH).
Study design and treatment regimens. This was a longitudinal cohort study design where each
patient was followed for 16weeks of anti-TB treatment. The treatment regimens for patients with RR/
MDR-TB were as follows: (i) an injectable but bedaquiline-free regimen composed of daily dosed kana-
mycin (15mg/kg), levofloxacin (750mg for patients weighing,50 kg and 1,000mg for those
weighing$50 kg), pyrazinamide (25mg/kg), ethionamide (750mg), and cycloserine (750mg); (ii) an all-
oral based regimen composed of bedaquiline (400mg daily for 2weeks, and then 200mg thrice per
week), linezolid (600mg/day), levofloxacin, pyrazinamide, and ethionamide; (iii) an injectable bedaqui-
line-containing regimen composed of kanamycin, bedaquiline, levofloxacin, pyrazinamide, and ethiona-
mide; and (iv) a standard fixed-dose combination containing rifampin (150mg), isoniazid (75mg), pyra-
zinamide (400mg), and ethambutol (275mg), termed RHZE, for patients with DS-TB. Patients
weighing,50 kg received three tablets of RHZE and those weighing$50 kg received four tablets of
RHZE per day.
Study setting. Patients were recruited at Kibong’oto Infectious Diseases Hospital, a national center
of excellence for clinical management of drug resistant (DR)-TB located in the Siha district of the
Kilimanjaro region in Tanzania (25). TB-MBLA testing was performed at the National Institute for Medical
Research, Mbeya Medical Research Centre branch, given that laboratory’s prior experience with the
assay.
Sample size determination. The numbers of patients required to determine differences in bacteri-
cidal activity over time in 4 treatment regimens were calculated as previously reported by Guo et al.
(28). We assumed a Spearman correlation of 0.51 and a baseline M. tuberculosis burden of 5.5 log10
eCFU/ml, as well as daily M. tuberculosis decline and decay rate of 0.42 and 0.05 log10 eCFU/ml, respec-
tively (14, 16). Hence, at least 7 patients were needed per regimen to reach a power of 80% with a two-
sided type I error of 5%. Adjusting for least 25% of patients who were likely to be lost to follow-up, not
evaluated due to negative microbiological results at baseline, and/or died, a minimum of 37 patients
was desirable for sampling and analyzing at the end of 4months of treatment.
Sputum collection, processing, and culturing of M. tuberculosis. One sample of approximately
5 ml of early morning sputum was collected from each patient for laboratory testing at day 0 (baseline)
and at days 3, 7, 14, 28, 56, 84, and 112 of anti-TB treatment. Before culturing, sputum was homogenized
using a sterile magnetic stirrer at room temperature for 30 min. Then, 1ml of homogenized sputum was
treated using 4 ml of 4 M guanidine thiocyanate (GTC) containing 1 M Tris-HCl (pH 7.5) and 1% (vol/vol)
of b-mercaptoethanol, and was frozen at 280°C in order to preserve the M. tuberculosis RNA from
degrading. The M. tuberculosis culture was performed on LJ slants from the remaining sputum samples
collected at six time points from days 0, 14, 28, 56, 84, and 112 of treatment, as per previous description
(29). In brief, sputum was decontaminated by N-acetyl-L-cysteine (NALC)-NaOH, and finally resuspended
in 1ml of phosphate buffer. A total of 200ml of decontaminated sputum was inoculated into two LJ
slants and incubated for up to 8weeks to detect mycobacterial growth. Incubated LJ slants were read
on a weekly basis and were deemed negative if there was no growth at week 8.
RNA extraction and RT-qPCR for TB-MBLA. M. tuberculosis quantification by TB-MBLA was per-
formed as described by Gillespie et al. (30). In summary, M. tuberculosis RNA in 1ml of homogenized spu-
tum preserved in 4 ml of guanidine thiocyanate (GTC) at 280°C was extracted using the RNA pro kit
(FastRNA Pro BlueKit; MP Biomedical, CA, USA) as instructed by the manufacturer. The extract was
treated with DNase I enzyme (TURBO DNA-free kit; Ambion, CA, USA) to remove DNA from the dead
cells. The M. tuberculosis 16S rRNA, a biomarker for viable cells, was amplified and quantified by RT-qPCR
using specific primers and probes. The Cq was translated to bacterial load (estimated CFU per ml [eCFU/
ml]) using a standard curve on a Rotor gene Q 5plex platform (Qiagen). The cutoff for TB-MBLA positivity
was a 30 Cq value that corresponds to 1.0 log10 eCFU/ml, beyond which the test was considered nega-
tive (16, 30).
Statistical analysis. Data were recorded in a clinical case report form, entered, and cleaned before
statistical analysis. Patients who completed 8 treatment visits and had positive pretreatment TB-MBLA
results were analyzed and visualized in R softward version 4.0.2 (http://www.R-project.org). Continuous
variables, such as age, body mass index (BMI) in kg/m2, and time to TB-MBLA negativity were described
by the median with the 25th and 75th interquartile range (IQR), and were compared across different regi-
mens using a Kruskal-Wallis test. Accordingly, proportions for HIV status, gender, cavitary lung disease
on chest, and previous TB treatment were compared across different regimens using a chi-square or
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 3
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
TABLE 1 Fitting and selection of a reliable polynomial nonlinear mixed effects model for repeated measuresa
Polynomial models (degree)b Intercepts (log10 eCFU/ml) ICC SD AIC Likelihood ratio test P value
Nonpolynomial (model 1) 3.00 0.54 0.81 722.89 1 versus 2 , 0.001
Quadratic (model 2) 2.99 0.63 0.67 634.63
Cubic (model 3) 3.00 0.65 0.63 611.59 2 versus 3 , 0.001
Quartic (model 4) 3.20 0.67 0.61 592.7 3 versus 4 , 0.001
Quintic (model 5) 2.89 0.68 0.60 588.58 4 versus 5 0.020
aICC, intraclass correlation coefficient; SD, standard deviation; AIC, Akaike information criterion.
bModel 4 had the lowest AIC and within variability (SD) but high ICC values, the key selection criteria for a reliable model, and hence it was used to modelM. tuberculosis
killing rates.
Fisher’s exact test. Using baseline bacterial load, chest cavity, HIV, silicosis, and gender as fixed effects,
the rate of M. tuberculosis killing (log10eCFU/ml) was fitted on quartic polynomial nonlinear mixed effects
(NLME) for repeated measures as previously described (31, 32). Individual patients were accounted for
random effect. A model was reliably selected if it had low Akaike information criterion but high intraclass
correlation coefficient (Table 1). The mean difference in M. tuberculosis load, due to two different regi-
mens received by patients at the end of 4months of treatment, was compared using one-way analysis
of variance (ANOVA) and Tukey’s test for repeated measures (33). An injectable regimen without beda-
quiline was used as a reference regimen. The median time to TB-MBLA and culture conversion to nega-
tive was estimated using the Kaplan-Meier method, and was compared across different regimens using
a log rank test (34). Cox proportional hazards regression models were used to estimate the hazard ratios
(HR) for M. tuberculosis killing, and was adjusted for the effects of HIV, baseline bacillary load, cavitary
disease, silicosis, gender, history of treatment for drug-sensitive TB, and clearance rate. We computed an
overall mean M. tuberculosis load of 4.0 log10 eCFU/ml, and it was used to categorize a patient’s bacterial
load as “high bacterial load” versus “low bacterial load” depending on whether patients had detectable
M. tuberculosis above or below this mean, respectively. A P value of,0.05 was considered significant. A
95% confidence interval (CI) of the mean clearance rate and HR was included.
RESULTS
Population. Of 59 patients enrolled, 37 patients produced a total of 296 serial sputa
for final analysis. Reasons for exclusion and patient’s distribution are outlined in Fig. 1.
Among 296 serial sputa analyzed, 104 sputa came from 13 patients who received an
injectable but bedaquiline-free regimen, 72 sputa were from 9 patients who received
an injectable bedaquiline-containing regimen, 64 from 8 patients who received an all-
oral bedaquiline-based regimen, and 56 sputa from 7 patients who were treated for
drug-sensitive TB with conventional RHZE. Clinical and demographic parameters are
presented in Table 2. Twenty-seven (73%) out of 37 patients were male. Their median
(IQR) age was 37 (32 to 49) years. Patients who received standard RHZE treatment were
younger than those who received RR/MDR-TB treatment regimens (P=0.038). Also, 11
(30%) patients were living with HIV with a CD4 T cell count of 208 (95% CI 144 to 272)
cells/ml. More patients with HIV received an all-oral than injectable-based treatment
regimen (P=0.001).
Mycobactericidal activities of different regimens over time. The M. tuberculosis
load measured by TB-MBLA and culturing in Fig. 2 decreased significantly over time
(R = 20.77, P, 0.001). The mean M. tuberculosis load in log10 eCFU/ml (95% CI) was
reduced from 5.19 (4.40 to 5.78) at baseline to 3.10 (2.70 to 3.50) at day 14, then to
2.52 (2.13 to 2.90) at day 28, 1.88 (1.53 to 2.22) at day 56, and ,1.36 (1.03 to 1.70) at
day 84 through 112 of treatment. The overall mean daily M. tuberculosis killing was
20.24 (95% CI 20.39 to 20.08) log10 eCFU/ml, and it varied with treatment regimen
(Table 3, P, 0.001). An injectable bedaquiline-containing regimen had the highest
mean M. tuberculosis killing rate, followed by an all-oral bedaquiline-based regimen
compared to the injectable but bedaquiline-free reference regimen (Table 3, P=0.019).
Kanamycin-containing regimens in Fig. 3 had rapid bactericidal activity at day 14, but
this was not translated into long-term bactericidal effect (P, 0.001). An all-oral beda-
quiline-based regimen had a sharp decline after day 28.
Median time to M. tuberculosis killing. There was strong positive correlation in
time to sputum conversion between TB-MBLA and culture (r = 0.46 [95% CI 0.36 to
0.55]; P, 0.001). The overall median time to sputum TB-MBLA conversion to negative
was 56 (IQR 28 to 84) days. The median times to TB-MBLA conversion to negative were
28, 42, and 84 days among patients on injectable bedaquiline, an all-oral bedaquiline-
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 4
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Monitoring MDR-TB Treatment Response by TB-MBLA Journal of Clinical Microbiology
FIG 1 Recruitment and patient distribution into different treatment regimens. Patients with drug-sensitive (DS)
and rifampin/multidrug-resistant (RR/MDR)-TB were recruited and treated using different anti-TB treatment
regimens. Regimens included (i) standard RHZE composed of rifampin, isoniazid, pyrazinamide, and
ethambutol; (ii) injectable bedaquiline (BDQ)-free regimen composed of kanamycin (KAN), levofloxacin (LFX),
pyrazinamide (PZA), ethionamide (ETH), and cycloserine (CS); (iii) injectable BDQ-containing regimen composed
of KAN, BDQ, LFX, PZA, and ETH; and (iv) all-oral BDQ regimen contained BDQ, LFX, linezolid (LZD), PZA, and
ETH. Among other criteria, 11 out of 59 patients recruited had negative TB molecular bacterial load assay (TB-
MBLA) results at baseline and were excluded from the final analysis.
based regimen, and injectable but bedaquiline-free regimens, respectively. Irrespective
of treatment regimen, 92% (34/37) of patients had negative culture results compared
to 65% (24/37) of negative TB-MBLA at day 56 (P=0.037). The number of patients who
converted to sputum negative by culture and TB-MBLA per treatment regimen is
shown in Fig. 4. Among 13 patients who received the injectable but bedaquiline-free
regimen, 2 and 7 of them remained culture and TB-MBLA positive, respectively,
whereas all 8 patients who received injectable bedaquiline-containing regimens had
negative LJ culture and TB-MBLA at day 56 (Fig. 4A to D). Favorably, all patients on
injectable bedaquiline for MDR-TB and standard RHZE regimen for DS-TB had negative
TABLE 2 Socio-demographic and clinical characteristics of patients per treatment regimena
Variable All RHZE (n=7) Injectable± BDQ (n=21) All-oral BDQ (n=9) P value
Median age (IQR) 37 (32–49) 30 (29–33) 42 (34–54) 36 (33–44) 0.038
Male (%) 27 (73) 4 (57) 18 (86) 5 (56) 0.125
Chest cavity, n (%) 29 (78) 7 (100) 14 (67) 8 (89) 0.163
Probable TB, n (%) 34 (92) 7 (100) 18 (86) 9 (100) 0.568
HIV positive, n (%) 11 (20) 0 (0) 3 (14) 8 (89) 0.001
TB/Silicosis, n (%) 7 (19) 1 (14) 4 (19) 2 (22) 0.731
Malnourished, n (%) 22 (59) 4 (57) 11 (52) 7 (78) 0.432
Retreatment, n (%) 23 (62) 5 (71) 14 (67) 4 (44) 0.528
Median BMI (IQR) 18 (15–19) 17 (15–20) 18 (16–20) 17 (15–18) 0.301
Median days spent before care (IQR) 84 (60–196) 85 (68–93) 84 (56–196) 88 (68–365) 0.778
aBDQ, bedaquiline; BMI, body mass index; injectable6 BDQ, kanamycin with or without BDQ; IQR, interquartile range. Probable TB was defined as the presence of
radiological changes, including cavity, infiltrates, nodules, hilar lymphadenopathy, and aortopulmonary window adenopathy on chest radiograph. P value was computed
to compare RHZE, injectable6 BDQ, and kanamycin with or without BDQ regimens.
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 5
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
FIG 2 M. tuberculosis killing during the first 4 months of anti-TB treatment. The plots show M. tuberculosis (Mtb) kinetics as measured by
TB-MBLA during treatment with different anti-TB regimens. The dotted line is the cutoff value for a positive TB-MBLA test. (A) Overall time-
dependent decline of M. tuberculosis load in estimated CFU per 1ml (eCFU/ml) of sputum between patients as measured by TB-MBLA. (B)
Delineation of this decline as measure by both TB-MBLA and Lowenstein-Jensen culture. Overall, bacterial load at baseline has a strong positive
correlation with median time to sputum conversion to negative by both TB-MBLA and culture. Patients with higher bacterial load at baseline had a
later culture conversion to negative than those with lower baseline loads. (C to F) M. tuberculosis decline in eCFU/ml among patients treated with
standard RHZE (C); injectable bedaquiline-free regimen containing kanamycin (KAN), levofloxacin (LFX), pyrazinamide (PZA), ethionamide (ETH), and
cycloserine (CS) (D); injectable bedaquiline-containing regimen was composed of KAN, bedaquiline (BDQ), LFX, PZA and ETH (E); and an all-oral
bedaquiline regimen containing BDQ, LFX, linezolid (LZD), PZA, and ETH (F).
TB-MBLA at days 56 and 84, respectively. Compared to 31% (4/13) of patients who
received an injectable but bedaquiline-free regimen, only 11% (1/9) of those who received
an all-oral bedaquiline-containing regimen remained positive TB-MBLA but negative LJ
culture at day 112 of treatment (Fig. 4A and B versus Fig.4E and F; P=0.283).
Hazard ratios of M. tuberculosis killing. The overall mean M. tuberculosis load
log10 eCFU/ml at baseline was 5.19 (95% CI 4.40 to 5.78), and was similar in all patients
TABLE 3Mean dailyM. tuberculosis killing rates (log10 eCFU/ml) and corresponding burden at day 0 (baseline) and day 112 of treatment
MeanM. tuberculosis killing rates
Unadjusted model for Adjusted model for Mean (95% CI)
covariates covariates M. tuberculosis load
Day 0
Treatment regimensc Rates (95% CI) P value Rates (95% CI) P value (baseline)a Day 112b
1. Reference (injectable BDQ-free) 20.18 (20.27 to20.08) 20.17 (20.23 to20.12) 4.73 (4.13–5.32) 2.77 (2.51–3.04)
2. Injectable with bedaquiline 20.48 (21.25 to10.28) 0.239 20.62 (21.05 to20.20) 0.019 4.63 (3.95–5.47) 2.08 (1.81–2.36)
3. All-oral bedaquiline 20.26 (20.48 to11.00) 0.507 20.35 (20.65 to20.13) 0.054 5.36 (4.65–6.08) 2.47 (2.20–2.74)
4. Standard RHZE 20.23 (20.57 to11.02) 0.593 20.29 (20.78 to10.22) 0.332 5.17 (4.36–5.99) 2.51 (2.18–2.85)
aBaseline meanM. tuberculosis load in all regimens were comparable (ANOVA, P=0.453).
bP values for mean differences inM. tuberculosis load for regimens by pairwise comparison at day 112 were as follows: regimens 1 and 2, P, 0.001; regimens 2 and 3,
P=0.031; regimens 1 and 3, P=0.077; and regimens 2 and 4, P=0.040.
cReference regimen was the injectable bedaquiline (BDQ)-free regimen composed of kanamycin (KAN), levofloxacin (LFX), pyrazinamide (PZA), ethionamide (ETH), and
cycloserine (CS). Injectable bedaquiline regimen was composed of KAN, BDQ, LFX, PZA, and ETH. All-oral bedaquiline regimen contained BDQ, LFX, linezolid (LZD), PZA, and
ETH. RHZE contained rifampin, isoniazid, PZA, and ethambutol (E). Covariates adjusted included baseline bacterial load, cavity, gender, HIV, and silicosis;M. tuberculosis
killing rates varied among regimens.
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 6
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Monitoring MDR-TB Treatment Response by TB-MBLA Journal of Clinical Microbiology
FIG 3 Kaplan-Meier curves showing median time to M. tuberculosis killing in patient sputum per treatment regimen. The
dotted lines denote the median time to TB-MBLA conversion from positive to negative. Bedaquiline-containing regimens
had short median time to TB-MBLA conversion to negative compared to the injectable but bedaquiline-free regimen
containing kanamycin (KAN), levofloxacin (LFX), pyrazinamide (PZA), ethionamide (ETH), and cycloserine (CS). Injectable
bedaquiline-containing regimen was composed of KAN, bedaquiline (BDQ), LFX, PZA, and ETH. The all-oral bedaquiline
regimen was composed of BDQ, LFX, linezolid (LZD), PZA, and ETH. Standard RHZE was composed of rifampin, isoniazid,
PZA, and ethambutol.
treated with any of the 4 regimens (Table 3, P=0.453). The mean M. tuberculosis load
(log10 eCFU/ml) among female patients was 5.6 (95% CI 5.0 to 6.2) log10 eCFU/ml com-
pared to 4.7 (95% CI 4.3 to 5.2) log10 eCFU/ml among male patients (P= 0.017). Patients
with chest cavity had mean M. tuberculosis load of 5.26 (95% CI 4.45 to 5.87) compared
to 4.40 (95% CI 3.91 to 4.75) log10 eCFU/ml in those without cavity (P=0.080).
Adjusting for bacterial load, initial killing rate, silicosis, chest cavity, HIV status, and gen-
der, the hazard ratios (HR) for M. tuberculosis killing were 12.37 (95% CI 2.87 to 53.30;
P=0.001) and 14.31 (95% CI 3.49 to 58.65; P, 0.001) for patients who received an all-
oral bedaquiline versus injectable bedaquiline-containing regimens, respectively
(Table 4). Bacterial load at baseline strongly correlated positively with median time to
sputum conversion to negative measured by both TB-MBLA and culture (r=0.48 [95%
CI 0.18 to 0.69]; P=0.003). High M. tuberculosis load and TB with silicosis were inde-
pendent predictors of slow M. tuberculosis killing compared to low M. tuberculosis load
and TB without silicosis (Table 4, P# 0.033).
DISCUSSION
This study shows for the first time to our knowledge that the killing rates of M. tu-
berculosis in patients treated for RR/MDR-TB, as well as those with concomitant TB with
silicosis, varies with treatment regimens. As measured by TB-MBLA, M. tuberculosis
decreased significantly over time on treatment, and this kinetic correlated with what
was observed using LJ culture medium. For decades, culture has been used as a
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 7
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
FIG 4 Number of patients who converted to negative by TB-MBLA and Lowenstein-Jensen culture during the first 4months of treatment with different
anti-TB regimens. The overall sputum conversion from positive to negative TB-MBLA and LJ culture results had the same trend in four different regimens.
At recruitment (day 0), all 37 patients had positive results for TB by TB-MBLA and culture (MBLA1, LJ1). Both TB-MBLA and culture tests were negative
(MBLA2, LJ2) at days 56 and 84, respectively, in all patients on either injectable plus bedaquiline (B) or standard RHZE (D) composed of rifampin,
isoniazid, PZA, and ethambutol. A total of 3 patients who received the injectable bedaquiline-free regimen (A), together with 1 patient on the all-oral
bedaquiline regimen (C), remained TB-MBLA positive but culture negative (MBLA1, LJ2).
routine microbiological tool for monitoring drug-resistant TB treatment response (15,
35), but in many settings with endemic TB, culture is unavailable or limited to special-
ized centers. Importantly, culture results can take up to 8weeks from the time of spu-
tum collection, which delays patient care if a treatment decision is made based on a
result from a specimen collected 2 months earlier. Given the continued decentraliza-
tion of RR/MDR-TB services in Tanzania and elsewhere, monitoring treatment response
in laboratories capable of performing qPCR, such as with Xpert MTB/RIF, will allow lab-
TABLE 4 Hazard ratio (HR) ofM. tuberculosis killing in a Cox proportion-hazard model
Unadjusted model Adjusted model
Predictor variablea HR (95% CI) P value HR (95% CI) P value
Male gender 0.86 (0.40–1.85) 0.705 2.44 (0.82–7.24) 0.109
TB/silicosis 0.20 (0.10–0.88) 0.028 0.12 (0.03–0.49) 0.003
TB/HIV 2.26 (1.07–4.77) 0.033 0.88 (0.31–2.50) 0.813
Cavitary disease 0.38 (0.17–0.86) 0.021 0.85 (0.17–2.70) 0.790
Positive chest x-ray 0.57 (0.17–1.88) 0.354 0.23 (0.03–1.62) 0.790
HighM. tuberculosis load 0.72 (0.54–0.97) 0.033 0.26 (0.13–0.54) ,0.001
Retreatment 1.02 (0.51–2.05) 0.958 0.59 (0.24–1.44) 0.248
All-oral bedaquiline 1.58 (0.61–4.04) 0.344 12.37 (2.87–53.30) 0.001
Injectable-bedaquiline 4.63 (1.64–13.09) 0.004 14.31 (3.49–58.65) ,0.001
Standard RHZE 1.43 (0.53–3.89) 0.482 3.25 (0.90–11.73) 0.072
High initialM. tuberculosis killing rate 5.96 (2.03–17.48) 0.009 4.81 (1.39–16.65) 0.013
aAll-oral bedaquiline regimen was composed of bedaquiline (BDQ), levofloxacin (LFX), linezolid (LZD),
pyrazinamide (PZA), and ethionamide (ETH). Injectable bedaquiline is a modified regimen composed of
kanamycin (KAN), BDQ, LFX, PZA, and ETH. Standard RHZE included rifampin (H), isoniazid (H), PZA, and
ethambutol (E).
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 8
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Monitoring MDR-TB Treatment Response by TB-MBLA Journal of Clinical Microbiology
oratory assays to impact treatment decisions closer to the point-of-care. Therefore, this
study in RR/MDR-TB complements the growing evidence supporting the application of
TB-MBLA in routine clinical management (14, 16, 36).
Interestingly, our findings suggest that bactericidal activity at day 14 may not be a
suitable predictor of the long-term efficacy of a regimen, particularly when that regi-
men contains bedaquiline. In this cohort at day 14, more than 75% of people had a
positive TB-MBLA and more than half had a positive culture result. However, between
14 and 56 days we observed substantial M. tuberculosis killing in those treated with a
bedaquiline-containing regimens, suggesting that evaluation of bactericidal activity be
performed later, such as at day 56, for modern RR/MDR-TB regimens. Using culture,
one previous phase 2b clinical trial reported high bactericidal activity of a bedaquiline-
containing regimen in patients with DS- and RR/MDR-TB (37). However, detectable M.
tuberculosis beyond day 56 in our study supports this trial’s argument that day 56 is
unreliable as an indicator of a regimen’s ability to either predict long-term treatment
outcomes or shorten treatment duration (37). This further raises the question of
whether TB-MBLA may in fact be a superior predictor to culture.
Another important finding from this study of TB-MBLA is that M. tuberculosis
killing kinetics were regimen dependent. Overall, there was rapid and prominent
killing of M. tuberculosis at day 14 for patients who received kanamycin regardless
of receipt of bedaquiline. However, superior activity of kanamycin-containing regi-
mens at day 14 had no long-term bactericidal effect. As a result, 3 patients on the
injectable but bedaquiline-free regimen remained positive by TB-MBLA but nega-
tive by LJ culture after 4 months of treatment. On the other hand, patients who
received an all-oral bedaquiline-containing regimen achieved these rates of killing
at or after 1 month of treatment. This observation concurs with previous reports
that the bactericidal activity of bedaquiline in MDR-TB is delayed at the beginning,
but accelerates later in therapy (38). Usually, recovery of M. tuberculosis by TB-
MBLA correlates better with Mycobacteria Growth Indicator Tube (MGIT) liquid
culture than with LJ solid culture, which may partially explain the discrepancy
between the two tests at month 4 of treatment (16). This argument supports pre-
vious findings that culturing M. tuberculosis on LJ recovers a lower yield than in
MGIT liquid culture (39). Nonetheless, our findings, as measured by TB-MBLA, fit
with the pharmacodynamical understanding that kanamycin and other aminogly-
coside/polypeptides that are active against mycobacteria will primarily exert their
effect against those extracellular organisms that are rapidly dividing, and these
may be more abundant early in the treatment course (40, 41).
The shorter overall time to sputum conversion to negative, as measured by TB-
MBLA and conventional culture, for all patients who received bedaquiline regard-
less of kanamycin further supports the argument that bedaquiline should be a cor-
nerstone of regimens designed to shorten the duration of MDR-TB treatment (42).
The conventional injectable but bedaquiline-free regimen has been in practice for
decades, even though more than 40% of patients treated with this regimen had
unfavorable outcomes in settings where TB is endemic (43). Aminoglycosides such
as kanamycin are no longer part of the current MDR-TB treatment regimens, not
due to lack of bactericidal activity, as our data would suggest the contrary in the
early treatment period, but rather because of the significant toxicity and patient
intolerance that leads to treatment interruption (24, 44). From a microbiological
perspective alone, as demonstrated in this study and others, such as Mpagama et
al. (45), and also in a more patient-centered approach, our results demonstrate
the potential importance of finding more tolerable substitutes for kanamycin that
can match the early bactericidal effect.
The main strength of this study is that we utilized TB-MBLA to model killing rates
among patients with RR/MDR-TB and those with TB/silicosis. We have shown that
patients with TB/silicosis had slower M. tuberculosis killing rates by TB-MBLA compared
to those with TB and without silicosis. This low rate of killing could partially be
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 9
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
attributed to the underlying pulmonary pathophysiology, which can include progres-
sive massive fibrosis (46, 47) and a blunted local host immune response to M. tubercu-
losis infection (46). We observed a similarly lower rate of M. tuberculosis killing among
patients with RR/MDR-TB who had high initial bacterial load, which is consistent
with previous studies of TB-MBLA kinetics from patients with drug-sensitive TB (14,
16, 36). In this study, approximately 1 and 4 out of 10 patients had, respectively,
positive LJ culture and positive TB-MBLA at day 56. This supports the previous
argument that TB-MBLA is more sensitive compared to agar-based Loewenstein-
Jensen culture, in which the M. tuberculosis population gets lost due to contamina-
tion at later time points (18). Limitations of the study include the timing of end-
points, which were limited to 4months, such that predicting long-term treatment
success was beyond the scope of this study. Nevertheless, modeling M. tuberculosis
killing for 4months as we accomplished here has been used as a marker for treat-
ment failure and relapse in several observational studies (35, 48), and exceeds the
duration of monitoring used in other trials of RR/MDR-TB regimens that have
employed conventional culture-based techniques (37). Additionally, this study had
no control over the treatment regimens prescribed. However, given the feasibility
of TB-MBLA and the comparability of this study’s findings to prior studies with TB-
MBLA in drug-susceptible TB (16), we plan to apply TB-MBLA systematically within
an ongoing operational research protocol for injectable-free RR/MDR-TB treatment
in Tanzania that employs standardized regimens over various treatment durations.
Lastly, because of the small number of patients per treatment regimen, these find-
ings should be cautiously inferred to other RR/MDR-TB populations. Nevertheless, a
longitudinal cohort design in this study allowed control of variabilities between
patients, as well as intrapatient tracking of each regimen’s bactericidal activities
over time (28, 49).
In conclusion, patients who received bedaquiline-containing regimens exhibited
higher M. tuberculosis killing rates and had shorter time to sputum TB-MBLA and
culture conversion to negative. While both kanamycin-containing regimens had
superior bactericidal activity during the first 2 weeks of RR/MDR-TB treatment, the
addition of bedaquiline allowed for improved killing after 1month of therapy.
Together, these findings provide insight into formulating optimal all-oral bedaqui-
line-containing regimens with the best potential to shorten duration of MDR-TB
treatment (37, 44, 50). Given that TB-MBLA does not require laboratory procedures
associated with culture and the prolonged time to receive a culture-based result,
we envision it can be used to make regimen adjustments in the presence of anti-TB
drug susceptibility testing results.
ACKNOWLEDGMENTS
This study received financial support from the EDCTP2 program supported by the
European Union project (grant number TMA2016SF-1463-REMODELTZ) and DELTAS
Africa Initiative (Afrique One-ASPIRE/DEL-15-008). The Afrique One-ASPIRE is funded by
a consortium of donors, including the African Academy of Sciences, Alliance for
Accelerating Excellence in Science in Africa, the New Partnership for Africa's
Development Planning and Coordinating Agency, the Wellcome Trust (107753/A/15/Z),
and the UK Government. The funding bodies had no role in the conceptualization,
methodology, data interpretation, or writing of the manuscript.
We acknowledge Batuli Mono, Taji Mnzava, Joseph Kachala, and Bibie Said of KIDH
for their assistance with recruitment and data collection from study participants. We
also thank Elisha S. Juma and Sarapia P. Malya of KIDH, and Emmanuel Sichone and
Joseph John of NIMR Mbeya for assisting with laboratory work. In addition, we also
acknowledge the KIDH administration for granting permission to conduct this study.
We have no conflicts of interest to declare.
P.M.M., E.A.M., E.S., W.S., K.K.A., S.M., and S.G.M. conceived and designed the study.
P.M.M. and B.M. acquired data. P.M.M., B.M., E.A.M., E.S., W.S., and S.G.M. interpreted the
clinical and TB-MBLA results. P.M.M., K.K., E.A.M., P.P.J.P., W.S., and S.G.M. analyzed the
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 10
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Monitoring MDR-TB Treatment Response by TB-MBLA Journal of Clinical Microbiology
data. P.M.M. drafted the manuscript and responded to all coauthors’ inputs. S.H.G.,
K.K.A., S.M., N.E.N., and S.K.H. reviewed the manuscript. All authors wrote, approved,
and agreed to be accountable for all scientific aspects in the final version of the
manuscript.
REFERENCES
1. Mitnick CD, White RA, Lu C, Rodriguez CA, Bayona J, Becerra MC, Burgos 13. Desjardin LE, Perkins MD, Wolski K, Haun S, Teixeira L, Chen Y, Johnson JL,
M, Centis R, Cohen T, Cox H, D’Ambrosio L, Danilovitz M, Falzon D, Ellner JJ, Dietze R, Bates J, Cave MD, Eisenach KD. 1999. Measurement of
Gelmanova IY, Gler MT, Grinsdale JA, Holtz TH, Keshavjee S, Leimane V, sputum Mycobacterium tuberculosis messenger RNA as a surrogate for
Menzies D, Migliori GB, Milstein MB, Mishustin SP, Pagano M, Quelapio MI, response to chemotherapy. Am J Respir Crit Care Med 160:203–210.
Shean K, Shin SS, Tolman AW, Van Der Walt ML, Van Deun A, Viiklepp P, https://doi.org/10.1164/ajrccm.160.1.9811006.
Ahuja SD, Andreev YG, Ashkin D, Avendano M, Banerjee R, Bauer M, 14. Honeyborne I, McHugh TD, Phillips PPJ, Bannoo S, Bateson A, Carroll N,
Benedetti A, Brand J, Chan ED, Chiang CY, DeRiemer K, Dung NH, Enarson Perrin FM, Ronacher K, Wright L, Van Helden PD, Walzl G, Gillespie SH.
D, Flanagan K, Flood J, García-García L, Gandhi N, Granich RM, Hollm- 2011. Molecular bacterial load assay, a culture-free biomarker for rapid
Delgado MG, Collaborative Group for Analysis of Bacteriology Data in and accurate quantification of sputum Mycobacterium tuberculosis bacil-
MDR-TB Treatment, et alet al. 2016. Multidrug-resistant tuberculosis treat- lary load during treatment. J Clin Microbiol 49:3905–3911. https://doi
ment failure detection depends on monitoring interval and microbiologi- .org/10.1128/JCM.00547-11.
cal method. Eur Respir J 48:1160–1170. https://doi.org/10.1183/13993003 15. Rockwood N, Du Bruyn E, Morris T, Wilkinson RJ. 2016. Assessment of
.00462-2016. treatment response in tuberculosis. Expert Rev Respir Med 10:643–654.
2. Park JH, Choe J, Bae M, Choi S, Jung KH, Kim MJ, Chong YP, Lee SO, Choi https://doi.org/10.1586/17476348.2016.1166960.
SH, Kim YS, Woo JH, Jo KW, Shim TS, Kim MY, Kim SH. 2019. Clinical char- 16. Sabiiti W, Azam K, Farmer ECW, Kuchaka D, Mtafya B, Bowness R,
acteristics and radiologic features of immunocompromised patients with Oravcova K, Honeyborne I, Evangelopoulos D, McHugh TD, Khosa C,
pauci-bacillary pulmonary tuberculosis receiving delayed diagnosis and Rachow A, Heinrich N, Kampira E, Davies G, Bhatt N, Ntinginya EN, Viegas
treatment. Open Forum Infect Dis 6:ofz002. https://doi.org/10.1093/ofid/ S, Jani I, Kamdolozi M, Mdolo A, Khonga M, Boeree MJ, Phillips PPJ, Sloan
ofz002. D, Hoelscher M, Kibiki G, Gillespie SH. 2020. Tuberculosis bacillary load, an
3. Das P, Ganguly SB, Mandal B. 2019. Sputum smear microscopy in tubercu- early marker of disease severity: the utility of tuberculosis Molecular Bac-
losis: it is still relevant in the era of molecular diagnosis when seen from terial Load Assay. Thorax 75:606–608. https://doi.org/10.1136/thoraxjnl
the public health perspective. Biomed Biotechnol Res J 3:77–79. https:// -2019-214238.
doi.org/10.4103/bbrj.bbrj_54_19. 17. Shiferaw MB, Yismaw G. 2019. Magnitude of delayed turnaround time of
4. van Zyl-Smit RN, Binder A, Meldau R, Mishra H, Semple PL, Theron G, laboratory results in Amhara Public Health Institute, Bahir Dar, Ethiopia.
Peter J, Whitelaw A, Sharma SK, Warren R, Bateman ED, Dheda K. 2011. BMC Health Serv Res 19:240. https://doi.org/10.1186/s12913-019-4077-2.
Comparison of quantitative techniques including Xpert MTB/RIF to evalu- 18. Mtafya B, Sabiiti W, Sabi I, John J, Sichone E, Ntinginya NE, Gillespie SH.
ate mycobacterial burden. PLoS One 6:e28815. https://doi.org/10.1371/ 2019. Molecular bacterial load assay concurs with culture on NaOH-
journal.pone.0028815. induced loss of Mycobacterium tuberculosis viability. J Clin Microbiol 57:
5. Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, Tahirli R, Gler e01992-18. https://doi.org/10.1128/JCM.01992-18.
MT, Blakemore R, Worodria W, Gray C, Huang L, Caceres T, Mehdiyev R, 19. World Health Organization. 2020. Global tuberculosis report 2020. World
Raymond L, Whitelaw A, Sagadevan K, Alexander H, Albert H, Cobelens Health Organization, Geneva, Switzerland.
F, Cox H, Alland D, Perkins MD. 2011. Feasibility, diagnostic accuracy, 20. Leveri TH, Lekule I, Mollel E, Lyamuya F, Kilonzo K. 2019. Predictors of
and effectiveness of decentralised use of the Xpert MTB/RIF test for di- treatment outcomes among multidrug resistant tuberculosis patients in
agnosis of tuberculosis and multidrug resistance: a multicentre imple- Tanzania. Tuberc Res Treat 2019:3569018. https://doi.org/10.1155/2019/
mentation study. Lancet 377:1495–1505. https://doi.org/10.1016/S0140 3569018.
-6736(11)60438-8. 21. World Health Organization. 2014. Companion handbook to the WHO
6. World Health Organization. 2016. The use of molecular line probe assays guidelines for the programmatic management of drug-resistant tubercu-
for detection of resistance to second line anti-tuberculosis drugs: policy losis. World Health Organization, Geneva, Switzerland.
guidance. World Health Organization, Geneva, Switzerland. 22. Harris T, Bardien S, Simon Schaaf H, Petersen L, de Jong G, Fagan JJ. 2012.
7. Nicol MP. 2013. Xpert MTB/RIF: monitoring response to tuberculosis Aminoglycoside-induced hearing loss in HIV-positive and HIV-negative
treatment. Lancet Respir Med 1:427–428. https://doi.org/10.1016/S2213 multidrug-resistant tuberculosis patients. S Afr Med J 102:363–366.
-2600(13)70133-4. https://doi.org/10.7196/samj.4964.
8. Theron G, Venter R, Calligaro G, Smith L, Limberis J, Meldau R, Chanda D, 23. Perumal R, Abdelghani N, Naidu N, Yende-Zuma N, Dawood H, Naidoo K,
Esmail A, Peter J, Dheda K. 2016. Xpert MTB/RIF results in patients with Singh N, Padayatchi N. 2018. Risk of nephrotoxicity in patients with drug-
previous tuberculosis: can we distinguish true from false positive results? resistant tuberculosis treated with Kanamycin/capreomycin with or with-
Clin Infect Dis 62:995–1001. https://doi.org/10.1093/cid/civ1223. out concomitant use of tenofovir-containing antiretroviral therapy. J
9. Nikolayevskyy V, Miotto P, Pimkina E, Balabanova Y, Kontsevaya I, Acquir Immune Defic Syndr 78:536–542. https://doi.org/10.1097/QAI
Ignatyeva O, Ambrosi A, Skenders G, Ambrozaitis A, Kovalyov A, .0000000000001705.
Sadykhova A, Simak T, Kritsky A, Mironova S, Tikhonova O, Dubrovskaya 24. World Health Organization. 2019. WHO consolidated guidelines on drug-
Y, Rodionova Y, Cirillo D, Drobniewski F. 2015. Utility of propidium mono- resistant tuberculosis treatment. World Health Organization, Geneva,
azide viability assay as a biomarker for a tuberculosis disease. Tuberculo- Switzerland.
sis (Edinb) 95:179–185. https://doi.org/10.1016/j.tube.2014.11.005. 25. Mbelele PM, Aboud S, Mpagama SG, Matee MI. 2017. Improved perform-
10. Kayigire XA, Friedrich SO, Karinja MN, van der Merwe L, Martinson NA, ance of Xpert MTB/RIF assay on sputum sediment samples obtained from
Diacon AH. 2016. Propidium monoazide and Xpert MTB/RIF to quantify presumptive pulmonary tuberculosis cases at Kibong’oto infectious dis-
Mycobacterium tuberculosis cells. Tuberculosis 101:79–84. https://doi eases hospital in Tanzania. BMC Infect Dis 17:808. https://doi.org/10
.org/10.1016/j.tube.2016.08.006. .1186/s12879-017-2931-6.
11. Cangelosi GA, Meschke JS. 2014. Dead or alive: molecular assessment of 26. Theron G, Peter J, Richardson M, Warren R, Dheda K, Steingart KR. 2016.
microbial viability. Appl Environ Microbiol 80:5884–5891. https://doi.org/ GenoType MTBDRsl assay for resistance to second-line anti-tuberculosis
10.1128/AEM.01763-14. drugs. Cochrane Database Syst Rev 9:CD010705. https://doi.org/10.1002/
12. Li R, Tun HM, Jahan M, Zhang Z, Kumar A, Fernando D, Farenhorst A, 14651858.
Khafipour E. 2017. Comparison of DNA-, PMA-, and RNA-based 16S rRNA 27. Robertson LC, Al-Haddad M. 2013. Recognizing the critically ill patient.
Illumina sequencing for detection of live bacteria in water. Sci Rep Anaesth Intensive Care Med 14:11–14. https://doi.org/10.1016/j.mpaic
7:5752. https://doi.org/10.1038/s41598-017-02516-3. .2012.11.010.
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 11
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana
Mbelele et al. Journal of Clinical Microbiology
28. Guo Y, Logan HL, Glueck DH, Muller KE. 2013. Selecting a sample size for 39. Diriba G, Kebede A, Yaregal Z, Getahun M, Tadesse M, Meaza A, Dagne Z,
studies with repeated measures. BMC Med Res Methodol 13:100. https:// Moga S, Dilebo J, Gudena K, Hassen M, Desta K. 2017. Performance of
doi.org/10.1186/1471-2288-13-100. Mycobacterium Growth Indicator Tube BACTEC 960 with Lowenstein-Jen-
29. Tripathi K, Tripathi PC, Nema S, Shrivastava AK, Dwiwedi K. 2014. Modified sen method for diagnosis of Mycobacterium tuberculosis at Ethiopian
Petroff’s method: an excellent simplified decontamination technique in National Tuberculosis Reference Laboratory, Addis Ababa, Ethiopia. BMC
comparison with Petroff’s method. Int J Recent Trends Sci Technol Res Notes 10:181. https://doi.org/10.1186/s13104-017-2497-9.
10:461–464. 40. Krause KM, Serio AW, Kane TR, Connolly LE. 2016. Aminoglycosides: an
30. Gillespie H, Stephen SW. 2017. Mybacterial load assay. Methods Mol Biol overview. Cold Spring Harb Perspect Med 6:a027029–18. https://doi.org/
1616:89–105. https://doi.org/10.1007/978-1-4939-7037-7_5. 10.1101/cshperspect.a027029.
31. Rustomjee R, Lienhardt C, Kanyok T, Davies GR, Levin J, Mthiyane T, 41. Motta I, Calcagno A, Bonora S. 2018. Pharmacokinetics and pharmacoge-
Reddy C, Sturm AW, Sirgel FA, Allen J, Coleman DJ, Fourie B, Mitchison netics of anti-tubercular drugs: a tool for treatment optimization? Expert
DA, Bah-Sow OY, Diop H, Fielding K, Gninafon M, Mitchison D, Lienhardt Opin Drug Metab Toxicol 14:59–82. https://doi.org/10.1080/17425255
C, Odhiambo J, Perronne C, Portaels F, Rustomjee R, Ramjee A, Master I, .2018.1416093.
Olowolagba A, Chinappa T, Osburne G, Bamber S, Pala AS, Pillay L, Tembe 42. Doan TN, Cao P, Emeto TI, McCaw JM, McBryde ES. 2018. Predicting the out-
C, Mpangase P, Hadebe T, Ngcobo CP, Mkhize Z, Dlamini CN, Gill L, Dube comes of new short-course regimens for multidrug-resistant tuberculosis
T, Saul M, Merle C, Suma KF, Gatifloxacin for TB (OFLOTUB) study team. using intrahost and pharmacokinetic-pharmacodynamic modeling. Antimi-
2008. A phase II study of the sterilising activities of ofloxacin, gatifloxacin crob Agents Chemother 62:1–11. https://doi.org/10.1128/AAC.01487-18.
and moxifloxacin in pulmonary tuberculosis. Int J Tuber Lung Dis 43. World Health Organization. 2019. Global tuberculosis report 2019. World
12:128–138. Health Organization, Geneva, Switzerland.
32. Movshovitz-Hadar N, Shmukler A. 1991. A qualitative study of polyno- 44. World Health Organization. 2018. Rapid communication: key changes to
mials in high school. Int J Math Educ Sci Technol 22:523–543. https://doi treatment of multidrug- and rifampicin-resistant tuberculosis. World
.org/10.1080/0020739910220404. Health Organization, Geneva, Switzerland.
33. Hazra A, Gogtay N. 2016. Biostatistics series module 3: comparing groups: 45. Mpagama SG, Ndusilo N, Stroup S, Kumburu H, Peloquin CA, Gratz J,
numerical variables. Indian J Dermatol 61:251–260. https://doi.org/10 Houpt ER, Kibiki GS, Heysell SK. 2014. Plasma drug activity in patients on
.4103/0019-5154.182416.
treatment for multidrug-resistant tuberculosis. Antimicrob Agents Che-
34. Gillespie SH, Crook AM, McHugh TD, Mendel CM, Meredith SK, Murray SR,
mother 58:782–788. https://doi.org/10.1128/AAC.01549-13.
Pappas F, Phillips PPJ, Nunn AJ, REMoxTB Consortium. 2014. Four-month
46. Kone
cný P, Ehrlich R, Gulumian M, Jacobs M. 2018. Immunity to the dual
moxifloxacin-based regimens for drug-sensitive tuberculosis. N Engl J
threat of silica exposure and mycobacterium tuberculosis. Front Immunol
Med 371:1577–1587. https://doi.org/10.1056/NEJMoa1407426.
35. Goletti D, Lindestam Arlehamn CS, Scriba TJ, Anthony R, Cirillo DM, Alonzi 9:3069. https://doi.org/10.3389/fimmu.2018.03069.
T, Denkinger CM, Cobelens F. 2018. Can we predict tuberculosis cure? 47. Skowron
ski M, Halicka A, Barinow-Wojewódzki A. 2018. Pulmonary tuber-
What tools are available? Eur Respir J 52:1801089. https://doi.org/10 culosis in a male with silicosis. Adv Respir Med 86:121–125. https://doi
.1183/13993003.01089-2018. .org/10.5603/ARM.2018.0019.
36. Honeyborne I, Mtafya B, Phillips PPJ, Hoelscher M, Ntinginya EN, 48. Ahmad N, Ahuja SD, Akkerman OW, Alffenaar J-WC, Anderson LF, Baghaei P,
Kohlenberg A, Rachow A, Rojas-Ponce G, McHugh TD, Heinrich N, Pan Bang D, Barry PM, Bastos ML, Behera D, Benedetti A, Bisson GP, Boeree MJ,
African Consortium for the Evaluation of Anti-tuberculosis Antibiotics. Bonnet M, Brode SK, Brust JCM, Cai Y, Caumes E, Cegielski JP, Centis R, Chan
2014. The molecular bacterial load assay replaces solid culture for meas- P-C, Chan ED, Chang K-C, Charles M, Cirule A, DalcolmoMP, D'Ambrosio L, de
uring early bactericidal response to antituberculosis treatment. J Clin Vries G, Dheda K, Esmail A, Flood J, Fox GJ, Fréchet-Jachym M, Fregona G,
Microbiol 52:3064–3067. https://doi.org/10.1128/JCM.01128-14. Gayoso R, Gegia M, Gler MT, Gu S, Guglielmetti L, Holtz TH, Hughes J,
37. Tweed CD, Dawson R, Burger DA, Conradie A, Crook AM, Mendel CM, Isaakidis P, Jarlsberg L, Kempker RR, Keshavjee S, Khan FA, Kipiani M, Koenig
Conradie F, Diacon AH, Ntinginya NE, Everitt DE, Haraka F, Li M, van SP, Koh W-J, Kritski A, Collaborative Group for the Meta-Analysis of Individual
Niekerk CH, Okwera A, Rassool MS, Reither K, Sebe MA, Staples S, Patient Data in MDR-TB treatment-2017, et al. 2018. Treatment correlates of
Variava E, Spigelman M. 2019. Bedaquiline, moxifloxacin, pretomanid, successful outcomes in pulmonary multidrug-resistant tuberculosis: an indi-
and pyrazinamide during the first 8 weeks of treatment of patients with vidual patient data meta-analysis. Lancet 392:821–834. https://doi.org/10
drug-susceptible or drug-resistant pulmonary tuberculosis: a multi- .1016/S0140-6736(18)31644-1.
centre, open-label, partially randomised, phase 2b trial. Lancet Respir 49. Schober P, Vetter TR. 2018. Repeated measures designs and analysis of
Med 7:1048–1058. https://doi.org/10.1016/S2213-2600(19)30366-2. longitudinal data: if at first you do not succeed-try, try again. Anesth
38. Nguyen TVA, Cao TBT, Akkerman OW, Tiberi S, Vu DH, Alffenaar JWC. Analg 127:569–575. https://doi.org/10.1213/ANE.0000000000003511.
2016. Bedaquiline as part of combination therapy in adults with pulmo- 50. Silva DR, Mello F. C d Q, Migliori GB. 2020. Shortened tuberculosis treat-
nary multi-drug resistant tuberculosis. Expert Rev Clin Pharmacol ment regimens: what is new? J Bras Pneumol 46:e20200009. https://doi
9:1025–1037. https://doi.org/10.1080/17512433.2016.1200462. .org/10.36416/1806-3756/e20200009.
April 2021 Volume 59 Issue 4 e02927-20 jcm.asm.org 12
Downloaded from http://jcm.asm.org/ on April 8, 2021 at Univ of Ghana