Using biomarkers to predict TB treatment duration (Predict TB): a prospective, randomized, noninferiority, treatment shortening clinical trial

Background

Tuberculosis (TB) is one of the top three global causes of infectious diseases and one of the top ten global causes of death, recently surpassing even HIV/AIDS¹. Treatment of drug sensitive TB (DS-TB) is long, typically requiring six months with good adherence to achieve cure and prevent relapse after therapy is stopped. However, treatment adherence and completion rates are not optimal, which could probably be improved by shorter and more effective treatments. Multiple studies conducted over the past 40 years have attempted to reduce the treatment duration of TB. In the 1970s, successful combination chemotherapy lasted 24 months, based on a series of studies conducted by the British Medical Research Council (BMRC). Follow-up studies in the early 1980s successfully reduced treatment duration to 6 months using pyrazinamide and rifampin, achieving relapse rates of 1–2%. Trials that reduced the duration to below 6 months experienced increasing relapse rates, roughly 12% at 4 months and up to 20% at 3 months², establishing 6 months as the accepted treatment duration for all cases of DS-TB.

Three recent randomized-controlled trials attempted to shorten treatment using a 4-month experimental arm with a fluoroquinolone substituted for one of the four standard drugs^3–5. In all three trials, the 4-month treatment arms had relapse rates of approximately 15–20%, significantly higher than the standard of care 6-month arms but similar to the BMRC 4-month treatment trials (12%, 95% CI 9-16 among 364 patients) conducted 30+ years earlier². These trials suggest that, with currently available drugs, roughly 80–85% of drug-sensitive TB patients are cured with 4 months of standard therapy but 15–20% will relapse if not treated for at least 6 months and, further, that a subset of those with less severe disease could be cured at 4 months. Identifying this subset prospectively could lead to: (1) new treatment guidelines for eligible lower risk patients; (2) criteria for selection of high-risk patients that might be candidates for Phase 2b studies with novel regimens with proposed shorter durations; and (3) quantitative estimates of the rate of change of markers associated with durable cure at specific timepoints to establish milestones for even shorter regimens. The hypothesis that patients with less severe disease can be cured earlier was tested in a separate treatment shortening trial, in which only those with less severe disease and good initial treatment response were randomized to 4 vs. 6 months of treatment⁶. In this trial, less severe disease was defined as absence of pulmonary cavities on baseline chest x-ray, and good treatment response was defined as sputum culture conversion to negative by 2 months of treatment. Only participants who met these 2 criteria were randomized to 4 vs. 6 months of treatment with standard of care drugs. This trial was stopped early by its Data and Safety Monitoring Board (DSMB) because those in the 6-month arm had a relapse rate of 1.6% while the 4-month arm had a significantly higher relapse rate of 7% (P<0.01). Despite this trial not meeting its target, the disease stratification criteria did increase the treatment success rate of the 4-month arm from 80–85% in the prior non-stratified trials to 93% (95% CI 89%, 96%), approaching 6-month treatment relapse rates. If the stratification criteria could be further refined, a successful 4-month treatment arm may be feasible. The hypothesis of the Predict TB trial is that a combination of radiographic characteristics at baseline, the rate of change of these features at one month, and markers of residual bacterial load at 4 months will identify patients with tuberculosis who are cured within 4 months (16 weeks) of standard treatment.

Methods

Study design

This is a prospective, multicenter, randomized, phase 2b, noninferiority clinical trial of pulmonary DS-TB participants conducted at 4 sites in Henan Province, China (Henan Provincial Chest Hospital, Xinmi City Institute of Tuberculosis Prevention and Control, Kaifeng City Institute of Tuberculosis Prevention and Control, and Zhongmu County Health and Epidemic Prevention Station) and 5 sites in and around Cape Town, South Africa (Stellenbosch University, University of Cape Town Lung Institute, University of Cape Town South African Tuberculosis Vaccine Initiative, Khayelitsha Site B, and TASK Applied Science). Inclusion criteria are:

• 1) Age 18 to 65 years, with body weight from 35 kg to 90 kg

• 2) Not been treated for active TB within the past 3 years

• 3) Not yet on TB treatment

• 4) Xpert positive for Mycobacterium tuberculosis

• 5) Rifampin-sensitive pulmonary tuberculosis as indicated by Xpert

• 6) Laboratory parameters within previous 14 days before enrollment:

  • a. Serum AST and ALT <3x upper limit of normal (ULN)

  • b. Creatinine <2x ULN

  • c. Hemoglobin >7.0 g/dL

  • d. Platelet count >50 x10⁹ cells/L

• 7) Able and willing to return for follow-up visits

• 8) Able and willing to provide informed consent to participate in the study

• 9) Willing to undergo an HIV test

• 10) Willing to have samples, including DNA, stored

• 11) Willing to consistently practice a highly reliable, non-hormonal method of pregnancy prevention (e.g., condoms) during treatment if participant is a premenopausal female, unless she has had a hysterectomy or bilateral tubal ligation or her male partner has had a vasectomy

Exclusion criteria are:

• 1) Extrapulmonary TB, including pleural TB

• 2) Pregnant or desiring/trying to become pregnant in the next 6 months or breastfeeding

• 3) HIV infected

• 4) Unable to take oral medications

• 5) Diabetes as defined by point of care HbA1c ≥6.5%, random glucose ≥200 mg/dL (or 11.1 mmol/L), fasting plasma glucose ≥126 mg/dL (or 7.0 mmol/L), or the presence of any anti-diabetic agent (including traditional medicines) as a concomitant medicine

• 6) Disease complications or concomitant illnesses that may compromise safety or interpretation of trial endpoints, such as known diagnosis of chronic inflammatory condition (e.g. sarcoidosis, rheumatoid arthritis, connective tissue disorder)

• 7) Use of immunosuppressive medications, such as TNF-alpha inhibitors or systemic or inhaled corticosteroids, within the past 2 weeks

• 8) Use of any investigational drug in the previous 3 months

• 9) Substance or alcohol abuse that in the opinion of the investigator may interfere with the participant’s adherence to study procedures

• 10) Any person for whom the physician feels this study is not appropriate

Eligible participants who sign the informed consent are started on standard of care treatment with fixed-dose combination tablets composed of isoniazid (H), rifampin (R), pyrazinamide (Z), and ethambutol (E) during the initial 8-week intensive phase and HR during the subsequent continuation phase, using national weight-based guidelines (Table 1). PET/CT scans are done at baseline, week 4, and either week 16 or 24. Participants are followed through month 18 and a fourth PET/CT scan is done if recurrent TB develops during follow-up. All PET/CT scans are read by 2 readers using MIM Software (v. 6.6 or higher, MIM Software Inc, Cleveland, Ohio, USA). Scans with discrepant arm assignments by the initial 2 readers are read by a third reader. All PET/CT scan readers underwent a prequalification training process with 15 practice baseline and week 4 scans to ensure consistency among all readers in how the scans are read (see Supplementary File 5 for PET/CT reading SOP). The practice PET/CT scans were obtained from a previously conducted study at Stellenbosch University funded by the Bill and Melinda Gates Foundation (Mycobacterium tuberculosis biomarkers for diagnosis and cure, OPP51919).

Table 1. Adult weight-based dosing guidelines for intensive phase and continuation phase TB treatment using fixed-dose combination (FDC) tablets by country.

These are the dosing guidelines used in the Predict TB study and are taken from the South African national TB guidelines³⁹ and the Chinese fixed drug combination tablet package insert from the Shenyang Hongqi Pharmaceutical Company, Ltd.

	Intensive Phase (initial 8 weeks)	Continuation Phase (weeks 9–24)
	HRZE (75/150/400/275 mg)	HR (75/150 mg)	HR (150/300 mg)
Weight	China and South Africa	South Africa
30–37 kg	2 tablets daily	2 tablets daily	--
38–54 kg	3 tablets daily	3 tablets daily	--
55–70 kg	4 tablets daily	--	2 tablets daily
>70 kg	5 tablets daily	--	2 tablets daily
		China
<50 kg		--	individual drug tablets (non-FDC)
≥50 kg		--	2 tablets daily

Note: H=isoniazid; R=rifampin; Z=pyrazinamide; E=ethambutol

Participants are stratified into treatment arms by the early treatment completion criteria, composed of baseline PET/CT criteria, the change in these measurements at week 4, and a minimum adherence dose count and Xpert cycle threshold as a marker of residual sputum bacterial load at week 16 (Table 2). Those with more severe disease (who do not meet all early treatment completion criteria) are placed in Arm A (standard of care arm, treatment completion at week 24). Those with less severe disease (who meet all early treatment completion criteria) are randomized either to Arm B (standard treatment duration of 24 weeks) or Arm C (standard treatment shortened to 16 weeks) at week 16. The third PET/CT scan is done at week 16 for participants in Arms B or C and randomized to either week 16 or 24 for participants in Arm A. All participants are followed to week 72 for final treatment outcomes (Figure 1), with sputum, blood, and urine samples collected per the schedule in Table 3. Study enrollment began in Cape Town in June 2017 and is expected to begin in Henan in October 2017. Enrollment is expected to take about 3 years and the study is expected to complete within 5 years.

Table 2. Predict TB early treatment completion criteria.

These are the criteria used at baseline, week 4, and week 16 to stratify enrolled participants to Arm A or Arm B vs C. Participants must meet all criteria to be eligible for randomization to Arm B vs C at week 16. Participants who do not meet all criteria are placed into Arm A.

Category	Criteria
Radiographic criteria	Baseline PET/CT:     •  No total lung collapse of a single side, AND     •  No pleural effusion, AND     •  No single cavity air volume on CT scan >30 mL, AND     •  CT scan hard volume (-100 to +100 HU density) <200 mL, AND     •  PET total activity <1500 units Week 4 PET/CT:     •  All individual cavities decrease by >20% (unless cavity <2 mL),        AND     •  CT scan hard volume does not increase by >10% unless the        increase is <5 mL, AND     •  PET total activity does not increase by >30% unless the        increase is <50 units
Bacterial load criterion	Week 16 Xpert cycle threshold ≥30
Adherence criterion	Minimum of 100 doses received by week 16

Figure 1. Predict TB study schematic.

Table 3. Predict TB study timeline.

Study enrollment began in Cape Town in June 2017 and is expected to begin in Henan in October 2017. Enrollment is expected to take about 3 years and the study is expected to complete within 5 years.

	Screening	D0	W1V (D7)	W2 (D14)	W4 (D28)	W8D (D56)	W12 (D84)	At Week 16	W16 (D112)	W20 (D140)	W24 (D168)	W36 (D252)	W48 (D336)	W60 (D420)	W72 (D504)	TB Recurrence
Main Study Informed Consent (Plus Genetic and HIV in RSA)	X							RANDOMIZE to Arm B or C
Medical History/ Focused History	X	X	X	X	X	X	X		X	X	X	X	X	phone call	X	X
Physical Exam/ Focused Physical Exam	X	X	X	X	X	X	X		X	X	X	X	X		X	X
Sputum Collectionξ
Smear/Culture	XXH	X	X	X	X	X	X	then	X	X	X	X	X		X	X
GeneXpert	X	(X)S			X	X			XE		X (arms A and B only)					X
Biomarkers		X		X	Xg	Xg			Xg		Xg		Xg		Xg	Xg
Saliva		X			X				X		X					X
Blood collection
CBC/Chems/LFTG	X							and if not assigned to Arm A,								X
Biomarkers		X	X	X	X	X			X		X		X		X	X
Pregnancy TestP (serum at screening, urine for others)	xh(serum)
HIV Testing	X															X
Plasma drug levels										X (all arms)
Finger Stick		X			X				Arms B/C and randomized A		Randomized for scan in Arm A only					X
Urine collection
Biomarkers		X	X	X	X	X		Review treatment completion criteria	X		X		X		X	X
Pregnancy TestP (serum at screening, urine for others)	x (can do if desired before CXR)	X			X				Arms B and C only		Arm A only					X
FDG-PET/CTW		X			X				Arms B/C and randomized A		Randomized for scan in Arm A only					X
Adherence monitoring			X	X	X	X	X		X	Arms A and B only	Arms A and B only

^ξAdditional sputum may be collected if contaminated or otherwise compromised

A subject could be called back for this additional sputa collection, if necessary.

^HSputum at screening will also be used for screening Xpert

^SMay not be done if within 7 days of screening

^hPregnancy testing from screening may be used for the D0 PET/CT scan if D0 is within 2 days of the screen

^GThese will be performed at any visit if clinically significant.

^PBefore any PET/CT scan or CXR is done, a pregnancy test will be done for applicable females. If the pregnancy test is positive, the PET/CT scan will not be performed.

^EFor those eligible for randomization to Arms B/C

^DAt week 8, ethambutol and pyrazinamide will be discontinued

^WPET/CT scan windows: baseline w/i 7 days after treatment initiation; W4 must be at least 4 wks after baseline scan with a -3/+7 d window; W16 and 24 scan w/i 14 d of visit; relapse ASAP, but w/i 2 wks of recurrence

^VVisit windows: Week 1–2: +/- 3 days; Week 4–24: +/- 7 d, noting that Weeks 16 and 24 should be as close as possible to actual date; Week 36–72: +/- 30 days.

^gIf sputum is not available for biomarkers, it will not be a protocol deviation.

Discussion

With currently available drugs for TB, all published trials attempting to shorten TB treatment below 6 months have failed. The trial that came closest to success randomized only those with less severe disease and favorable early treatment response (baseline chest x-ray without cavity and a month 2 sputum culture that had converted to negative). Multiple tuberculosis biomarkers potentially predictive of treatment outcomes are being studied to improve the risk stratification of patients¹⁴. From a microbiological standpoint, sputum culture conversion at 2 months of treatment is most commonly used to predict non-relapsing cure¹⁵ but its true predictive ability is poor, with one meta-analysis showing a pooled sensitivity and specificity for predicting relapse of 40% (95% CI 25%-56%) and 85% (95% CI 77%-91%), respectively¹⁶. A review of data from the BMRC trials from the 1970s and 1980s found only a weak correlation (R²=0.36) for this marker as a surrogate for treatment failure and relapse, depending on factors such as geographic location, baseline disease and cavity status, and concomitantly used medications¹⁷. Further evidence against using culture conversion as a surrogate for predicting treatment outcome was recently demonstrated from the phase 3 TB treatment shortening trial REMoxTB⁴, where subsequent analyses of the culture data collected demonstrated poor correlation with treatment outcome whether analyzed at a single time point (2 months) or over time (time to culture conversion or time to culture positivity)¹⁸.

The ability of early radiographic changes to predict subsequent treatment outcomes in TB has been recognized for over 50 years¹⁹ and prior studies have identified baseline cavity on chest x-ray as a risk factor for relapse^20–22. However, chest x-rays are not sensitive for cavities, particularly smaller ones; more recent analyses of radiographic biomarkers have moved beyond chest x-ray to 2-deoxy-2-[¹⁸F]-fluoroglucose (FDG) positron emission tomography/computed tomography (PET/CT) as an early marker of treatment response and possibly as a marker for relapse at the end of treatment. CT scans produce more detailed lung morphology than chest x-rays and PET scans provide additional information on inflammatory activity. In macaques, changes on PET/CT scans correlate with TB disease activity and treatment response^23,24. Our group has analyzed human PET/CT data from a randomized clinical trial using metronidazole in the treatment of pulmonary multi-drug resistant tuberculosis (MDR-TB) participants²⁵. As a substudy within the overall MDR-TB study, we performed PET/CT scans at 0 and 2 months and CT scans at 0, 2, and 6 months of treatment and correlated these changes with final treatment outcomes 30 months after treatment start (6 months after the end of therapy). PET changes at 2 months and CT changes at 6 months appeared to be more sensitive to predict final treatment outcomes than sputum culture conversion at 2 months, although these differences were not statistically significant²⁶. These results support the potential of PET/CT imaging biomarkers as possible surrogate endpoints in clinical trials, and larger cohorts are needed to confirm these results.

We developed the radiographic early treatment completion criteria for the Predict TB trial (Table 2) (unpublished study; National Institutes of Health, Bethesda, MD, USA) using a cohort of 100 pulmonary DS-TB participants from Cape Town who received PET/CT scans at baseline, 1 month, and 6 months while on standard therapy through the national TB treatment program²⁷. The participants were treated for 6 months, then followed through 18 months for final treatment outcomes. In total, 92 participants had complete PET/CT scan data, Xpert MTB/RIF cycle thresholds, and treatment outcomes. After 18 months of follow-up, 73 were considered cured, 8 failed treatment, and 11 were restarted programmatically on TB treatment, defined as the participant restarting treatment during follow-up for any reason. Our radiographic early treatment completion criteria are divided into baseline disease burden and week 4 reduction in disease burden due to treatment, which is reflective of the Johnson 2009 study which also had a measure of baseline disease burden (baseline chest x-ray without cavity) and treatment response (month 2 sputum culture conversion).

Currently, the only direct measure of TB sputum bacterial load is sputum smear, which is not very sensitive. Alternative surrogate markers evaluated include the time to positivity (TTP) of a positive culture on liquid mycobacterial culture systems, with the shorter TTP indicating a higher bacterial load. Different studies have demonstrated some correlation between Mycobacteria Growth Indicator Tube (MGIT) TTP and sputum bacterial load but with poor specificity in predicting treatment outcomes^28,29. Thus, although time to culture conversion and MGIT TTP do correlate independently with treatment outcome, these markers do not discriminate well between high and low risk patients and therefore have only a limited role in predicting treatment outcomes of individual patients¹⁸. Another marker of sputum bacterial load is the GeneXpert cycle threshold. The GeneXpert assay is an automated rapid molecular diagnostic test for Mtb and resistance to rifampin with results provided directly from sputum within 2 hours³⁰. The test is run using a polymerase chain reaction and the number of cycles (cycle threshold) at which the amplification curve crosses the specified threshold is recorded, with a lower cycle threshold suggestive of a higher bacterial load. Three studies have correlated Xpert MTB/RIF cycle threshold with sputum smear status, with varying sensitivity and specificity levels depending on the cycle threshold cut point used^31–34. A fourth study found that Xpert MTB/RIF results correlated with smear grades, solid culture results, and liquid culture TTP (all p<0.0001) but Xpert MTB/RIF sputum positivity rates declined more slowly during treatment than sputum smear and culture results. Using the combined binary smear and culture results as a reference standard, sensitivity of Xpert MTB/RIF was excellent at 97.0% (95% CI 95.8-97.9) but specificity was poor at 48.6% (95% CI 45.0-52.2) as the assay is unable to differentiate viable, dormant, and non-viable Mtb bacteria³⁵.

In the study mentioned previously of 100 pulmonary DS-TB participants in Cape Town, a week 24 Xpert MTB/RIF cycle threshold of ≥30 predicted treatment failure with higher sensitivity and specificity than earlier time points³⁶. This is in contrast to the week 8 culture, which has lower sensitivity (for cure) than Xpert MTB/RIF cycle threshold at week 24: 61% (week 8 culture) vs 89% (Xpert MTB/RIF cycle threshold week 24, p<0.01). Estimates of specificity (for failure) for Xpert MTB/RIF cycle threshold week 24 was higher than week 8 culture (88% vs 50%), but the improvement was not statistically significant. The poor performance of week 8 culture data as a predictor of treatment outcome is similar to what was observed in the REMoxTB trial¹⁸.

The observation that a later test predicts outcomes better than an earlier test may be similar to findings in HIV infection, where baseline CD4 cell count is a strong predictor of mortality over time but current CD4 cell count is even stronger³⁷. This has been seen in TB too, where culture conversion status at month 6 predicts final treatment outcome significantly better than culture conversion status at month 2³⁸. Taken together, these results suggest that Xpert MTB/RIF cycle thresholds collected later may be able to replace an earlier microbiological culture in predicting treatment outcomes, with the major advantage of Xpert MTB/RIF over culture being the time to test result, with Xpert requiring 2 hours and culture up to 6 weeks. Thus, Xpert cycle threshold may be useful as a point-of-care test whereas culture cannot.

In summary, the Predict TB trial builds upon previous trial results, in particular the Johnson 2009 trial⁶. Instead of using a chest x-ray to determine baseline disease burden, we will use a PET/CT scan. Instead of using month 2 culture conversion as a measure of treatment response, we will use month 1 change in PET/CT scan disease burden and a month 4 Xpert MTB/RIF cycle threshold. Prior treatment shortening trials that randomized all subjects to shortened vs. standard treatment achieved treatment success rates in the 4-month arms of roughly 80–85%. Using a risk stratification approach, the Johnson study increased this to 93%. By refining their risk stratification parameters, we hypothesize that the treatment success rate in our 4-month arm will be non-inferior to our 6-month arm. If successful, our methodology could be extended to identify participants cured with even shorter regimens. We do not expect that PET/CT scans will become a risk stratification tool for global TB use due to its cost and availability limited to larger cities. Use of PET/CT scans would likely be limited to clinical trials but could be a method to stratify trial participants and possibly become a surrogate endpoint by which to reduce the number of participants needed in a Phase 2b trial, shorten overall trial duration or predict drug sterilizing activity such as in early bactericidal activity studies. These achievements could expand the number of regimens evaluated in Phase 2b trials prior to committing to an expensive and time-consuming Phase 3 study and thereby contribute to the likelihood of identifying optimal regimens. If the Predict TB trial is successful, other immunological biomarkers or transcriptional signatures that correlate with treatment outcome may be identified. These markers or signatures will likely be much cheaper and more widely available than PET/CT scans and more amenable to being scaled up globally.