Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

INTRODUCTION: Traditionally, single critical concentrations of drugs are utilized for Mycobacterium tuberculosis (Mtb) drug susceptibility testing (DST); however, the level of drug resistance can impact treatment choices and outcomes. Mutations at the katG gene are the major genetic mutations in multidrug resistant (MDR) Mtb and usually associated with high level resistance. We assessed the minimum inhibitory concentrations (MICs) of MDR or rifampin resistant (RR) and isoniazid (INH) resistant Mtb isolates to determine the quantification of drug resistance among key anti-tuberculosis drugs.
METHODS: The study was conducted on stored Mtb isolates collected as part of a national drug resistance survey in Ethiopia. MIC values were determined using Sensititre™ MYCOTB plates. A line probe assay (MTBDRplus) was also performed to identify genetic determinants of resistance for all isolates.
RESULTS: MIC testing was performed on 74 Mtb isolates including 46 MDR, 2 RR and 26 INH phenotypically resistant isolates as determined by the Löwenstein Jensen (LJ) method. Four (15%) INH resistant Mtb isolates were detected as borderline rifampin resistance (MIC = 1 μg/ml) using MYCOTB MIC plates and no rifampin resistance mutations were detected by LPA. Among the 48 MDR/RR TB cases, 9 (19%) were rifabutin susceptible (MIC was between ≤0.25 and 0.5μg/ml). Additionally, the MIC for isoniazid was between 2-4 μg/ml (moderate resistance) for 58% of MDR TB isolates and 95.6% (n = 25) of the isolates had mutations at the katG gene.
CONCLUSION: Our findings suggest a role for rifabutin treatment in a subset of RR TB patients, thus potentially preserving an important drug class. The high proportion of moderate level INH resistant among MDR Mtb isolates indicates the potential benefit of high dose isoniazid treatment in a high proportion of katG gene harboring MDR Mtb isolates.

Introduction

The effective management of drug resistant tuberculosis (TB) relies on the accurate detection of resistance to guide appropriate treatment [1]. Drug susceptibility testing (DST) is an important step in the design of effective treatment regimens as it guides clinical management of individual patients and is essential for TB control program planning [2]. Traditionally, a single critical concentration (CC) per drug has been utilized to determine if Mycobacterium tuberculosis (Mtb) isolates are resistant or susceptible to an individual drug; however, this approach limits the ability to detect borderline resistance and can result in resistance misclassification. Minimum inhibitory concentration (MIC) testing provides quantitative results that may help guide the therapeutic decision-making process [3, 4]. High level drug resistance indicates a drug is unlikely to have any clinical benefit; on the other hand, low-level drug resistance indicates a possible benefit of a drug either with standard or increased dosing from a different drug with the same class [3, 5].

The Sensititre™ MYCOTB plate (Trek Diagnostic Systems, Thermo Fisher Scientific, USA) was developed to determine the MICs of key first- and second-line TB drugs that includes ofloxacin, amikacin, moxifloxacin, linezolid, cycloserine, clofazimine, kanamycin, levofloxacin, rifampin, capreomycin, para-aminosalicylicacsid, isoniazid, tedizolid, and rifabutin. Previous reports have found the Sensititre™ MYCOTB to be accurate method for performing DST for both first- and second-line anti-TB drugs and result would be available within 14 days [6-8]. The results allow the clinician to adjust dose or choose among the best available alternatives to optimize therapy [3, 8, 9].

Certain mutations are more likely to confer high levels resistance whereas some may be found in low level resistance. Mutations at the inhA promoter are mostly conferring low-level resistance, while mutations in katG gene particularly at S315T are usually associated with high level resistance [10, 11]. The katG mutations are the major genetic mutations in MDR TB [12] and it had a wide range of MIC value that suggest some MDR patient might potentially benefited from high dose treatment [13].

Rifabutin is an alternative first line anti-TB drug for TB-HIV co-infected patients [14]. As indicated in previous studies, rifabutin based therapy had very low relapse rate for person co-infected with TB and HIV [15, 16]. Some RR TB are susceptible for rifabutin and those cases are more likely to harbor certain mutation than resistant cases for both rifampin and rifabutin [17-19]. The presence or absence of cross resistance of rifampin with rifabutin may assist in guiding the treatment choice for the rifabutin based therapy [20].

To our knowledge, in Ethiopia there is no previous data on MIC of key first line anti-TB drugs (isoniazid, rifampin and rifabutin) for Mtb isolates including multidrug or rifampin resistant (MDR/RR) and isoniazid (INH) resistant isolates. To help fill this knowledge gap, we conducted a study to determine MIC values of MDR/RR TB and INH resistance Mtb isolates collected from population-based drug resistance survey. The overall goal of our study is to provide information about the proportion of TB cases with low, moderate, and high-level resistance to INH and the proportion of rifabutin susceptible among RR Mtb isolates that could potentially benefit in MIC tailored regimen choice.

Materials and methods

We utilized stored Mtb isolates to profile the MIC values of MDR/RR TB and INH resistant Mtb isolates. The study population has been described in detail previously [21]. Briefly, we utilized stored MDR/RR and INH resistant Mtb isolates collected for a national drug resistance survey (DRS) conducted between November 2011 and June 2013. The DRS enrolled all TB suspects with sputum smear positive samples from 32 health facilities throughout Ethiopia. The 32 health facilities were purposely selected to include facilities in each region throughout the country and to provide a representative sample of Ethiopia. Phenotypic DST was performed on all Mtb isolates using the indirect proportion Lowenstein-Jensen (LJ) method for first-line drugs including INH (0.2 μg/ml) and rifampin (RIF) (40 μg/ml). Interpretation of results was based on the proportion of the growth on control and drug containing media [22, 23]. The DRS found 67 MDR, 5 RR and 70 INH resistant Mtb isolates. Among these isolates, we selected 74 Mtb isolates (46 MDR, 2 RR and 26 INH resistant TB) based on subculture growth characteristics i.e pure Mtb growth, Mtb growth with contamination, no growth (S1 Table). All available MDR/RR and previously treated INH resistant Mtb isolate that had sufficient growth were undergo with MIC testing [22].

Line probe assay

All stored MDR/RR and INH Mtb isolates had LPA testing performed. The Genotype MTBDRplus (Hain Lifescience, Nehren, Germany, version 2) assay was performed to evaluate for genetic determinants of resistance to rifampin and isoniazid. Extraction, amplification, detection, and result interpretation were performed as per the manufacturer’s instructions [24].

Minimum inhibitory concentration

After thawing frozen Mtb isolates, 100μl of vortexed allocate was subcultured on to Middlebrook 7H11 agar medium. Sensititre™ MYCOTB MIC plates were utilized for MIC testing for all included Mtb isolates. The MYCOTB plate contains lyophilized antibiotics of three key first-line drugs including isoniazid (0.25–4 μg/ml), rifampin (0.25–8 μg/ml) and rifabutin (0.25–8 μg/ml). MIC testing was performed according to the manufacturer’s recommendation. Briefly, colonies were scraped, and a 0.5 McFarland standard was prepared using a saline-Tween solution. After 15 minutes, 100 microliters were transferred to 11ml of Middlebrook 7H9 broth containing oleic acid albumin-dextrose-catalase and vortexed for 30s. Each well of the MIC plate was inoculated with 100 microliters of the reconstituted sample aliquot. Plates were then covered with plastic seals and incubated at 37°C, and visually monitored for growth at days 1, 2, 7, 10, 14 and 21. The MIC result was recorded on days 10 and subsequently on 21 if growth was not sufficient for interpretation. Blood agar and Middlebrook 7H10 plates were also inoculated with suspension to check contamination and read after 24 and 48 hours.

We classified Mtb isolates as either susceptible or resistant based on reported CCs [6, 7, 9, 25]. The lowest concentration with no visible growth was viewed using a magnified mirror and recorded as the MIC value. Mtb isolates were defined as resistant if the MIC was greater than the CC or susceptible if the MIC was less than or equal to the CC. For INH, we categorized into the following 3 groups based on MIC values: low level resistance (≤0.25μg/ml to 0.5μg/ml), moderate resistance (1μg/ml≥ MIC ≤4μg/ml) and high level resistance (MIC>4μg/ml). The upper cutoff values of moderate level resistance were defined based on a clinical trial report in which patients with such levels of resistance were having a comparable early bactericidal activity as sensitive strains [26].

Ethical approval

We use a stored isolates. The study obtained ethical approval from the Addis Ababa University Ethics Committee.

Statistical analysis

Descriptive statistics were used to describe the MIC values of MDR, RR and INH resistant Mtb isolates.

Results

Study isolates and patients

A total of 74 Mtb isolates from unique patients including 46 MDR, 2 RR and 26 INH phenotypically resistant isolates had MIC testing using MYCOTB Sensititre™ plates performed. Among the 74 unique individuals from which the samples came from, 58% were male and the mean age was 30 years. Forty six percent of patients (n = 34) had a history of prior anti-TB treatment with first-line drugs (2RHZE/4RH). Ninety five percent (n = 70) of the patients had human immunodeficiency virus (HIV) testing performed and 33% (n = 23) were HIV positive. Over 50% (n = 13) of the HIV positive individual had a history of prior anti-TB treatment (2RHZE/4RH).

MIC testing

Among the 46 MDR, 2 RR and 26 INH resistant Mtb isolates, MYCOTB Sensititre™ MIC plate confirmed resistance in 46, 2 and 24 cases respectively (Table 1). Two INH resistance Mtb isolates as defined by LJ method were detected as susceptible using Sensititre™ MYCOTB MIC plate (MIC≤0.25 μg/ml) and mutations confirming INH resistance were detected using LPA. Four (15%) INH resistant Mtb isolates as defined by LJ method were borderline rifampin resistant TB at CC = 0.5 μg/ml using MYCOTB MIC plates and no rifampin resistance mutations were detected by LPA. The Mtb isolates with borderline rifampin resistant were recovered from patients with newly diagnosed TB.

Table 1

Minimum inhibitory concentration of rifampin, rifabutin and isoniazid among multidrug resistant (MDR), rifampin resistant (RR) and isoniazid (INH) resistant Mycobacterium tuberculosis isolates.

Drugs	DST result	MIC in μg/ml	Total N	Total %	Newly diagnosed, N	Retreatment, N
Rifampin	MDR	>8	46	100	21	27
	RR TB	>8	2	100	1	1
	INH resistant	0.5	4	15	4	0
		≤0.25	22	85	15	7
Rifabutin	MDR	>8	18	39	4	14
		8	7	15	3	4
		4	9	20	9	0
		2	2	4	1	1
		1	2	4	1	1
		0.5	1	2	0	1
		≤0.25	7	15	2	5
	RR TB	2	1	50	0	1
		≤0.25	1	50	1	0
	INH resistance	≤0.25	26	100	19	7
Isoniazid	MDR	>4	19	41	8	11
		4	25	54	10	15
		2	2	4	2	0
	INH resistance	>4	9	35	1	8
		4	9	35	5	4
		2	5	8	4	1
		0.5	1	4	1	0
		≤0.25	2	8	1	1

DST: drug susceptibility testing

Rifabutin MIC distribution

The MIC of Mtb isolates to rifabutin and the corresponding mutation profiles are summarized in Table 2. Sixty two percent of the mutations occurred at codon 530–533 and 93% of rifabutin resistant Mtb isolate had MIC ≥ 4μg/ml. Among 48 MDR/RR Mtb isolates, 9 isolates (19%) were found to be rifabutin-susceptible (MIC≤0.5μg/ml), including 6 patients with a prior history of TB treatment and 4 from persons living with HIV. Six (66%) of the 9 rifampin-resistant and rifabutin-susceptible Mtb isolates were classified as rifampin resistance due to the absence of wild-type probes only. Furthermore, 33% of the rifampin-resistant and rifabutin-susceptible Mtb isolates had mutations detected by the MDRTBplus probe in the 513–519 codon regions.

Table 2

Line probe assay result and minimum inhibitory concentration of rifabutin among multidrug resistant (MDR) and rifampin resistant (RR) Mycobacterium tuberculosis isolates.

DST result	Wild type absent and mutation developed on MTBDRplus probe	MIC in μg/ml
		≤0.25	0.5	1	2	4	8	>8
MDR	Susceptible	0	0	0	1	0	0	1
	W3W4	2	0	0	0	1	0	0
	W3W4MUT1	0	1	0	0	0	0	0
	W4W7MUT1MUT2B	0	0	1	0	0	0	0
	W7	4	0	0	0	0	0	1
	W7MUT2A	1	0	0	0	0	0	1
	W7MUT2B	0	0	0	0	0	0	2
	W8	0	0	0	0	1	0	1
	W8MUT3	0	0	1	1	6	7	12
	MUT3	0	0	0	0	1	0	0
RR	Susceptible	0	0	0	1	0	0	0
	W7	1	0	0	0	0	0	0

DST: drug susceptibility testing

Low and moderate level isoniazid Mtb

Among 46 MDR Mtb isolates, 58% (n = 27) had moderate level (MIC = 2–4 μg/ml) isoniazid resistance. Most (n = 25, 95.6%) moderate level isoniazid resistant Mtb isolates had mutations in the katG gene; of the remaining two isolates, one had mutations at the inhA gene mutations and the other isolates had no mutations detected by the LPA (Table 3). Among 26 INH resistant Mtb isolates, 3 (12%) had low level resistance (MIC ≤0.5μg/ml) including 2 with mutations detected by the MTBDRplus in the inhA gene.

Table 3

Frequency of isoniazid resistance Mycobacterium tuberculosis isolates with minimum inhibitory concentration and line probe assay result.

DST result	MIC in μg/ml	Total	Wild type absent and mutation developed on MTBDRplus probe
			Susceptible	inhAW1MUT1	katGW1	katGW1MUT1	katGMUT1
INH resistant	≤0.25	2	0	1	0	0	1
	0.5	1	0	1	0	0	0
	2	5	2	0	0	3	0
	4	9	0	0	0	9	0
	>4	9	0	0	0	8	1
MDR	2	2	0	0	0	2	0
	4	25	1	1	2	21	0
	>4	19	1	0	0	17	1

DST: drug susceptibility testing

Discussion

Utilizing a national representative sample, we carried out the first study evaluating MIC testing of Mtb isolates in Ethiopia with results highlighting potential roles for rifabutin and high dose INH treatment in many patients with MDR TB. Our study reports borderline rifampin resistance among INH resistant Mtb, low level rifabutin resistance among RR Mtb and moderate level isoniazid resistance among katG gene harboring MDR Mtb isolates. Data on rifabutin susceptible RR Mtb would assist in guiding the treatment choice for the rifabutin-based therapy especially in HIV co-infected TB patients [15, 20]. Additionally, detections of borderline rifampin resistance using Sensititre™ MYCOTB MIC plate which mostly missed using phenotypic methods are vital to interrupt transmission [27-29]. Besides, information on the level of INH resistance is important to guide the treatment choice with high dose INH for MDR Mtb with katG mutations [26].

Rifabutin is considered as an alternative treatment option in place of rifampin in some persons with HIV and TB due to fewer drug-drug interactions with certain antiretroviral drugs [14]. In our study, 19% (n = 9) of the Mtb isolates that were MDR/RR were susceptible to rifabutin and 44% (n = 4) of them were person living with HIV; a finding that is in line with previous studies [17-19]. Studies show that the treatment outcomes and the rate of acquired resistance of rifabutin susceptible MDR TB patients can be improved by rifabutin-based therapy [16, 30]. A clinical trial on MDR TB patients who had been treated using individualized regimens reports a significantly higher treatment success rate for rifabutin based therapy (85.7%) than control group (52.4%) [20]. Another clinical trial indicates that TB-HIV co-infected patient who takes rifabutin twice a week had low relapse rate (1 of 20) than rifampin-based therapy (8 of 9) [15]. Additionally, mutations at codon 514, 516 and 522 are usually associated with rifabutin susceptibility even though the mutations alone had not always classified such cases [31]. In our study, we found that 33% rifampin resistant but rifabutin susceptible was detected at codon 513–519; this is in line with previous studies [17-19]. Overall, our finding showed that the MIC of rifabutin susceptible MDR/RR Mtb isolates were between ≤0.25 and 0.5μg/ml which indicates the value of MIC determination in detecting those cases than the traditional DST which uses a single CC as well as frontline genotypic methods [31].

Borderline rifampin resistance is mostly associated with low-level rifampin resistance and can be missed by growth-based methods [27-29]. Our finding showed that 15% INH resistant Mtb isolates as defined by LJ method were borderline rifampin resistance at CC = 0.5 μg/ml (MIC = 1 μg/ml) using Sensititre™ MYCOTB MIC plate. We did not detect rifampin mutations using LPA suggesting the mutation might occur outside of rifampicin resistance-determining region. The WHO review indicates that a couple of rifampin borderline resistance was detected as susceptible using LJ method at the current CC (40 mg/L). Compare to LJ method, 36% of mutations at codon I572F and 28% of mutation at the six rifampicin resistance-determining region were detected as susceptible using LJ method [31]. The treatment outcome of patients with borderline rifampin resistance has been associated with treatment failures [32] and WHO recommends a MDR-TB regimen for their treatment [31]. Our findings indicate the importance of MIC in detecting borderline rifampin resistance which is mostly difficult to detect using phenotypic and frontline genotypic methods [27–29, 31].

World Health Organization recommended the inclusion of high dose isoniazid in MDR TB regimen to increase the treatment success [33]. Although the clinical efficacy of high dose isoniazid is not fully understood [34, 35]. In our study, the MIC of isoniazid was moderate level (1μg/ml≥ MIC ≤4μg/ml) for just over half of all MDR TB isolates, which is consistent with other studies [36, 37]. We found that 95.6% of moderate level isoniazid resistant TB had mutations in the katG gene and only one isolate had mutations at the inhA gene. A previous study reports MDR TB patients with inhA mutation have MIC value ranges from 0.05–4μg/ml and that they had comparable early bactericidal activity as sensitive strains. The high dose treatment(10–15 mg/kg daily) procedure a similar early bactericidal activity for participant with inhA resistance TB compared to isoniazid sensitive TB participant who were treated with standard dose (5mg/kg daily) [26]. As noted, the treatment outcome of low to moderate level isoniazid resistance with high dose isoniazid treatment would be increased if the treatment is guided by MIC results [38, 39]. Our finding suggests a high proportion of katG harboring MDR TB patients could benefit from high dose INH if MIC testing is performed.

Limitations of our study include the following: First, the MIC was performed in selected isolates especially for INH resistance. Additionally, treatment data were not available precluding us from evaluating outcomes based on certain phenotypic and genetic resistance. Therefore, a large scale study that includes treatment outcome and adverse events would provide additional needed data to inform evidence-based guidelines on the use of rifabutin and high dose isoniazid in the treatment of MDR-TB.

Conclusions

We profile the MIC of rifampin, rifabutin and isoniazid using MYCOTB Sensititre™ MIC plate. Our findings suggest a potential role for rifabutin treatment in certain patients with RR TB and further studies are needed to explore the utility of providing a rifabutin for RR TB patients. Moreover, over half of MDR TB isolates had moderate level isoniazid resistant indicates the potential benefit of high dose isoniazid treatment in a high proportion of katG gene harboring MDR Mtb isolates if aligned with MIC values. Furthermore, additional studies on MIC testing are important to guiding management and allowing for personalized treatment in low-income countries like Ethiopia.

Subculture growth characteristics of multidrug resistant (MDR), rifampin resistant (RR) and isoniazid (INH) resistant Mycobacterium tuberculosis isolates in newly diagnosed and previously treated TB patients.

No growth indicates Mtb did not grow; not retrieved indicates the Mtb isolate could not be located and therefore, subculture was not performed; insufficient growth indicates that the colony count was between 3 and 10.

(DOCX)

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27 Apr 2022

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30 Jun 2022

Dear PLoS One,

We appreciate the time and effort of the editor and reviewers in providing a detailed review of our manuscript. We have provided a point-by-point response below to outline how we have addressed the editor and reviewers’ comments. We thank you for the consideration of our work,

Sincerely,

Muluwork Getahun on behalf of the study team

Response to Reviewers

Reviewer #1:

1. Comment: This article on "Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis isolates" was very well written, concise and easy to follow. In the context of Ethiopia, this type of study is highly significant. Since there was significant number of patients were identified with HIV positive, it was not discussed about the possible outcome/impact of HIV on MDR TB. However, please review the attached file for detailed comments.

Response: Thank you for this comment. To highlight the topics of HIV and MDR-TB we have made revisions in the revised manuscript as follows.

Page 9 line 187-189: “Among 48 MDR/RR Mtb isolates, 9 isolates (19%) were found to be rifabutin-susceptible (MIC≤0.5µg/ml), including 6 patients with a prior history of TB treatment and 4 from persons living with HIV.”

Page 10 line 214-215: “Rifabutin is considered as an alternative treatment option in place of rifampin in some persons with HIV and TB due to fewer drug-drug interactions with certain antiretroviral drugs.”

Page 10 line 226-227: “Another clinical trial indicates that TB-HIV co-infected patients who takes rifabutin twice a week had low relapse rate (1 of 20) compared to those on rifampin-based therapy (8 of 9) (Burman et al., 2006).”

Reviewer #2:

Major comments:

2. Comment: Study sample size: The manuscript provides drug susceptibility data for 74 samples isolated from 32 health facilities from different locations of Ethiopia. However, the given sample size is far too small for making a representation of the TB drug susceptibility profile of Ethiopia. The authors could consider increasing the sample size.

Response: We included all the available MDR/RR Mtb isolates, all previously treated isoniazid resistant Mtb and 40% of newly diagnosed isoniazid resistant Mtb isolates that were collected from the national drug resistance survey. The remaining 22 newly diagnosed isoniazid Mtb isolates did not undergo testing with MIC due to the shortage of MIC plates

3. Comment: Based on their observation, the authors recommend considering the use of rifabutin and high dose INH for the treatment of MDR-TB. It seems plausible. However, despite the available references on the use of rifabutin and high dose INH for treatment of MDR-TB, I feel that the sample size of the current study and the fraction (9 out of 48 MDR isolates, 19%) is yet very small to conclude this point.

Response: We agree with the reviewer’s comment. While our study highlights the potential role for rifabutin for MDR TB patients the small sample size precludes broad generalizability. Based on the reviewer’s comment and to make this point clear, we have added in the following sentence to the limitations paragraph in the revised manuscript.

Page 12 line 264-267: “Therefore, a large scale study that includes treatment outcome and adverse events would provide additional needed data to inform evidence-based guidelines on the use of rifabutin and high dose isoniazid in the treatment of MDR-TB. “

4. Comment: The title of the manuscript looks very general. I would suggest revising the manuscript title to include the specific geographical location that is the country name.

Response: Based on the reviewer’s comment, we have revised the title of the manuscript to include the location of the work. The new title of the manuscript (line 1, page 1) is “Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia”

Minor comments:

5. Comment. Add the full form of RFB in the first place

Response: Thank you for this comment. In response to this comment, we are now using “rifabutin” rather than RFB throughout the manuscript.

6. Comment L94: Underwent

Response: We have revised this sentence in the revised manuscript. Page 6 line 130: “All stored MDR/RR and INH-resistant Mtb isolates had LPA testing performed.”

7. Comment L100: Subcultured

Response: We have revised this sentence in the revised manuscript. Page 6 line 136-137: “After thawing frozen Mtb isolates, 100 µl of vortexed allocate was subcultured on to Middlebrook 7H11 agar medium.

Reviewer #3:

8. Comment: My major concern is the use of LPA which; by now have several modifications after 2011 for determining the resistance of drugs. However, authors use a latest version of LPA which was modified in 2017/2018 but I am not sure if there are additional guidelines by Hains in 2019 to calculate the resistance of these drugs? Author should clarify this and give a detail about it.

Response: Our study utilized the latest version of the Hains LPA test, and there have been no further modifications since that time.

9. Comment: What is the concentration used for sub-culturing these strains on 7H11 plates?

Response: We used 100 µl of vortexed Mtb isolates, and we have highlighted this in the manuscript as outlined below.

Page 6 line 136-137: “After thawing frozen Mtb isolates, 100 µl of vortexed allocate was subcultured on to Middlebrook 7H11 agar medium.”

10. Comment: What is the difference between no growth on subculture and not retrieved group?

Response: No growth indicates that the subculture was performed but Mtb did not grow. While, not retrieved indicates the isolates were not located and available and therefore, a subculture was not performed. We have made the following revision in the revised manuscript:

Page 26 line 499-501: “No growth indicates Mtb did not grow; not retrieved indicates the Mtb isolate could not be located and therefore, subculture was not performed; insufficient growth indicates that the colony count was between 3 and 10.”

11. Comment: I am curious to know if there are inoculum differences when the strains were scrapped off from the 7H11 plate to resuspend for McFarland standard? For example, how much cfu/ml would it be in the borderline strains?

Response: We used a 0.5 McFarland standard for all isolates which equates to CFU/ml of 1.5 x 108. This ensured that all inoculum sizes were the same.

12. Comment: The detection rate of different type of mutations, for example mutations at different SNP’s have authors considered for detection rate, when determining the type of mutation?

Response: We have outlined the mutations identified by LPA in Table 2 and have revised the following sentences:

Page 9 line 185-186: The MICs of Mtb isolates to rifabutin and the corresponding mutation profiles are summarized in Table 2. The identified mutation profiles are described under comment 14.

13. Comment: I understand the authors mentioned about the data regarding treatment is not accessible but important to know that the mutation outside Rifampicin determining region in those two borderline mutant strains is a result of treatment failure/lost follow up.

Response: The borderline mutant strains were present among patients with newly diagnosed INH-resistant TB. The following revision was made based on the reviewer’s comment:

Page 8 line 182-183: The Mtb isolates with borderline rifampin resistant were recovered from patients with newly diagnosed TB.

14. Comment: Is there any possibility of predicting that mutations will occur in which region and whether they are correlated with the MIC? Are there any models? Authors may give description about it.

Response: Mutations at codon 530-533 likely had a higher MIC to rifabutin. The information is described in the revised manuscript as follows:

Page 9 line 186-187: Sixty two percent of the mutations occurred at codon 530-533 and 93% of rifabutin resistant Mtb isolate had MIC ≥ 4µg/ml.

Page 9 line 190-192: Six (67%) of 9 rifampin-resistant and rifabutin-susceptible Mtb isolates were classified as rifampin resistance due to the absence of wild-type probes only.

15. Comment: Are the suspects in the newly diagnosed category have a known TST and IGRA status?

Response: Thank you for this comment. Unfortunately, we did not have this clinical information on TST and IGRA results. Additionally, it should be noted that the TST and IGRA are not routinely performed among persons with suspected TB in Ethiopia.

16. Comment: I am always skeptical about using high concentrations of drugs, authors mention the use high concentrations of INH for treatment. Unfortunately, mechanistically it remains poorly understood. Also, it is important to note that INH is poorly penetrant and its possible to have side effects and emergence of persisters later. However, I do not deny that there are studies done but those TB treated subjects I am not sure if they followed up which remains a cause of concern.

Response: We appreciate the reviewer’s comments and agree with this concern. Regardless of MIC testing results, high dose isoniazid is often part of an empiric MDR TB treatment regimen in low income countries such as Ethiopia. As noted, the treatment outcome of low to moderate level isoniazid resistance with high dose isoniazid treatment would be increased if the treatment is guided by MIC results (Moulding, 1981, Lange et al., 2014). Our study highlights the potential role of the use of high dose INH treatment in many patients with MDR TB if an MIC test guided approach is implemented.

Additionally, a large scale phase II clinical trial on “High-Dose Isoniazid Among Adult Patients With Different Genetic Genetic Variants of INH-Resistant Tuberculosis (TB)” would provide the needed evidence. (https://clinicaltrials.gov/ct2/show/NCT01936831?cond=isoniazid&draw=3&rank=24)

Based on the reviewer’s comment, the manuscript has been revised as follows:

Page 12 line 264-267: Therefore, a large scale study that includes treatment outcome and adverse events would provide additional needed data to inform evidence-based guidelines on the use of rifabutin and high dose isoniazid in the treatment of MDR-TB.

Submitted filename: Response to Reviewers.docx

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30 Aug 2022

Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

PONE-D-22-08463R1

Dear Dr. Getahun,

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Additional Editor Comments (optional):

Authors have included all the points in revised manuscript as suggested by reviewers! Such revision has been verified by original reviewers and suggested for acceptance of the current version, threfore this highly important research Article on MDR MTB isolates and rifamicin and isoniazid MIC determination , alternative treatment options for MDR TB be accepted for publication!

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: No

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #2: Yes: Jees Sebastian

Reviewer #3: No

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4 Sep 2022

PONE-D-22-08463R1

Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

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