Date:
Subject:
19 March 2010
Manuscript resubmisson (Manuscript ID 1828005310331098)
Dear Editor,
We thank you, the editorial board and the reviewers for critically appraising our manuscript entitled
“Validation of the GenoType® MTBDRplus assay for diagnosis of multidrug resistant tuberculosis
in South Vietnam”.
We revised our manuscript after carefully considering all comments. Please find attached our response to
both the concerns raised by the editorial board and by the two reviewers. We have addressed each item
carefully and are confident that our paper now meets the requirements.
We herewith resubmit our manuscript for publication as research paper in the BMC Infectious Diseases.
All authors have seen and approved the final version, and approved submission to the BMC Infectious
Diseases. None of the authors has any conflict of interest to disclose.
We hope that you will consider publication of this manuscript in your journal.
On behalf of the co-authors,
Yours sincerely,
Edine W. Tiemersma, PhD
Research Unit
KNCV Tuberculosis Foundation
PO Box 146
2501CC Den Haag
The Netherlands
Phone +31 70 427 09 60
Fax +31 70 358 40 04
E-mail: tiemersmae@kncvtbc.nl
Authors’ reply to comments of the reviewers
We have carefully considered all comments of the three reviewers and have incorporated these
wherever possible. Below, we specify our answers to specific comments and concerns raised by the
reviewers. Since the first referee had no comments or concerns to our manuscript, only those of
referees 2 and 3 are given here.
Reply to comments and concerns raised by Referee 2
Major Compulsory Revisions.
As this manuscript is focusing on the validation of GenoType(R) MTBDRplus assay, authors should
confirm the result by sequencing every sample. Authors need to show the concordance of the results of
GenoType(R) MTBDRplus assay and gene mutation of their samples in their laboratory setting.
We do not agree with the referee that we should have confirmed all results of the MTBDRplus assay by
sequencing. Rather than validating the test itself, which has been done in many published studies1, the
purpose of our study was to validate the test to diagnose MDR- and RIF-resistant TB in the Vietnamese
setting. This is relevant beyond the Vietnamese since the sensitivity of the assay depends on the
distribution of gene mutations in the bacterial population, and this may display geographic variation.
Since resistance to rifampicin (RIF) determines which treatment the patient will be allocated to (firstline or MDR-TB treatment), we considered achieving a high sensitivity and specificity of RIF resistance
of crucial importance. We do not see the need to confirm expected results by sequencing and therefore
we have only sequenced those isolates with repeatedly discordant results for RIF to check for
mutations that are not covered by the MTBDRplus assay.
Taking this comment from the reviewer into consideration we now more clearly state our purpose in
the Background of the manuscript (last paragraph):
“The National Tuberculosis Control Program of Vietnam intends to use this test in support of Programmatic
Management of DR-TB (PMDT) on sputum specimens of all MDR-TB suspects (i.e., those failing category 1
treatment and those being smear-positive after 3 months of category 2 treatment). The Genotype® MTBDRplus
assay will be used to select patients with rifampicin resistant isolates for PMDT. Therefore, we assessed the test’s
sensitivity and specificity in diagnosing MDR-TB at the laboratory of Pham Ngoc Thach Hospital (PNTH) using
a geographically representative set of M. tuberculosis isolates with known phenotypic resistance patterns from
the South of Vietnam.”
Reply to comments and concerns raised by Referee 3
Please use line numbers for future submissions.
Proposed revision has been incorporated.
In Abstract: Please describe the % of specificity & sensitivity.
These proportions were specified in the abstract, but we admit that this may have been a bit unclear
due to the wording. We propose to change the text of the abstract to:
“The sensitivity of the GenoType® MTBDRplus was 93.1% for rifampicin, 92.6% for isoniazid and 89% for the
combination of both; its specificity was 100%.”
In Background:
1. Epidemiology of TB in the country needs to be mentioned.
See Hilleman et al. Int J Tuberc Lung Dis 2005;9:1161–7; Hillemann et al. J Clin Microbiol 2005;43:3699–3703;
Brossier et al. J Clin Microbiol 2006;44:3659–64; Cavusoglu et al. J Clin Microbiol 2006;44:2338–42; Makinen et al.
J Clin Microbiol 2006;44:350-2; Miotto et al. J Clin Microbiol 2006;44:2485–2491 and J Clin Microbiol.
2008;46:393-4; Somoskovi et al. J Clin Microbiol 2006;44:4459–4463; Huang et al. J Clin Microbiol 2009;47:2520–
4.
1
Authors’ reply to comments of the reviewers
We have added a short paragraph describing the epidemiology of TB in (the South of) Vietnam (as 2nd
paragraph of the Background):
“Vietnam ranks 14th among the countries with the highest burden of TB and the incidence of TB is highest in the
southern part of the country (WHO 2009). In 2005, South Vietnam notified 29,789 new smear positive cases
yielding an estimated prevalence of 92.8 per 100,000 (NTP, unpublished data, 2006).”
2. Is the assay intended for screening of all suspected or only for high-risk TB and/or MDR-TB cases? If
the assay is for PMDT, please describe the drug-resistance rate of TB cases in the study setting.
In future, the MTBDRplus genotype assay will be used for PMDT to select those patients who need to
receive MDR-TB treatment, as is described in the last paragraph of the Background. All patients failing
category 1 treatment or being smear-positive after 3 months of category 2 treatment and chronic
patients (i.e. those not responding on category 2 treatment) are eligible. Two studies have reported
resistance rates among new and retreatment patients in the South of Vietnam. The first was conducted
in Ho Chi Minh City between 1998 and 2000. INH resistance was highly prevalent (25% among new
and 54% among previously treated patients), RIF resistance occurred at rates of 4% and 27%
respectively, whereas MDR-TB occurred in 4% and 25% of new and previously treated patients,
respectively (Quy et al., 2006), whereas MDR-TB among chronic patients was as high as 80% (Quy et
al., 2003). Another study, including patients from 40 different clinics in the south of Vietnam in 2001,
found slightly lower resistance rates (Huong et al., 2006).
Proposed changes to text (2nd paragraph of the Background):
“M. tuberculosis resistance to INH is common (16-25% among new patients) (Huong, Lan et al. 2006; Quy, Buu
et al. 2006). Among patients experiencing a first episode of TB these two studies reported MDR-TB rates of 2
and 4%, and of 23% and 27% among previously treated patients, respectively (Huong, Lan et al. 2006; Quy, Buu
et al. 2006), whereas 80% chronic patients had MDR-TB (Quy, Lan et al. 2003).”
We have made the purpose of testing this assay’s validity more explicit in the last paragraph of the
Background:
“The National Tuberculosis Control Program of Vietnam intends to use this test in support of Programmatic
Management of DR-TB (PMDT) on sputum specimens of all MDR-TB suspects (i.e., those failing category 1
treatment and those being smear-positive after 3 months of category 2 treatment). The Genotype® MTBDRplus
assay will be used to select patients with rifampicin resistant isolates for PMDT. Therefore, we assessed the test’s
sensitivity and specificity in diagnosing MDR-TB at the laboratory of Pham Ngoc Thach Hospital (PNTH) using
a geographically representative set of M. tuberculosis isolates with known phenotypic resistance patterns from
the South of Vietnam.”
In Methods
1. It is not clear how the study population was determined. Only limited samples were tested, it might
have some selection bias. Please clarify exactly how 111 isolates were selected from those 1,044
isolates. Are those 1,044 isolates mainly sampled from South Vietnam?
This is described in the first two paragraphs of the Methods section. We propose to clarify this by
adding the following text (underlined) to these paragraphs:
“Of these, 1,044 (57%) specimens were collected in the South of Vietnam and were tested in PNTH (910 from
new patients and 134 from previously treated patients). (…) Isolates from all 30 new and 29 re-treatment cases
that were either identified as MDR-TB (n=55) or resistant to rifampicin (n=4) by phenotypic DST were included
in this study. In addition, from the isolates that were susceptible to all tested first-line drugs from new and retreatment patients, we randomly selected 52 isolates so that the total number of tested strains was 111.”
Random selection was performed by ordering the pan-susceptible isolates (n=669) by study number
(in increasing order) and then taking each 12th isolate.
2. It is puzzling that there were no INH mono-resistant isolates. Is this the case? It might be helpful to
briefly state why there were no INH mono-resistant isolates included in the study.
Authors’ reply to comments of the reviewers
In fact, 4.6% of the isolates was resistant to INH only. Since we aimed to assess the test’s suitability
in terms of sensitivity and specificity for future use in the PMDT program, which selects patients based
on resistance to RIF (either to RIF alone or in combination with INH), we did not enrich the sample by
purposefully including INH mono-resistant isolates. This is now explained in the Background of the
manuscript (see also our answer under Background, comment 2).
3. Please describe methods for species identification (Page 6) and for NTM identification (Innolipa).
We added a paragraph on species identification to the Methods section:
“M. tuberculosis was identified on the basis of a positive niacine reaction (Kent & Kubica, 1985). Further species
identification was performed using Innolipa Mycobacteria v2 (Innogenetics, Gent, Belgium) according to the
manufacturer’s specifications if a discrepancy was found between initial species identification and results from
the MTBDRplus assay (i.e., no hybridization with the TUB band).”
4. The lab has the sequencing capacity for RIF resistant gene. Why authors did not perform INH
resistant-gene sequencing to resolve the discordant INH DST results?
The purpose of our study was to evaluate the sensitivity and the specificity of the GenoType®
MTBDRplus assay as compared to conventional DST, which is currently used in routine practice with
the ultimate aim to replace the currently used conventional DST with the rapid MTBDRplus assay for
screening for MDR-TB. Since resistance to rifampicin (RIF) alone determines which treatment the
patient will be allocated to (first-line or MDR-TB treatment), we considered achieving a high sensitivity
and specificity of RIF resistance of crucial importance. INH-resistance is of lesser importance in this
respect and we did not check for INH discordances.
In Results
1. Please define “WT”, “MUT”, “TUB” and “RRDR” in Methods.
Proposed revision has been incorporated in the manuscript.
“For each gene, the GenoType® MTBDRplus assay tests for presence of so-called wild-type (WT) and mutant
(MUT) probes, the first comprising the most important resistant areas of the respective genes and the second
some of the most common resistance mediating mutations. Next to that, the TUB zone hybridizes with amplicons
generated from all members of the Mycobacterium complex and can thus serve for species identification.”
2. Please omit MUT 2 before H526L. In the GenoType assay, MUT 2 only specifically includes MUT 2A
(H526Y) & 2B (H526D).
Proposed revision has been incorporated in the manuscript.
3. Please clearly state those 4 isolates RIF resistant or susceptible in the text (page 9, line 13).
These were phenotypically RIF resistant isolates, which is now clarified in the text.
4. The primary culture and DST were done by a national reference laboratory, PNTH, with an external
quality assurance system. The authors use re-cultured isolates for this study. Could it be a mixed
culture in the initial isolation, and NTM outgrew M. tuberculosis during subsequent subculture. What
was the clinical picture of that case?
There is always the possibility that the isolate contains a mixed culture, which is indicated in the text.
For the interest of the reviewer, we have looked up the clinical picture of this patient. Since a
description of this clinical picture in the text would deduct too much from the messages of our
manuscript, we have not included it in the text. The patient was a 59-year old man who was previously
treated for TB (approx. one year earlier) and was cured (i.e. smear-negative) after 8 months of
Authors’ reply to comments of the reviewers
standard NTP category 1 treatment. He returned to a district TB unit with hemoptysis and was found to
excrete AFB-positive bacilli by smear examination. After treatment with category 2 treatment, he was
cured.
5. Since the 1st-line DST result of MAIS was MDR, did authors exclude the isolate before performing
statistical analysis?
We did not exclude this isolate in the submitted manuscript because the isolate was originally found to
contain MDR M. tuberculosis based on presence of AFB-negative bacilli, a positive niacin test and DST.
We agree with the reviewer that we should exclude this isolate in any calculations comparing the
MTBDRplus assay with conventional DST. However, since re-culturing and subsequent spoligotyping of
several colonies indicated that one of the colonies originating from this isolate was an M. tuberculosis,
we included this isolate in Table 3 and in the section discussing the results of the mixed infection
analysis. The following changes are made to the results section to explain this; first paragraph:
“Of 111 isolates tested, there was one MDR strain that lacked the ‘TUB’ band. Although this strain was earlier
identified as M. tuberculosis, further species identification identified the strain as M. avium-intracellulare
(MAIS). Spoligotyping after re-culturing this isolate showed that it was a mixture of M. tuberculosis and MAIS.
Since the GenoType® MTBDRplus assay did not identify this strain as MDR-TB, all analyses describing the
assay’s performance include 110 isolates, of which 58 were resistant to rifampicin by conventional DST.”
Last paragraph:
“With this rapid assay four possible mixtures were detected, although one of these was not identified as M.
tuberculosis due to a lacking ‘TUB’ band by the assay and was later identified to contain MAIS. These four
phenotypically rifampicin resistant isolates were demonstrated to carry mutations in the rpoB gene and/or in the
katG gene or the inhA promoter region, but did not lack hybridization on any of the wild type probes. By using
DNA fingerprinting one of these isolates was confirmed to be a mixture of two MTB strains (spoligotype T1 and
an undefined type; RFLP type T1 and Beijing), and the isolate lacking the ‘TUB’ band (number 12647) was
identified as a mixture of a MTB strain (spoligotype U) and a non-tuberculous mycobacterium (note that
spoligotyping yielded a weak signal for M. tuberculosis for one single colony culture of this isolate) (Table 3,
Figure). In the two remaining samples mixed bacterial populations could not be detected. After spoligotyping
which revealed no differences as it has a very low resolution among Beijing strains, we also performed IS6110
RFLP typing on single colony cultures of each of the two samples and found they all had identical banding
patterns (Table 3).”
In the discussion (fore-last paragraph):
“The GenoType® MTBDRplus assay may not be sensitive enough to be used for species identification in case of
mixed bacterial populations, since the M. tuberculosis and M. avium mixture revealed no ‘TUB’ band.”
6. The strain number and % described for INH resistant TB did not match those in Table 3. It is not
easy to follow the descriptions in the first paragraph of Page 10. Rewrite is recommended.
We rewrote the paragraph as follows:
“Among 50 INH resistant TB strains as identified by the GenoType® MTBDRplus test, katG mutations occurred
in 43 (86%) and inhA mutations in 9 strains (18%). Two of the 43 (5%) strains with a katG codon 315 mutation
had an additional mutation in the inhA promotor region. The most frequently observed katG mutation was katG
S315T1 (in 38 of 43 strains, 90.5%), whereas katG S315T2 (2.3%) and unknown mutations (i.e., no hybridization
to the katG WT nor to either of the mutation probes, 9.3%) occurred less frequently. All 9 strains with a mutation
in the inhA promotor region had a inhA C15T mutation (table 3).”
7. For this evaluation, it is more important to determine the mixed culture of resistant and susceptible
isolates rather than mixed genotypes. Isolates with different genotypes could harbour the same drugresistant mutations. Genotyping (spoligotyping & RFLP used in this study) alone was not able to find
out a mixed culture of resistant and susceptible isolates with identical genotypes. For those two
isolates, authors could try to use other molecular methods.
Authors’ reply to comments of the reviewers
Indeed, there is a theoretical possibility of mixed cultures of resistant and susceptible strains despite
identical spoligo- and RFLP-types. However, this possibility is remote: approximately 35% of the
strains circulating in this part of Vietnam are of the Beijing genotype, 50% are of the East-AfricanIndian (EAI) genotype and 15% of remaining genotypes (Buu et al, Int J Tuberc Lung Dis 2009; Buu et
al, Emerg Infect Dis 2009). While the Beijing strains have identical spoligo-patterns, they display
marked variation in RFLP patterns; the EAI genotypes indeed share the same RFLP pattern but display
at least 3 distinguishable spoligo-patterns (Buu et al, unpublished data). In addition, the Beijing strains
are markedly more likely to be multidrug resistant than the EAI strains (Buu et al, Int J Tuberc Lung
Dis 2009). Therefore the probability of mixed infection with a multidrug resistant strain and a RIF/INH
susceptible strain of identical spoligo and RFLP pattern is very small. We therefore propose not to
include a discussion on this issue.
8. Need to define how spoligotypes were designated.
We added the following text to the Methods section (including references):
“The Beijing genotype was defined by spoligotyping as any isolate without Direct Repeat spacers 1–34 and the
presence of ≥3 of the spacers 35–43 (Kremer et al., J Clin Microbiol 2004). Other genotypes were defined as
described by Brudey et al (BMC Microbiol 2006), including the Vietnam genotype (EAI-VNM) that belongs to
the East African Indian genotype family of M. tuberculosis and is the most frequent genotype in this study site
(Buu et al., Int J Tuberc Lung Dis 2009).”
In Discussions
1. The sensitivity of this assay for INH and MDR determined in this study is higher than a few recent
studies (JCM 2008, Vol 46, 3426-3428; JCM 2009, Vol 47, 2520-2524).
Akpaka et al. (J Clin Microbiol 2008) indeed report a low sensitivity of the MTBDRplus assay for INHresistant (34.6%) and MDR-TB (29.4%). Although it does not become clear from the paper on which
number of isolates these proportions are calculated, these sensitivities are probably significantly lower
than those reported in our paper. As the authors suggest in their paper, INH mutations occurring in the
Caribbean region may not sufficiently be covered by the MTBDRplus test. Another possible explanation
that the authors mention was the DST method used for INH resistance identification. The sensitivities
reported by Huang et al (J Clin Microbiol 2009) are a bit lower than we report, but the confidence
intervals that we could calculate from their data overlap with ours and thus show no statistical
differences (see table below).
Sensitivity
Isoniazid
% (95% CI)
MDR
% (95% CI)
our study (N=55 MDR isolates)
92.6% (82.1-97.9)
88.9% (77.4-95.8)
Huang et al., J Clin Microbiol 2009
(N=242 MDR isolates)
81.8% (76.4-86.5)
78.5% (72.8-83.5)
We have included these two papers in our discussion.
2. A brief explanation of the significance of the major genotypes of M. tuberculosis in the region might
be helpful, e.g. drug susceptibility patterns, unique resistance mutations.
We added the following paragraph to the Background section of the manuscript:
“Genetically, approximately half of the strains belongs to the East-African Indian clade whereas the other half are
of the Beijing genotype, which was found to be strongly associated with (multi-)drug resistance (Anh et al,
Emerg Infect Dis 2000; Buu et al, Int J Tuberc Lung Dis 2009).”
In Conclusion
Authors’ reply to comments of the reviewers
The impact of implementing this rapid assay for the local TB control program was not clearly described.
Is this assay intended to be used for any suspected TB or MDR-TB cases?
As specified above, this assay will be used for any TB patient suspected of MDR-TB, i.e. those failing
category 1 treatment (still being smear-positive at the end of the treatment) and those being smearpositive after 3 months of category 2 treatment. A PMDT program has just started in the hospital and
all patients being found with MDR-TB will be put on MDR-TB treatment based on the results of the
MTBDRplus test. The impact of implementation of this rapid test will be studied in a separate
demonstration project, in which the yield of the test in terms of time and cost saved (both on the side
of the patient and the hospital) will be taken into account.
Tables
1. Table 1 & 2 could be omitted. The contents could be described in the text.
We agree that Table 1 is a bit redundant and therefore deleted it from the manuscript. Although the
contents of Table 2 can indeed be captured in the text, we propose to keep this table in the
manuscript, as it gives a quick overview of the key results of the study, including 95% confidence
intervals.
2. Table 3, please try to modify the Table format. It looks like a raw data set. In this study, 55 MDR
isolates were analyzed; however, there were 57 in Table 3.
The difference in numbers follows from Table 1 and the first paragraph in the Results section. Indeed,
55 isolates that had been identified by conventional DST to contain MDR-TB were analyzed (of which
one turned out to be a non-TB isolate) supplemented with 4 RIF mono-resistant isolates, thus including
a total of 59 isolates with any resistance. The MTBDRplus identified 57 isolates with either resistance to
RIF, INH, or both, of which 1 was subsequently identified as MAIS (no hybridization with the TUBband) and was excluded for further analysis. Thus, 56 strains are presented in Table 2 (formerly Table
3). We re-arranged the Table as follows:
Table 2. Mutation patterns following from the GenoType ® MTBDRplus assay
rpoB mutations
D516V
D516V
D516V
H526D
H526Y
S531L
S531L
S531L
S531L
S531L
S531L
H526Y + S531L
unknown *
unknown *
unknown *
unknown *
---
katG mutations
inhA mutations
Frequency
Proportion
S315T1
-1
0.9
unknown *
-1
0.9
-C15T
1
0.9
S315T1
-1
0.9
S315T1
-5
4.6
unknown *
-2
1.8
S315T1
C15T
1
0.9
S315T1
-16
14.6
S315T2
-1
0.9
-C15T
4
3.6
--2
1.8
--1
0.9
S315T1
-13
11.8
--3
1.8
unknown *
C15T
1
0.9
-C15T
1
0.9
S315T1
-1
0.9
-C15T
1
0.9
Total number of strains with any mutations
56
50.9
---54
49.1
Total number of strains
110
100
*unknown mutation: no hybridization to one or more of the wildtype probes nor to any of the mutation probes
Authors’ reply to comments of the reviewers
3. Table 4, if “TUB” is negative for the strain 12467, according to manufacture’s manual, it cannot be
evaluated by this test system. Therefore, it makes no sense to list GenoType results in Table 4. In fact,
The GenoType assay is a PCR-based assay, TUB zone can still be positive for a sample with mixed
culture of M. tuberculosis and NTM.
We agree with the reviewer and therefore omitted this strain from all analyses in the Results section
and in the Table.
Figure
In Page 24, please revise NMT to NTM. The quality of the figure is too poor to see.
We have now added a figure with improved quality and have changed NMT to NTM.