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BMC Infectious Diseases logoLink to BMC Infectious Diseases
. 2014 Feb 1;14:54. doi: 10.1186/1471-2334-14-54

Comparative evaluation of three immunochromatographic identification tests for culture confirmation of Mycobacterium tuberculosis complex

Kinuyo Chikamatsu 1,, Akio Aono 1, Hiroyuki Yamada 1, Tetsuhiro Sugamoto 1, Tomoko Kato 1,2, Yuko Kazumi 1, Kiyoko Tamai 3, Hideji Yanagisawa 3, Satoshi Mitarai 1,2
PMCID: PMC3916065  PMID: 24484470

Abstract

Background

The rapid identification of acid-fast bacilli recovered from patient specimens as Mycobacterium tuberculosis complex (MTC) is critically important for accurate diagnosis and treatment. A thin-layer immunochromatographic (TLC) assay using anti-MPB64 or anti-MPT64 monoclonal antibodies was developed to discriminate between MTC and non-tuberculosis mycobacteria (NTM). Capilia TB-Neo, which is the improved version of Capilia TB, is recently developed and needs to be evaluated.

Methods

Capilia TB-Neo was evaluated by using reference strains including 96 Mycobacterium species (4 MTC and 92 NTM) and 3 other bacterial genera, and clinical isolates (500 MTC and 90 NTM isolates). M. tuberculosis isolates tested negative by Capilia TB-Neo were sequenced for mpt64 gene.

Results

Capilia TB-Neo showed 100% agreement to a subset of reference strains. Non-specific reaction to M. marinum was not observed. The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% (99.6% for M. tuberculosis, excluding M. bovis BCG) for clinical MTC isolates and 100% for NTM isolates tested, respectively. Two M. tuberculosis isolates tested negative by Capilia TB-Neo: one harbored a 63-bp deletion in the mpt64 gene and the other possessed a 3,659-bp deletion from Rv1977 to Rv1981c, a region including the entire mpt64 gene.

Conclusions

Capilia TB-Neo is a simple, rapid and highly sensitive test for identifying MTC, and showed better specificity than Capilia TB. However, Capilia TB-Neo still showed false-negative results with mpt64 mutations. The limitation should be recognized for clinical use.

Keywords: Capilia TB-Neo, Mycobacterium tuberculosis complex identification, mpt64 gene

Background

Tuberculosis remains a major threat to global health, and therefore, rapid identification of the causative M. tuberculosis complex (MTC) is critical. Liquid culture detection is now widely used for managing HIV-co-infected and drug-resistant tuberculosis, and liquid culture can improve the recovery of acid-fast bacilli and decreases the time to detection. However, because other non-tuberculosis mycobacterium (NTM) species may also grow, it is important to identify MTC from positive culture for rapid and appropriate management of tuberculosis.

Several methods are available to identify mycobacteria. Conventional biochemical tests are generally time-consuming [1,2] and not surely reproducible, while more recently developed techniques involving molecular biology [3-7] or high-performance liquid chromatographic analysis of mycolic acid [8] are accurate and rapid, but these require expensive devices. In contrast, immunochromatographic species identification tests, Capilia TB (TAUNS, Izunokuni, Japan), SD BIOLINE TB Ag MPT64 rapid (Standard Diagnostics, Inc., Korea) and BD MGIT™ TBc Identification Test (Becton, Dickinson and Company, USA) have been adopted as a cheap, rapid, and accurate alternative in clinical laboratories around the world [9-11]. However, false positives to Mycobacterium marinum, Staphylococcus aureus[9,12], and false negatives in MPB64 mutants [10,13] have occasionally been reported. Capilia TB-Neo (TAUNS, Izunokuni, Japan), an improved version of Capilia TB, has recently been developed to overcome these problems. In this study, we evaluated the performance of Capilia TB-Neo with reference strains and clinical isolates. Any false-negative MTC clinical isolate detected by Capilia TB-Neo were further investigated relative genes.

Methods

Reference strains and clinical isolates

Reference strains of 96 Mycobacterium species and subspecies (4 MTC and 92 NTM) and 3 other genera with acid-fastness (Nocardia asteroids, Rhodococcus equi and Rhodococcus aichiense) were used for the evaluation (Table 1). A total of 500 MTC and 90 NTM clinical isolates (10 M. abscessus, 4 M. chelonae, 13 M. fortuitum, 8 M. gordonae, 15 M. avium complex, 7 M. intracellulare, 3 M. nonchromogenicum, 5 M. scrofulaceum, 4 M. xenopi, 15 M. kansasii, 1 M. gastri, 2 M. peregrinum, 1 M. intermedium, 1 M. szulgai, and 1 M. marinum) were selected to provide a representative sample of the isolates available from Miroku Medical Laboratory Co., Ltd. (Saku, Japan) from 2009 to 2010, and the collection from the Ryoken survey in 2002 and 2007. The clinical isolates were collected from patients as a part of routine examination. No ethical approval was required for this type of laboratory based study only using isolates. Reference strains and clinical isolates were cultured with OADC-supplemented Middlebrook 7H9 broth (Becton, Dickinson and Company, USA) and 2% Ogawa medium (Kyokuto Pharmaceutical Industrial Co., Japan) at 37°C or 30°C.

Table 1.

List of reference strains and the results of identification of MTC by using Capilia TB-Neo, SD MPT64, and TBc ID

Species Strain Capilia TB-Neo SD MPT64 TBc ID Species Strain Capilia TB-Neo SD MPT64 TBc ID
M. tuberculosis H37Rv
ATCC27294
+
+
+
M. interjectum
ATCC51457
-
-
-
M. africanum
ATCC25420
+
+
+
M. intermedium
ATCC51848
-
-
-
M. bovis
ATCC19210
+
+
+
M. intracellulare
ATCC13950
-
-
-
M. microti
ATCC19422
+
+
+
M. kansasii
ATCC12478
-
-
-
M. abscessus
ATCC19977
-
-
-
M. kubicae
ATCC700732
-
-
-
M. acapulcensis
ATCC14473
-
-
-
M. lactis
ATCC27356
-
-
-
M. agri
ATCC27406
-
-
-
M. lentiflavum
ATCC51985
-
-
-
M. aichiense
ATCC27280
-
-
+
M. madagascariense
ATCC49865
-
-
-
M. alvei
ATCC51304
-
-
-
M. malmoense
ATCC29571
-
-
-
M. asiaticum
ATCC25276
-
-
-
M. marinum
ATCC00927
-
-
+
M. aurum
ATCC23366
-
-
-
M. moriokaense
ATCC43059
-
-
-
M. austroafricanum
ATCC33464
-
-
-
M. mucogenicum
ATCC49650
-
-
-
M. avium subsp. avium
ATCC25291
-
-
-
M. neoaurum
ATCC25795
-
-
-
M. avium subsp. paratuberculosis
ATCC19698
-
-
-
M. nonchromogenicum
ATCC19530
-
-
-
M. avium subsp. “suis”
ATCC19978
-
-
-
M. novum
ATCC19619
-
-
-
M. avium subsp. silvaticum
ATCC49884
-
-
-
M. obuense
ATCC27023
-
-
-
M. branderi
ATCC51789
-
-
-
M. paraffinicum
ATCC12670
-
-
-
M. brumae
ATCC51384
-
+
-
M. parafortuitum
ATCC19686
-
-
-
M. celatum
ATCC51131
-
-
-
M. peregrinum
ATCC14467
-
-
-
M. celatum II
ATCC51130
-
-
-
M. petroleophilum
ATCC21497
-
-
-
M. chelonae chemovar niacinogenes
ATCC35750
-
-
-
M. phlei
ATCC11758
-
-
-
M. chelonae subsp. chelonae
ATCC35752
-
-
-
M. porcinum
ATCC33776
-
-
-
M. chitae
ATCC19627
-
-
+
M. poriferae
ATCC35087
-
-
-
M. chlorophenolicum
ATCC49826
-
-
-
M. pulveris
ATCC35154
-
-
-
M. chubuense
ATCC27278
-
-
-
M. rhodesiae
ATCC27024
-
-
-
M. confluentis
ATCC49920
-
-
-
M. scrofulaceum
ATCC19981
-
-
-
M. conspicuum
ATCC700090
-
-
-
M. senegalense
ATCC35796
-
-
-
M. cookii
ATCC49103
-
-
-
M. septicum
ATCC700731
-
-
-
M. diernhoferi
ATCC19340
-
-
-
M. shimoidei
ATCC27962
-
-
-
M. duvalii
ATCC43910
-
-
-
M. shinshuense
ATCC33728
-
-
-
M. engbaekii
ATCC27353
-
-
-
M. simiae
ATCC25275
-
-
-
M. flavescens
ATCC14474
-
-
-
M. smegmatis
ATCC19420
-
-
-
M. fortuitum subsp. acetamidolyticum
ATCC35931
-
-
-
M. smegmatis
ATCC700084
-
-
-
M. fortuitum subsp. fortuitum
ATCC06841
-
-
-
M. sphagni
ATCC33027
-
-
-
M. fortuitum subsp. fortuitum
ATCC49403
-
-
-
M. szulgai
ATCC35799
-
-
-
M. gadium
ATCC27726
-
-
+
M. terrae
ATCC15755
-
-
-
M. gallinarum
ATCC19710
-
-
-
M. terrae
DSMZ43540
-
-
-
M. genavense
ATCC51234
-
-
-
M. terrae
DSMZ43541
-
-
-
M. gilvum
ATCC43909
-
-
-
M. terrae
DSMZ43542
-
-
-
M. goodii
ATCC700504
-
-
-
M. thermoresistibile
ATCC19527
-
-
-
M. gordonae
ATCC14470
-
-
-
M. tokaiense
ATCC27282
-
-
-
M. gordonae group B 19
KK33-08
-
-
-
M. triplex
ATCC700071
-
-
-
M. gordonae group C 19
KK33-53
-
-
-
M. triviale
ATCC23292
-
-
-
M. gordonae group D 19
KK33-46
-
-
-
M. vaccae
ATCC15483
-
-
-
M. haemophilum
ATCC29548
-
-
-
M. valentiae
ATCC29356
-
-
-
M. hassiacum
ATCC700660
-
-
-
M. wolinskyi
ATCC700010
-
-
-
M. heckeshornense
DSMZ44428
-
-
-
M. xenopi
ATCC19250
-
-
-
M. heidelbergense
ATCC51253
-
-
-
Nocardia asteroides
ATCC19247
-
-
-
M. hiberniae
ATCC49874
-
-
-
Rhodococcus equi
ATCC6939
-
-
-
          Rhodococcus aichiense ATCC33611 - - -

Identification of mycobacteria

Mycobacterium species of the clinical isolates were identified using one or more of the following approaches: (i) the DNA or RNA amplification kits Cobas Amplicor PCR (Roche Diagnostics, Japan) and TRC Rapid (Tosoh Bioscience, Japan); (ii) the DNA-DNA hybridization DDH Mycobacteria Kit (Kyokuto Pharmaceutical Industrial Co., Japan); and (iii) 16S rRNA gene sequencing, supplementary [7]. The isolates identified as MTC were further examined by multiplex PCR analysis of cfp32, the region of difference (RD) 9, and RD12 according to the method of Nakajima et al. [14]. When MTC species other than M. tuberculosis sensu stricto were detected, they were further characterized with respect to RD1, RD4, RD7, and MiD3 [15]. If M. bovis Bacillus Calmette-Guerin (BCG) was identified, additional multiplex PCR analyses were performed to test for RD2, RD14, RD15, RD16, and SenX3-RegX3 to distinguish sub-strains of BCG [16,17]. The multiplex PCR amplification was performed using a Type-it Microsatellite PCR Kit (QIAGEN, Japan). Each PCR reaction contained 1.0 μl of DNA template, 6.25 μl of Type-it multiplex PCR Master mix, 1.25 μl of Q-solution, 0.25 μl of each primer (10 pmol/μl) and an appropriate amount of molecular grade water for a total reaction volume of 13 μl. The thermal profile was as follows: (i) 95°C (5 min); (ii) 28 cycles of 95°C (0.5 min), 58 or 55°C (1.5 min), 72°C (0.5 min); and (iii) a final extension step at 68 or 60°C (10 or 30 min). The amplified products were analyzed by 3% agarose gel electrophoresis. The expected RD loci for each MTC species are summarized in Table 2.

Table 2.

Oligonucleotide primers used in PCR and direct sequencing

Target gene Primer ID Nucleotide sequence (5'-3') Size (bp) Ref. no.
MTC identification
 
 
 
 
 16S rRNA
285
GAGAGTTTGATCCTGGCTCAG
1028
7
 
264
TGCACACAGGCCACAAGGGA
 
 
 
259
TTTCACGAACAACG GACAA
591
 
cfp32
Rv0577F
ATGCCCAAGAGAAGCGAATACAGGCAA
786
14
 
Rv0577R
CTATTGCTGCGGTGCGGGCTTCAA
 
 
 RD9
Rv2073cF
TCGCCGCTGCCAGATGAGTC
600
14
 
Rv2073cR
TTTGGGAGCCGCCGGTGGTGATGA
 
 
 RD12
Rv3120F
GTCGGCGATAGACCATGAGTCCGTCTCCAT
404
14
 
Rv3120R
GCGAAAAGTGGGCGGATGCCAG
 
 
 RD1
ET1
AAGCGGTTGCCGCCGACCGACC
 
15
 
ET2
CTGGCTATATTCCTGGGCCCGG
 
 
 
ET3
GAGGCGATCTGGCGGTTTGGGG
 
 
 RD4
Rv1510F
GTGCGCTCCACCCAAATAGTTGC
1033
15
 
Rv1510R
TGTCGACCTGGGGCACAAATCAGTC
 
 
 RD7
Rv1970F
GCGCAGCTGCCGGATGTCAAC
1116
15
 
Rv1970R
CGCCGGCAGCCTCACGAAATG
 
 
 MiD3
IS1561F
GCTGGGTGGGCCCTGGAATACGTGAACTCT
530
15
 
IS1561R
AACTGCTCACCCTGGCCACCACCATGGACT
 
 
Distinguish sub-strains of BCG
 
 
 
 RD2
RD2l
CCAGATTCAAATGTCCGACC
 
16
 
RD2r
GTGTCATAGGTGATTGGCTT
 
 
 RD14
RD14l
CAGGGTTGAAGGAATGCGTGTC
 
16
 
RD14r
CTGGTACACCTGGGGAATCTGG
 
 
 RD15
RD8l
ACTCCTAGCTTTGCTGTGCGCT
 
16
 
RD8r
GTACTGCGGGATTTGCAGGTTC
 
 
 RD16
RD16nf
ACATTGGGAAATCGCTGCTGTTG
 
17
 
RD16nr
GGCTGGTGTTTCGTCACTTC
 
 
SenX3-RegX3
C3
GCGCGAGAGCCCGAACTGC
 
16
 
C5
GCGCAGCAGAAACGTCAGC
 
 
Sequencing
 
 
 
 
mpt64 (Rv1980c)
mpb64W-F
ACTCAGATATCGCGGCAATC
1061
this study
 
mpb64W-R
CGATCACCTCACCTGGAGTT
 
 
 Rv1977
Rv1977F
GTTTCCCGAGATCAGCTCAA
348
this study
 
Rv1977R
ATCTCGTCGTGTGTCACCAG
 
 
 Rv1981c
Rv1981F
GATCGAATGCAGGCTGGTAT
399
this study
  Rv1981R ACTACTACCGCGGTGACGAC    

Capilia TB-Neo, SD MPT64, and TBc ID

The validation of Capilia TB-Neo (TAUNS, Izunokuni, Japan) was conducted using the aforementioned reference strains as well as MTC and NTM clinical isolates. In addition, SD BIOLINE TB Ag MPT64 rapid (SD MPT64: Standard Diagnostics, Inc. Korea) and BD MGIT™ TBc Identification Test (TBc ID: Becton, Dickinson and Company, USA), detect MPT64 which is the same as MPB64, were tested using reference strains and NTM clinical isolates. Each test was performed according to the manufacturer’s instructions. Briefly, clinical isolates growing on Ogawa medium were suspended in 1 ml of sterile distilled water, and the suspension subjected to the test. Similarly, positive liquid cultures of reference strains (McFarland No. 1 to 2) were directly subjected to each test. Positive test results were indicated by a red line in the test area after 15 min.

Sequencing of the mpt64 gene

Any false-negative M. tuberculosis isolate detected by Capilia TB-Neo was further analyzed by sequencing mpt64 and surrounding genes by using the primers listed in Table 2. Each PCR reaction contained 1.0 μl of DNA template, 12.5 μl of Type-it multiplex PCR Master mix, 2.5 μl of Q-solution, 0.5 μl of each primer (10 pmol/μl) and an appropriate amount of molecular grade water for a total reaction volume of 25 μl. The thermal profile was as follows: (i) 95°C (5 min); (ii) 30 cycles of 95°C (0.5 min), 62°C (1.5 min), 72°C (1 min); and (iii) final extension at 60°C (10 min). The amplified product was analyzed by 3% agarose gel electrophoresis and was purified using Mag Extractor (TOYOBO, Japan). The purified DNA products were subjected to direct sequencing using an ABI 377 automatic sequencer (Applied Biosystems, USA) and BigDye Terminator Cycle Sequencing v 3.1 (Applied Biosystems, USA), according to the manufacturer’s instructions. DNA sequences of mpt64 from each isolate were compared with M. tuberculosis H37Rv by using Genetyx-win ver. 5.2 (Genetyx Co., Japan).

Results

Each of the three kits (Capilia TB-Neo, SD MPT64, and TBc ID) was tested using the 99 reference strains. Capilia TB-Neo correctly produced positive results for four MTC (M. tuberculosis, M. africanum, M. bovis, and M. microti) and negative results for 92 NTM and 3 non-mycobacterial species (other genera) with acid-fastness, while SD MPT64 and TBc ID generated several false positives (Table 1). The sensitivity and specificity of Capilia TB-Neo to reference strains were 100%.

Of the 500 MTC clinical isolates tested, 497 were identified as MTC by Capilia TB-Neo. The other 3 isolates that tested negative by using Capilia TB-Neo also tested negative by using SD MPT64 and TBc ID. All three kits produced negative results for all 90 NTM clinical isolates examined. Thus, The sensitivity and specificity of Capilia TB-Neo to the clinical isolates were 99.4% and 100%, respectively.

The multiplex PCR system identified 492 M. tuberculosis isolates out of 500. Five isolates, which were cfp32-, RD9-, RD4-, RD7-, and MiD3-positive, but RD12-negative, were initially identified as M. canettii. However, colonies of these isolates showed a consistent rough surface on solid medium, and subsequent sequencing of hsp65 indicated that the isolates had the genotype of M. tuberculosis sensu stricto (data not shown). These isolates were collected from different areas of Japan. Consequently, 497 isolates were identified as M. tuberculosis. The remaining 3 isolates were deficient in RD1, RD4, RD7, RD9, and RD12, and therefore were identified as M. bovis BCG. Two of these isolates were confirmed as M. bovis BCG Tokyo based on the unique size of RD16, and the third isolate had the same RD pattern as BCG Connaught and BCG Montreal, as for RD2, RD14, RD15, RD16 and SenX3-RegX3 (Figure 1). Among the 3 MTC isolates that tested negative by Capilia TB-Neo, 2 isolates were M. tuberculosis and the other was M. bovis BCG Connaught or BCG Montreal (Table 3).

Figure 1.

Figure 1

Multiplex PCR analysis of Mycobacterium bovis BCG sub-strains and clinical isolates. 1: BCG Pasteur (ATCC35734), 2: BCG Glaxo (ATCC35741), 3: BCG Copenhagen (ATCC27290), 4: BCG Russian (ATCC35740), 5: BCG Montreal (ATCC35735), 6: BCG Connaught (ATCC35745), 7: BCG Danish, 8: BCG Tokyo, 9: Sample 421, 10: Sample 467, 11: Sample 475, M: Size marker.

Table 3.

Results of PCR detection and Capilia TB-Neo of MTC with clinical isolates

Species interpretation (Number of isolates)
Banding pattern
Capilia TB-Neo
%
  cfp32 RD9 RD12 RD4 RD7 MiD3 RD1    
M. tuberculosis (490)
+
+
+
NT
NT
NT
NT
+
98.0
M. tuberculosis (2)
+
+
+
NT
NT
NT
NT
-
0.4
"M. canettii" (5)a
+
+
-
+
+
+
+
+
1.0
M. bovis BCG Tokyo (2)b
+
-
-
-
-
+
-
+
0.4
M. bovis BCG Connaught (1)c + - - - - + - - 0.2

aConfirmed to be M. tuberculosis by hsp65  sequencing and morphology, bConfirmed by contracted RD16, cConfirmed by absence of RD2 and RD15, and contracted SenX3-RegX3, NT: Not tested.

Mutations in the mpt64 gene were detected by sequencing two M. tuberculosis isolates with negative results by Capilia TB-Neo. One isolate had a deletion of 63 bp from nucleotides 196 to 258 (amino acids position 43 to 63), and the other had a deletion of 3,659 bp from nucleotide 874 in Rv1977 to nucleotide 905 in Rv1981c, which included the whole mpt64 gene.

Discussion

In many industrialized countries, the ability to rapidly distinguish between MTC and NTM is critical in clinical practice. Indeed, the anti-tuberculosis drug resistance survey in Japan revealed that 19.3% of all clinical mycobacterial isolates are NTM [18], underscoring the importance of rapid and accurate detection of MTC from acid-fast bacillus-positive culture. The immunochromatographic assay kit for the identification of MTC is now widely used in many countries. Capilia TB-Neo is the improved version of Capilia TB, and has been subjected to few clinical evaluations. Here, we report good overall performance of the kit but with several limitations.

In this study, the sensitivity of Capilia TB-Neo was 99.4% to clinical MTC isolates or 99.6% excluding M. bovis BCG, while the specificity of the kit tested to clinical NTM isolates was 100%. However, the isolation of BCG could present a practical problem. The M. bovis BCG Tokyo strain is sporadically isolated in Japan as a complex of vaccination or bladder cancer therapy, and is identified as MTC with the kit [19]. Some BCG strains such as Connaught, Pasteur, and Tice lack RD2 including the mpt64 gene, but RD2 is conserved in others such as Tokyo, Moreau, and Russia [16]. This issue should be properly addressed to avoid confusion. Although it is difficult to discriminate BCG Tokyo from MTC with mpt64/mpb64, their differentiation would be an important advance in the development of a future TLC product. The weak false-positive reaction to M. marinum that was reported using Capilia TB [12] was not observed in this study, and resulted in better specificity. The minimum detection concentration of M. tuberculosis for Capilia TB-Neo was 105 CFU/ml (data not shown), which was one-tenth than that for the previous kit. There was a report that Cpilia TB-Neo was higher sensitivity than Capilia TB [20]. In summary, the overall performance of Capilia TB-Neo was better than Capilia TB in both sensitivity and specificity.

SD MPT64 and TBc ID were also tested with reference strains. Both SD MPT64 and TBc ID showed false-positive results against several NTM strains in this study. Kodama et al. [12] reported that no M. marinum strains grown on 2% Ogawa medium tested positive by using the Capilia TB, while all strains grown on 3 kinds of liquid medium, MGIT (Becton Dickinson, Japan), KRD medium (Japan BCG Laboratory, Japan) and Myco Acid (Kyokuto Pharmaceutical Industrial Co. Ltd., Japan), eventually displayed a positive reaction that intensified with time. Kodama et al. speculated that nonspecific antigen which could make complex with anti-MPB64 antibody may be produced in liquid mediums, but not on solid medium. Considering the effect of liquid culture, the original bacterial suspensions giving false-positive results, that were prepared from liquid and solid culture, were then re-tested before and after 10-fold dilution. Interestingly, none of these diluted strains tested positive in these kits, but bacterial concentrations were high enough for positive results in case of MTC. These results implied that a high concentration of bacterial antigens could induce non-specific reactions in SD MPT64 and TBc ID. The manufacturer’s instructions for the TBc ID indicate that this kit may be used up to 10 days after a positive MGIT alarm. This non-specific reaction should be properly addressed in clinical practice, and the users should perform morphological characterization with a microscope to identify cord formation.

Several mutations in the mpt64 gene produce a negative test result for M. tuberculosis isolates in the TLC assay using anti-MPB64 monoclonal antibodies. To date, these include a 63-bp deletion from nucleotide 196, a 1-bp deletion from nucleotide 266, a point mutation at position 388 or 402, IS6110 insertion mutation at position 177 or 501, a 176-bp deletion from nucleotide 512, and a 1-bp insertion at position 287 [10,13,21]. In our study, 2 M. tuberculosis isolates gave false-negative results by using the Capilia TB-Neo, SD MPT64, and TBc ID. One isolate had a deletion of 63 bp from nucleotide 196 in the mpt64 gene as reported previously, and the other isolate possessed a 3,659-bp deletion from nucleotide 874 in Rv1977 to 905 in Rv1981c, including the whole mpt64 gene. To the best of our knowledge, this is the first report of a large deletion in mpt64. A transposon site hybridization (TraSH) study [22] indicated that mpt64 is not essential for infection or in vitro growth of M. tuberculosis. This large deletion mutant supported the finding.

In summary, the TLC assay detecting MPB64 or MPT64 can be applied to specimens prepared from liquid and solid culture. It does not need special reagents, instruments, or complex techniques. Capilia TB-Neo tested in this study showed excellent sensitivity with perfect specificity.

Conclusions

Capilia TB-Neo showed high sensitivity and specificity with clinical mycobacterial isolates, and 100% specificity to reference strains. However, 2 M. tuberculosis isolates were tested negative by Capilia TB-Neo because of mutations in the mpt64 gene, and positive to certain BCG sub-strain. This study, therefore, serves to emphasize the importance of careful use of the kit and the complementary techniques such as morphological identification.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

KC carried out the TLC assays, molecular genetic studies, sequence alignment and drafted the manuscript. AA, HY, TS, and TK cultured clinical isolates. HY helped to draft the manuscript. YK prepared reference strains. KT and HY collected clinical isolates. SM was responsible for planning the study. All authors read and approved the final manuscript.

Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2334/14/54/prepub

Contributor Information

Kinuyo Chikamatsu, Email: chikamatsu@jata.or.jp.

Akio Aono, Email: aono@jata.or.jp.

Hiroyuki Yamada, Email: hyamada@jata.or.jp.

Tetsuhiro Sugamoto, Email: tsugamoto@jatahq.org.

Tomoko Kato, Email: dumbo22boko22@gmail.com.

Yuko Kazumi, Email: kazumi@jata.or.jp.

Kiyoko Tamai, Email: mml-idenshi@miroku-lab.co.jp.

Hideji Yanagisawa, Email: h-yanagisawa@miroku-lab.co.jp.

Satoshi Mitarai, Email: mitarai@jata.or.jp.

Acknowledgements

We thank TAUNS Co, Ltd (Izunokuni, Japan) for providing the Capilia TB-Neo, SD MPT64, and TBc ID.

References

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