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Abadia et al. BMC Infectious Diseases 2011, 11:110
http://www.biomedcentral.com/1471-2334/11/110

RESEARCH ARTICLE

Open Access

The use of microbead-based spoligotyping for
Mycobacterium tuberculosis complex to evaluate
the quality of the conventional method:
Providing guidelines for Quality Assurance when
working on membranes
Edgar Abadia1, Jian Zhang1, Viviana Ritacco2, Kristin Kremer3, Raymond Ruimy4, Leen Rigouts5,
Harrison Magdinier Gomes6, Atiná Ribeiro Elias6, Maryse Fauville-Dufaux7, Karolien Stoffels7,
Voahangy Rasolofo-Razanamparany8, Darío Garcia de Viedma9,10, Marta Herranz9,10, Sahal Al-Hajoj11,
Nalin Rastogi12, Carlo Garzelli13, Enrico Tortoli14, Philip N Suffys6, Dick van Soolingen3,15, Guislaine Refrégier1 and
Christophe Sola1,16*

Abstract
Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the
spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th,
2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique.
We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly
robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data
points. Samples were received from 11 international laboratories with a worldwide distribution.
Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA
extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping
participating centers. Information regarding how the classical Spoligotyping method was performed by center was
available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both
methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were
performed to validate the observed results.
Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 8090% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to
discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using
thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers
shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus
(Mostly spacers 15, 14, 18, 37, 39, 40).
Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most
centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study.
However performance was center-dependent, suggesting that training is a key component in quality assurance of
spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors
* Correspondence: christophe.sola@u-psud.fr
1
Institute of Genetics and Microbiology UMR8621, CNRS Université Paris-Sud
11 Universud, Campus d’Orsay, F-91405 Orsay-Cedex, France
Full list of author information is available at the end of the article
© 2011 Abadia et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Abadia et al. BMC Infectious Diseases 2011, 11:110
http://www.biomedcentral.com/1471-2334/11/110

Page 2 of 8

occur randomly either in the process of re-using membranes, or during the reading of the results and transferring
of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as
previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and
demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical
spoligotyping.

Background
Clustered Regularly Interspaced Palindromic Repeats
(CRISPRs) are a family of DNA repeats of 21 to 37 bp
that are separated by regularly sized, non repetitive
unique DNA spacer sequences [1]. CRISPRs are present
in the genomes of most bacteria (40%) and archaea
(90%) [2]. It is believed that some spacers originate from
mobile genetic elements [1] and it has been shown that
they confer ‘’immunity’’ against bacteriophages and plasmids [2-4].
Studies on the CRISPR region of Mycobacterium
tuberculosis complex (MTC) strains started in 1993 [5].
In 1995, spoligotyping was for the first time mentioned
in the article describing the Beijing genotype of Mycobacterium tuberculosis [6]. In 1997, Kamerbeek et al.
provided a standardized reverse line blot hybridization
method (spacer oligonucleotide typing = spoligotyping)
to genotype MTC complex strains based on polymorphism of this region, and soon after, corresponding membranes were commercialized [7,8]. Since then, numerous
studies have used spoligotyping often combined with
others markers to assess the diversity of MTC strains.
Spoligotyping appeared especially suitable as a simple
and cheap tool to distinguish genotype families of this
bacterium. Almost all international spoligotyping results
have been submitted to an international database,
SpolDB http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/ that has already been updated 4 times [8].
Recent studies point to the potential use of CRISPR loci
for molecular epidemiological studies of other pathogens
[9], and an increased knowledge of these bacterial genomic structures is likely to foster the development of new
high-throughput genotyping methods, either for studies
on population structure or molecular epidemiology.
While spoligotyping does not differentiate M. tuberculosis isolates with the same level of discrimination as
IS6110-RFLP (Restriction Fragment Length Polymorphism) [10] or MIRU-VNTR (Mycobacterial Interspersed
Repetitive Units-Variable Number of Tandem Repeats)
[11] it has several advantages [12]: i) it relies on a single
PCR amplification which requires much less DNA quantities than IS6110-RFLP so that even smear-positive sputum samples can be directly analyzed, ii) up to 40 DNA
samples can be analyzed within one day with the classical method and up to 186 with the novel microbeadbased spoligotyping that has proven very reliable [13,14],

iii) isolates with less than 6 copies of the IS6110 insertion element are better discriminated by spoligotyping
than by IS6110-RFLP typing, and iv) spoligotyping patterns can distinguish between main lineages and sublineages within the M. tuberculosis complex (MTC)
making them phylogenetically informative except in the
rare cases where the exact same deletions (covering the
exact same spacers) occurred by convergent evolution
[9,14-17].
The classical spoligotyping described by Kamerbeek et
al. revised by van Embden et al. is a robust method
with an intra-laboratory reproducibility over 90% in
well-trained laboratories [7,18]. However it can be
affected by several issues: (i) it relies on the quality of
the pre-prepared membrane and this has proven difficult
even when commercialized; (ii) spoligotyping does not
yield black or white results and reading is sometimes
subjective; only results double checked by experienced
staff seem reliable but this technically demanding procedure is not always implemented in specialized labs, (iii)
the repeated use of the same membrane may be the
cause of technical artifacts, (iv) data entry and classification are performed manually with an increased likelihood of errors during transcription [19]. All these issues
might have impaired reliability in international genetic
databases that have been compiled so far (SpolDB
projects).
The transfer of the classical spoligotyping method into
an advanced platform represents a technical progress
because it allows to produce a numerical raw result format and a standardized signal/noise cut-off determination, ensuring both a high throughput and an high
quality [13,14].
Hence, the aims of this study were to: (i) retrospectively assess the global quality of spoligotyping results
that have been produced in various laboratories worldwide during a decade by comparing their data with
those provided by the new, highly reliable system, (ii)
give a feedback to the laboratories about their data production quality, (iii) solve uncertainties on samples in
which centers are not confident, (iv) study if the DNA
extraction procedure may influence membrane-based
spoligotyping results, (v) assess the possible contribution
of the microbead-based technique to an increased quality of services in molecular epidemiological studies.
Altogether, this article identifies the main pitfalls in

Abadia et al. BMC Infectious Diseases 2011, 11:110
http://www.biomedcentral.com/1471-2334/11/110

CRISPR data production, at a time when tuberculosis
spoligotyping still inflates and is transferred towards
other micro-organisms.

Methods
Oligonucleotides

The capture probes for microbead-based spoligotyping
are identical to the ones from the membrane-based spoligotyping technique with minor modifications to correct
some sequences from the original set [7,18]. All capture
oligonucleotides (Eurogentec, Liège, Belgium) were
modified at the 5’ terminal amino group by a twelve carbon spacer linker to obtain the adequate free space
between the microspheres and the oligonucleotides
(increase of gyration radius).
Spoligotyping PCR protocol

PCR (25 μl total) was performed for 20 cycles for CTAB
DNA [7] and 25 cycles for thermolyzates using 2 μl of
tested DNA.
Hybridization

Was done in TMAC buffer at 52°C for 10 minutes as
described before by Zhang et al. [14].
Data analysis

For each spacer, hybridization signals were recorded as
RFI (Relative Fluorescence Intensity) values and were
transformed in a binary code (presence/absence) using a
signal/noise cut-off value of 2 times the lower values’
group. Octal codification and SpolDB4 lineage identification were assigned to each spoligotype. Spoligotypes
generated by the membrane technique were compared
to the Luminex generated spoligotypes, spoligotype by
spoligotype and spacer by spacer. The first comparison
allowed determining perfect matches (pm), i.e. obtaining
exactly the same spoligotype by both methods (identical
results for 43 over 43 spacers). The percentage of perfect matches in a center is calculated as the number of
isolates with pm divided by the total number of isolates
provided by the center. The second comparison dealing
with individual datapoints (or spacers) was represented
by the rate of difference (rd). This rd index indicates
the number of discordant data points for the total number of data points analyzed per center: e.g. if 8 of 100
DNAs tested show discordant results in 2 spacers the rd
value equals 8*2*100/(100*43) = 0.37%.
We also investigated if some spacers were more prone
to errors (see additional file 1). Problematic spacers
were defined as spacers having exhibited at least one
discrepancy in a center. Consecutive problematic spacers
are referred to as “blocks” of problematic spacers.

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Mycobacterial isolates, DNA extraction

DNA samples were provided by 11 centers that perform
membrane-based spoligoyping as a routine procedure: i)
Buenos Aires - Argentina (Servicio de Micobacterias,
Instituto Nacional de Enfermedades Infecciosas, ANLIS
“Carlos G. Malbran”, Buenos Aires, Argentina); ii) PisaItaly (Università di Pisa. Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia e Epidemiologia) and the Regional Reference Center for
Mycobacteria, Firenze; iii) Bilthoven - The Netherlands
(National Institute for Public Health and the Environment-RIVM); iv) Paris-France (Microbiology Laboratory,
Hôpital Bichat-Claude Bernard, AP-HP); v) MadridSpain (Servicio de Microbiología Clínica y Enfermedades
Infecciosas, Hospital General Universitario Gregorio
Marañón); vi) Pointe-à-Pitre - Guadeloupe (TB and
Mycobacteria Research Unit, Institut Pasteur de Guadeloupe); vii) Antananarivo-Madagascar (TB reference
laboratory, Institut Pasteur de Madagascar); viii) RiyadhSaudi Arabia (TB Research Unit, Comparative Medicine,
King Faisal Specialist Hospital and Research Center); ix)
Rio de Janeiro, Oswaldo Cruz Institute Laboratory of
Molecular Biology applied to Mycobacteria, Brazil; x)
Antwerp-Belgium, Mycobacteriology Unit, Prince Leopold Institute of Tropical Medicine, and xi) BrusselsBelgium, Scientific Institute of Public Health, National
Reference Centre of Tuberculosis and Mycobacteria.
These selected strains have a worldwide origin and
represent a wide range of MTC genotypes. DNA was
extracted using a thermolyzate or a cetyl-trimethylammonium bromide (CTAB) procedure. DNA samples
fulfilled the following criterion: samples representative
of hard to interpret spoligotypes and samples representative of the work flow, so we could evaluate the benefits of a new platform regarding the classical one on
these samples. Most centers had performed spoligotyping using commercial membranes (Ocimum, Hyderabad,
India) (except for the laboratories in Institut Pasteur of
Guadeloupe, Institut Pasteur of Madagascar, and the
University of Pisa). Samples were analyzed in a blind
way which means that we did not know the profiles
from the membrane-based spoligotyping until we had
everything processed by the microbead spoligotyping.
We named the centers from 1 to 11 for convenience
ranked according to their spoligotyping quality, so this
numbering is not correlated with the order in which
they are described by name to respect confidentiality.
The number of samples included in this study by Center
are as follows: 1 (49 samples), 2 (95 samples), 3 (98
samples), 4 (97 samples), 5 (87 samples), 6 (82 samples),
7 (120 samples), 8 (70 samples), 9 (59 samples), 10 (85
samples) and 11 (85 samples).

Abadia et al. BMC Infectious Diseases 2011, 11:110
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Statistical analysis

Homogeneity “Mann-Whitney” non-parametric U test
was performed in an Excel sheet and “Spearman rank
correlation” test (with Rho and p calculations) was performed using the online tool at http://www.u707.jussieu.
fr/biostatgv. df = degrees of freedom, relating the number of independent observations used to compute the
statistical parameters.

Results
Eight centers using commercial membranes and three
using in-house made membranes were included in the
study, totaling 927 isolates. We performed the
microbead-based spoligotyping on Luminex on the same
set of DNA samples as a new standard, even though it
is clear that only sequencing would provide full reference information on spacer sequences and genomic
structure of the Direct Repeat locus, especially in case of
doubtful hybridization results for some spacers
[13,14,18]. Centers were ranked from 1 to 11 according
to their performance. The seven best centers reached
over 90% of perfect match (pm = exactly the same spoligotype pattern over the 43 spacers), three centers
obtained between 87 and 84% concordance and one
laboratory only 51% (Figure 1). However, this specific
center had a high level of errors at a single spacer, and
when ignoring the errors at that spacer, it reached 88%
of pm (data not shown).

peper forper

100
90

Participating centers used either Cetyl-TrimethylAmmonium-Bromide (CTAB) extracted or heat-shock
extracted (thermolyzates) DNA, depending on the type
of studies they usually use the DNA for: the CTABbased extraction method is preferred for molecular analyses demanding high purity (e.g. IS6110-RFLP for
which CTAB is mandatory), whereas thermolyzates are
used in other cases due to its swiftness and ease of
implementation. Surprisingly, the best performing center
(Center 1) used only thermolyzate DNA, and Center 11
used only CTAB DNA (Figure 1). Altogether, centers
that used the thermolyzate DNA extraction procedure
performed as well as centers using the CTAB procedure
(nCTAB = 3, nThermolyzates = 6; Mann Whitney test: = U
= 6; p = 0.374).
Each spoligotype is a string of 43 characters so that in
total 39,861 data points were analyzed in this study. Our
comparative results showed 157 (0.39 %) data point discrepancies. We defined the rate of difference (rd) index
as the number of mismatches divided by the total number of analyzed data points in each center (see material
and methods). All centers except one exhibited a “rd“
below 1% (Figure 2). In addition, when ignoring the
errors due to the recurrently problematic spacers in
most centers, rd dropped to 0.38%. As expected, rd was
correlated to the rank of centers (Spearman rank correlation test Rho = 0.99; df = 9; p < 0.001): centers having
the highest numbers of erroneous spoligotype patterns
also had the highest number of individual spacers errors.
Still, specific centers had a relative low rd as expected
from their pm: centers 6 and 9 exhibited slightly higher
global quality (as measured by the pm) than centers 7
and 10 respectively (Figure 1), however, they had a

80
70

Performance %

60

PM

50
40
30
20
10
(11) CTAB

(10) Thermolyzates

(9) CTAB and Thermolyzates

(8) CTAB

(7) Thermolyzates

(6) Thermolyzates

(5) Thermolyzates

(4) Thermolyzates

(2) CTAB

(3) CTAB and Thermolyzates

(1) Thermolyzates

0

Centers and DNA extraction procedures

Figure 1 Relative quality of the classical membrane-based
spoligotyping by center. One hundred percent (100%
performance) quality is inferred when obtaining complete (identical)
concordance, i.e. 43/43 spacers, with the high-throughput based
spoligotyping for every isolate. The centers were numbered
according to their spoligotyping quality and these numbers are
shown between parentheses. DNA extraction procedure is also
mentioned for each center.

Figure 2 Datapoints discrepancies per center. Datapoints consist
in every spacer of each spoligotype pattern. The percentage of
differences among all datapoints is referred to as rd for rate of
difference.

Abadia et al. BMC Infectious Diseases 2011, 11:110
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Classical set of spacers

Discussion
We show in this study that different centers performing
membrane-based spoligotyping exhibit different inter
laboratory reproducibility rates ranging from 51 to 100%
and most of them reaching more than 90% of quality as
represented by their pm. One center had a specific
detection problem of spacer 39. Ignoring the errors
associated with this spacer, it reached a pm as high as
the other centers. The choice of the sample was initially
meant to include problematic specimens as well as sample representative of the work flow, however a separate
statistical analysis between the random work flow and

43
41
39
37
35
33
31
29
27
25
23
21
19
17
15
13
11
9
7
5
3
1
1

2

3

4

5

6

7

8

9

10

11

Centers

Figure 3 Problematic spacers by center as derived from the
comparison of spoligotypes generated by both methods.
Blocks of adjacent problematic spacers are circled.

50
45
40

Number of errors

35
30
25
20
15
10
5
0

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43

higher amount of individual discrepancies (as measured
by their rd, Figure 2). This indicates that, in these centers, the mismatches were often belonging to the same
spoligotype patterns. No statistical difference in the pm
relatively to the use of commercial or home-made membranes was observed (nHome = 3; nCommercial = 8; Mann
Whitney test: U = 10; p = 0.387 i.e. non significant).
We also looked for potentially problematic spacers
creating systematic mismatches in multiple centers.
Spacers that were wrongly identified by the classical
spoligotyping varied among centers (Figure 3). They
occasionally were located adjacent one to another and
part of the corresponding blocks were shared by two to
three centers (see circles, for instance for spacers 40 and
41, in Figure 3). However, most problematic spacers
were not generally shared. The spacer that most frequently introduced errors was spacer 39 (Figure 4) but
it was not detected among 42 isolates in one specific
center, likely due to a membrane production problem.
When ignoring these samples, spacers most prone to
introducing errors were spacers 15, 18, 14 and 40; and
to a lesser extent spacers 37, 8, 26, 29, 30, 33 and 41
(Figure 4).

Spacers

Figure 4 Number of datapoints genotyping errors per spacer.

hard-to-type samples was not feasible here (low effective
size), hence the discrepancies reported here are upper
limits and could have been lower if only random selection had occurred.
The reasons why spoligotyping errors occur can be
diverse. First, DNA quality associated to the extraction
procedure could be critical. In this study, however, the
DNA extraction procedure did not influence the performance of the membrane-based spoligotyping. Nevertheless, we have to keep in mind that we only tested CTAB
and thermolyzates and other extraction procedures were
not tested. Some centers repeatedly introduced genotyping errors at multiple spacers in the same samples.
Some samples may have been of inadequate quality for
all spacers to be sufficiently amplified. However, this
occurred both for centers using CTAB and thermolyzate
DNA, confirming that factors other than extraction procedure, such as the amount of extracted DNA, inclusion
of culture medium when preparing bacterial suspensions
or possible culture contaminations, may have influenced
PCR quality and hence modified the spoligotypes of
some samples.
Regarding artisanal process of membrane production,
an adequate quantity of specific probe concentrations
on the membrane could sometimes be defective which
would lead to the same spacer being recurrently problematic in the same center. Also, commercial membranes
might experience some spacer problems so internal
quality control must be done by each lab. Indeed, we
have observed that in Center 11, spacer 39 was affecting
the results of 42 strains. However, in other centers, such
problems were not observed. We advise positive and
negative controls to be included at randomly chosen
positions to detect such possible membrane production
problems; centers producing their own membranes
should also check their production adequately before
use.
Third, operator-dependent washing problems could
occur on any part of the membrane so that any spacer
could be wrongly scored for some isolates. Insufficient
washing can lead to false positive detection of a spacer.
As an example, spacers 14 or 37 were detected, although
they proved to be absent. In contrast, excessive washing

Abadia et al. BMC Infectious Diseases 2011, 11:110
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will level the signals of several probes down. All centers
used their membranes between 8 to 14 times which is
under the limit advised by membranes’ manufacturer
(up to 20 times). However, this might still be too high.
We advise users to always record the results with positive and negative controls included at randomly chosen
positions to detect the membrane fall down in quality,
and possible insufficient washes.
Fourth, there can be intrinsic problem due to the
CRISPR structure in MTC: some spacers constantly provide weaker signals that make the distinction between
positive and negative values more difficult. For instance,
DVR next to spacer 15 (DVR26) harbors a deletion of 4
nucleotides at its 5’ end as shown in highly diverse
strains [18]. Consequently, PCR primers do not hybridize properly around spacer 15 which likely leads to a
lower amplification level of this spacer.
The high-throughput spoligotyping method has the
advantage of being more sensitive to detect these slight
variations, given the 3D nature of the immobilized
probes, which provides more surface to attach more PCR
products. Moreover, unspecific hybridization is removed
more easily with the microbead technology, making the
detection of spacer 15 possible in any sample. Contrarily,
in our study, spacer 40 was detected several times by
membrane-based spoligotyping, but not by the
microbead-based technique (false positive on membrane).
Fifth, the hybridization reading from the film, and the
transfer of this information to a digital format have to
be handled manually in the membrane-based spoligotyping increasing the transcription error risk; the best procedure to reduce this risk is to have the results read in
duplicate by two independent readers, to check reading
mistakes and to solve potential discrepancies.
Reading errors are suppressed in the high-throughput
method due to an in-house designed automatized routine, which already provides a digital file with a suggested and objective interpretation according to
predefined thresholds (cut-off).
As the distribution of wrongly typed spacers is random (see additional file 1), low quality reading and
transfer of results may be the major problem in the classical spoligotyping. This can be particularly misleading
when errors concern spacers that are used to classify
strains into genotype level. For instance, spacer 18 has
been proposed to be highly informative to identify the
MTC “X” clade strains, and spacer 40 to define the T2
subclade [8,20].

Conclusions
CRISPR region genotyping in MTC, referred to as spoligotyping, revealed very useful for first line screening of
epidemiological analyses and remains a robust and
widely accepted genotyping method [21,15]. Since the

Page 6 of 8

technical improvement of microbead-based versus membrane-based spoligotyping method was previously
demonstrated in two independent settings, we took
microbead-based method as a new standard to study
retrospectively the quality of membrane-based spoligotyping results in 11 centers [13,14]. However, sequencing will remain the unique reference method for
CRISPR genomic structure in case of doubts. The performance of the membrane-based spoligotyping was
shown not to be influenced by the DNA extraction procedure. Overall, a relative good performance was
obtained in most centers. Even if globally reliable, we
report here that membrane-based spoligotyping suffers
some practical limitations which are overcome when
switching to the microbead-based format. This format
remains more expensive even though the higher
reagents cost is partly balanced by decreased add-ons
(high-throughput) and interpretation time.
A single center out of eleven had a systematic problem of membrane quality that was overlooked (non
reactivity of spacer 39). This issue was due to a problem
in membrane production. Other centers yielded errors
slightly more frequently at spacer 15, that is known to
be less amplified because of its neighboring modified
CRISPR sequence around this spacer. In contrast, the
increased sensitivity of the high-throughput method was
validated by the proper detection of this spacer [13,14].
Other genotyping errors by classical membrane-based
spoligotyping most likely are due to variation in interpretation of weaker spots and/or transferring of data
from the film to a digital file processes.
The high-throughput microbead-based method was
shown to have a better sensitivity due to proper detection of some spacers that can be misinterpreted by the
classical method, and a lower error rate due to automated interpretation and transcription. A new 68
spacers microbead-based spoligotyping format provides
an increase in discriminatory power for Principal
Genetic Group I clinical isolates, a feature that could be
especially useful in South-East Asia where the East-African Indian (EAI) clade predominates [14].
The launch of a new Luminex device (MagPix), with a
price drop, yet lower-plex, molecular diagnostics instrument (50Plex) that would be interesting for fields labs (e.g.
without requirement of air conditioning) could facilitate
the spreading of spoligotyping; however this will only happen if routine utilization (through a concomitant drop in
reagents prices) is done within acceptable cost limits for
public health and an improved quality of service.

Financial Competing interests
EA, JZ, GR and CS declare that they never received any
direct financial support by the Luminex Corp. or Luminex BV companies as well as no staff support was

Abadia et al. BMC Infectious Diseases 2011, 11:110
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provided by Luminex Corp. nor Luminex BV to perform
this study.

Page 7 of 8

2.

3.

Additional material
Additional file 1: Center’s distribution of problematic spacers. This
file highlights the problematic spacers shared by several centers (lines).

4.
5.

Acknowledgements
This study was supported by the ‘’Excellency Chair in microbiology of the
University Paris-Sud’’ granted to CS in September 2007 through a PhD (JZ)
and a postdoctoral grant (EA). EA is a senior mycobacteriology scientist of
the IVIC (Instituto Venezolano de Investigaciones Científicas, Caracas,
Venezuela). The work done at Institut Pasteur de Guadeloupe was partially
supported by United States National Institutes of Health - grant
R01LM009731. JZ was a PhD fellow of the Université Paris-Sud through the
‘’Ecole Doctorale Gènes Génomes Cellules’’. We are grateful to François
Topin, Luminex BV, The Netherlands, and to Luminex Corporation, Austin,
Texas for their support. Gilson S.A.S, Villiers-le-Bel, France, is also
acknowledged for providing automated multi-pipettes.
Author details
Institute of Genetics and Microbiology UMR8621, CNRS Université Paris-Sud
11 Universud, Campus d’Orsay, F-91405 Orsay-Cedex, France. 2Instituto
Nacional de Enfermedades Infecciosas ANLIS Carlos Malbrán, Vélez Sarsfield
563, 1281 Buenos Aires, Argentina. 3National Institute for Public Health and
the Environment, Bilthoven, The Netherlands. 4EA 3964 Université ParisDiderot & Microbiology Laboratory, Bichat-Claude Bernard Hospital AP-HP,
Paris, France. 5Mycobacteriology Unit, Prince Leopold Institute of Tropical
Medicine, 155 National Straat, 200 Antwerp, Belgium. 6Laboratory of
Molecular Biology applied to Mycobacteria, Oswaldo Cruz Institute, Rio de
Janeiro, Brazil. 7National Reference Centre of Tuberculosis and Mycobacteria,
Scientific Institute of Public Health, Brussels, Belgium. 8Unité des
Mycobactéries, Institut Pasteur de Madagascar, Antananarivo, Madagascar.
9
Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital
Gregorio Marañón, Madrid, Spain. 10CIBER Enfermedades Respiratorias
(CIBERES), Spain. 11Department of Comparative Medicine, King Faisal
Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 12Unité de la
Tuberculose et des Mycobactéries - WHO Supranational TB Reference
Laboratory, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe.
13
Dipartimento di Patologia Sperimentale Biotecnologie Mediche
Infettivologia ed Epidemiologia, Università di Pisa, I-56127 Pisa, Italy.
14
Regional Reference Center for Mycobacteria, Careggi Hospital, viale
Morgagni 85, 50134 Firenze, Italy. 15Department of Pulmonary Diseases and
Department of Microbiology, Radboud University Nijmegen, P.O. Box 9101,
6500 HB Nijmegen, The Netherlands. 16Unité de Génétique Mycobactérienne,
Institut Pasteur, 25-28 rue du Dr. Roux, F-75724 Paris-Cedex 15, France.

6.

7.

8.

1

Authors’ contributions
EA and JZ performed and analyzed the microbead-based spoligotypes, VR,
KK, RR, LR, HMG, AR, PS, MFD, KS, MH, VR, MH, DGV, SAAN, NR, CG, ET, DvS,
participated in sample recruitment and performed membrane-based
spoligotyping in their respective laboratories, GR performed the Statistical
analyses, CS conceived and coordinated the study, EA, GR and CS wrote the
manuscript. LR, VR, DvS, DGV contributed to the manuscript revision and all
authors approved the final version of the manuscript.

9.

10.

11.

12.

13.

14.

15.

16.

Competing interests
The authors declare that they have no competing interests.
Received: 18 October 2010 Accepted: 28 April 2011
Published: 28 April 2011
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Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/11/110/prepub
doi:10.1186/1471-2334-11-110
Cite this article as: Abadia et al.: The use of microbead-based
spoligotyping for Mycobacterium tuberculosis complex to evaluate the
quality of the conventional method: Providing guidelines for Quality
Assurance when working on membranes. BMC Infectious Diseases 2011
11:110.

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