Determination of circulating Mycobacterium tuberculosis strains and transmission patterns among pulmonary TB patients in Kawempe municipality, Uganda, using MIRU-VNTR.

Abstract

ABSTRACT:
Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping is a powerful tool for unraveling clonally complex Mycobacterium tuberculosis (MTB) strains and detection of transmission patterns. Using MIRU-VNTR, MTB genotypes and their transmission patterns among patients with new and active pulmonary tuberculosis (PTB) in Kawempe municipality in Kampala, Uganda was determined.
MIRU-VNTR genotyping was performed by PCR-amplification of 15 MTB-MIRU loci from 113 cultured specimens from 113 PTB patients (one culture sample per patient). To determine lineages, the genotypes were entered into the MIRU-VNTRplus database [http://www.miru-vntrplus.org/] as numerical codes corresponding to the number of alleles at each locus. Ten different lineages were obtained: Uganda II (40% of specimens), Uganda I (14%), LAM (6%), Delhi/CAS (3%), Haarlem (3%), Beijing (3%), Cameroon (3%), EAI (2%), TUR (2%) and S (1%). Uganda I and Uganda II were the most predominant genotypes. Genotypes for 29 isolates (26%) did not match any strain in the database and were considered unique. There was high diversity of MIRU-VNTR genotypes, with a total of 94 distinct patterns. Thirty four isolates grouped into 15 distinct clusters each with two to four isolates. Eight households had similar MTB strains for both index and contact cases, indicating possible transmission.
MIRU-VNTR genotyping revealed high MTB strain diversity with low clustering in Kawempe municipality. The technique has a high discriminatory power for genotyping MTB strains in Uganda.

Full text

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RESEARCH ARTICLE Open Access
Determination of circulating Mycobacterium
tuberculosis strains and transmission patterns
among pulmonary TB patients in Kawempe
municipality, Uganda, using MIRU-VNTR
Lydia Nabyonga1†, David P Kateete1, Fred A Katabazi1, Paul R Odong1, Christopher C Whalen2,
Katherine R Dickman1,3†and Joloba L Moses1*
Abstract
Background: Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR)
genotyping is a powerful tool for unraveling clonally complex Mycobacterium tuberculosis (MTB) strains and
detection of transmission patterns. Using MIRU-VNTR, MTB genotypes and their transmission patterns among
patients with new and active pulmonary tuberculosis (PTB) in Kawempe municipality in Kampala, Uganda was
determined.
Results: MIRU-VNTR genotyping was performed by PCR-amplification of 15 MTB-MIRU loci from 113 cultured
specimens from 113 PTB patients (one culture sample per patient). To determine lineages, the genotypes were
entered into the MIRU-VNTRplus database [http://www.miru-vntrplus.org/] as numerical codes corresponding to the
number of alleles at each locus. Ten different lineages were obtained: Uganda II (40% of specimens), Uganda I
(14%), LAM (6%), Delhi/CAS (3%), Haarlem (3%), Beijing (3%), Cameroon (3%), EAI (2%), TUR (2%) and S (1%).
Uganda I and Uganda II were the most predominant genotypes. Genotypes for 29 isolates (26%) did not match
any strain in the database and were considered unique. There was high diversity of MIRU-VNTR genotypes, with a
total of 94 distinct patterns. Thirty four isolates grouped into 15 distinct clusters each with two to four isolates.
Eight households had similar MTB strains for both index and contact cases, indicating possible transmission.
Conclusion: MIRU-VNTR genotyping revealed high MTB strain diversity with low clustering in Kawempe
municipality. The technique has a high discriminatory power for genotyping MTB strains in Uganda.
Background
Tuberculosis (TB) is a leading cause of morbidity and
mortality throughout sub-Saharan Africa, and Uganda
ranks sixteenth among countries with the highest bur-
den of disease [1]. Co-infection with HIV/AIDS and the
emergence of multi-drug resistant (MDR) Mycobacter-
ium tuberculosis (MTB) strains have made TB a major
public health problem [2]. The incidence of TB in
Uganda is estimated at 330 cases per 100,000 persons
per year, and this includes both HIV infected and non-
HIV infected patients [3]. TB prevalence in Uganda is
believed to be higher than reported due to lack of suffi-
cient healthcare; indeed, many people are not aware that
they are infected with MTB and this has led to low
levels of diagnosis and treatment [4]. Uganda also has
one of the lowest TB cure rates (32%) and high drug
default rate [1], which may lead to an increase in drug
resistance mutations.
Molecular genotyping tools for MTB such as IS6110-
based restriction fragment length polymorphism (RFLP),
“regions of difference” (RD) analysis, spoligotyping,
MIRU-VNTR, and single nucleotide polymorphism
(SNP) analysis have become invaluable in TB diagnosis
and investigations of disease transmission dynamics,
outbreaks and phylogenetics [5,6]. Of the MTB
* Correspondence: moses.joloba@case.edu
† Contributed equally
1Department of Medical Microbiology, School of Biomedical Sciences,
Makerere University College of Health Sciences, Kampala, Uganda
Full list of author information is available at the end of the article
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© 2011 Moses 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.

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genotyping tools, the gold standard is IS6110-RFLP, a
laborious method that requires large amounts of DNA
per isolate, and has poor inter-laboratory reproducibility
[7]. MIRU-VNTR, a faster genotyping method with dis-
criminatory power higher than that of IS6110 RFLP, has
recently been introduced [7,8]. MIRU-VNTR genotyping
is performed by amplifying a panel of 12, 15 or 24 loci
[9]; genotyping with a panel of 15 loci is best suited for
epidemiologic studies [7,10]. Additionally, MIRU-VNTR
can detect mixed MTB strains in a single sputum sam-
ple [7,11]. Due to its portable data format, MIRU-VNTR
can be used as a multi-purpose tool for strain identifica-
tion using a reference database [2]. However, the choice
of appropriate loci for MIRU-VNTR requires evaluation
in diverse MTB lineages in countries with high TB pre-
valence [7]. Mixed MTB infections in Ugandan patients
with pulmonary TB (PTB) were recently reported using
MIRU-VNTR [11], but genotypes/strains for the entire
patient population were not determined. In this study,
we aimed to determine the distribution and diversity of
MTB lineages in Kawempe municipality using MIRU-
VNTR genotyping, and assess the ability of the techni-
que to discriminate the predominant genotypes and
detect transmission in this community.
Methods
Patients, sample processing and cultures
This study was approved by the Joint Clinical Research
Centre (JCRC) Institutional Review Board (Kampala,
Uganda) and the University Hospitals Cleveland Institu-
tional Review Board (Cleveland, Ohio). Informed written
consent was obtained from patients who participated.
Sputum samples were collected from patients with at
least one positive culture for MTB, who were previously
enrolled in the Kawempe Community Household Con-
tact Study, an ongoing epidemiological study in Kam-
pala, Uganda, from which several papers have been
published [12-15]. Samples were collected consecutively
from October 2007 through February 2009, from
patients with PTB symptoms who reported not having
received treatment for TB in the preceding month.
Patient demographics, sample processing and cultures,
drug susceptibility testing and DNA extraction are
described in Dickman et al, 2010 [11]. Cultures were
confirmed as MTB by PCR-detection of a 500 bp frag-
ment of the IS6110, which is common in the members
of the MTB complex [16].
MIRU-VNTR PCR and data analysis
MIRU-VNTR genotyping was performed by PCR-ampli-
fication of a panel of 15 MTB MIRU loci using primers
described in the MIRU-VNTR standard protocol
[8,11,17]. To size amplicons, gel (3% agarose in TBE)
electrophoresis for three hours at 120 constant voltage
was performed. The allele calling table in the Supply
protocol [8,17] was used to assign the number of alleles
corresponding to the amplicon sizes. To determine
MTB strain lineages, relatedness or clustering, the
MIRU-VNTR genotypes (see additional file 1) were
matched with reference strains in the MIRU-VNTRplus
database (http://www.miru-vntrplus.org/), using a cate-
gorical coefficient of 1 and a distance cut off of < 0.3
that corresponds to a seven locus difference. Then, a
Neighbor Joining dendrogram was constructed from the
strains’ genotypes using the MIRU-VNTRplus online
program, on assumption of different evolutionary rates
(molecular clocks) for MTB MIRU loci [9].
A cluster was defined as two or more patients’ strains
with identical genetic patterns. Clusters were assumed
to have arisen from recent transmission, and the cluster-
ing rate was used to determine recent transmission of
MTB [18]. The minimum estimate of the proportion of
TB cases related to recent transmission was calculated
using the formula: (number of clustered patients - num-
ber of clusters)/total number of patients [18]. To deter-
mine the discriminatory power of MIRU-VNTR for this
patient population, the Hunter Gaston Discriminatory
Index (HGDI) [19] was calculated.
Results and Discussion
High strain diversity in Kawempe municipality
Between October 2007 and May 2009, 113 MTB cul-
tures from 113 patients with PTB were genotyped with
MIRU-VNTR using a panel of 15 loci [11]. Ten distinct
strains were identified; EAI, Delhi/CAS, Uganda I,
Uganda II, LAM, Haarlem, S, Beijing, Cameroon and
TUR. Isolates from 29 (26%) patients did not match any
strain in the database and were regarded unique (see
Table 1). These findings agree with a previous study
[20], which found strains of different lineages including
Delhi/CAS, LAM and Beijing in Rubaga municipality,
Table 1 Distribution of MTB lineages
Patients, N = 113
Lineage/strain Number (%)
Uganda I 16 (14)
Uganda II45 (40)
LAM6 (5)
Beijing3 (3)
Dehli/CAS 3 (3)
Haarlem3 (3)
Cameroon3 (3)
TUR 2 (2)
EAI2 (2)
S1 (1)
Unique29 (26)
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??????????????????
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Figure 1 Neighbor joining dendrogram showing clustering among the genotyped MTB strains. The 15 clusters are indicated with a
numerical brown font. The order of MIRU loci is as follows, left to right: 424, 577, 580, 802, 960, 1644, 1955, 2163b, 2165, 2401, 2996, 3192, 3690,
4952 and 4150.
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Uganda, using RD genotyping. Furthermore, in this
study, Uganda I and Uganda II were the predominant
lineages (at 14% and 40% prevalence, respectively), fol-
lowed by LAM (5%). An earlier study using RD genotyp-
ingreported MTB
“Uganda
predominant strain in Rubaga municipality [21]. The
Beijing, Dehli/CAS, Haarlem and Cameroon strains
were individually found in only 3% of the patients. One
MDR strain was unique while two with mono-resistance
to isoniazid were of EAI lineage. Another strain with
mono-resistance to streptomycin was unique.
There is high genetic diversity in Kawempe Municipal-
ity; the HGDI [19] was 0.996, which is very high and
comparable to that of Mulenga et al (0.988) in Ndola,
Zambia [22], an urban setting in an endemic country
similar to Uganda. The high genetic diversity of MTB
strains in Kawempe community could be a consequence
of reactivation of latent MTB infection, increased
human population/global travel and diversity in host
genetics [23].
genotype”
as the
MIRU-VNTR patterns
There was high diversity of MIRU-VNTR patterns
among the characterized isolates; a total of 94 distinct
patterns were identified, which included 15 clusters each
with two to four isolates. In total 34 (30%) isolates clus-
tered while 79 (70%) had unique patterns (see Figure 1).
The clustering rate was 17%, implying that the mini-
mum estimate of disease related to recent transmission
was 17% (see methods). This is low considering the high
population density, endemicity, poor housing and HIV/
AIDS prevalence in Kawempe municipality [4], which
are risk factors for TB transmission. Similar to the Casa-
blanca study [18], most disease in this community could
be due to reactivation of MTB infection rather than
recent transmission. Furthermore, the clustering rate
was low in comparison with an earlier study in Rubaga
municipality Kampala, Uganda [24], in which a high
clustering rate was reported using IS6110-RFLP geno-
typing. The differences in clustering rates between the
current and former study could be attributed to the
high discriminatory power of the 15 loci MIRU-VNTR
genotyping panel (HGDI of 0.996).
If true, the possible impact of the presence of low
transmission rate, and the implication that most dis-
ease in this community could be due to reactivation of
MTB infection could be highly influential with regards
to infection control measures and disease management.
Table 2 MTB transmission patterns in households
MIRU-VNTR locusa
PT IDLineageb
424577580 8029601644 19552163b 2165 2401299631923690 40524156
(J)
1 -1- 01
1 -1- 03
15 -1- 01
15 -1 - 03
20 -1- 01
20 -1- 02
20 -1- 07
33-1-01
23 -1- 01
23 -1- 03
35 -5- 01
35 -5- 06
Uganda II
Uganda II
2
2
3
3
2
2
2
2
4
4
2
2
5
5
6
6
5
5
5
5
2
3
2
2
2
2
1
1
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
3
3
3
3
3
3
3
3
3
3
3
3
8
8
3
3
3
3
3
3
3
3
1
1
5
5
3
3
3
3
3
3
3
3
2
2
4
4
2
2
2
2
2
2
2
2
2
2
2
2
3
3
4
4
4
4
4
4
4
4
3
3
2
2
2
2
2
2
2
2
1
1
4
4
4
4
4
4
5
5
5
5
5
5
7
7
3
3
4
4
4
4
4
4
3
3
4
4
3
3
3
3
3
3
3
3
2
2
3
3
7
7
2
2
4
4
4
4
7
7
9
9
2
2
3
3
2
2
2
2
2
2
4
4
(K)
(L)
Uganda II
Uganda II
Uganda II
Uganda II
(M)
(N)
(O)
36-1-01
36-1-03
41 -1- 01
41 -1- 02
93-1-01
100-1-01
70 -1- 01
70 -1- 03
Uganda II
Uganda II
Uganda I
Uganda I
Uganda I
Uganda I
Cameroon
Cameroon
3
3
2
2
2
2
1
1
4
4
5
5
5
5
5
5
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
2
2
2
2
2
2
2
2
4
4
3
3
3
3
5
5
2
2
2
2
2
2
3
3
5
5
3
3
3
3
3
3
8
8
7
7
7
7
5
5
4
4
2
2
2
2
2
2
(P)
(Q)
aThe numerical figures refer to the number of alleles per PCR-amplified MIRU-VNTR locus [8,17].
bBlanks for K, M and N indicate unique strains i.e., those without matching strain in the database. Letters in parenthesis [(J), (K), (L), (M), (N), (O), (P) and (Q)]
represent households.
Figures under PTID (Patient Identification) column refer to the index cases (ending with 1, e.g., 1-1-01) and contact cases (ending with a numerical value > 1, e.g.,
1-1-03). For (L) and (P), a similar strain was transmitted to patients in other households.
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Probably in future, treatment of latent MTB infections
should be considered as a control strategy in high
endemic areas as it is in industrialized settings. Never-
theless, TB transmission could still be high in
Kawempe municipality; the fact that this study only
looked at one culture sample per patient, more sam-
ples from more households (and more genotyping
methods done on each sample) will be needed for con-
clusive findings.
Six clusters (2, 5, 6, 9, 12 and 15, see Figure 1), each
with two isolates (12 isolates in total) involved mem-
bers of the same household, implying possible house-
hold transmission. Two clusters (4 and 10, see Figure
1), each with four isolates, involved participants within
the same household and those outside the household
(implying a common strain among them). Seven clus-
ters (1, 7, 8, 11, 13, 14 and 15, see Figure 1), each with
two isolates, were from epidemiologically unlinked par-
ticipants. Recent transmission of PTB was found in
only 17 patients (15%) who had epidemiologically
linked strains. Overall, 17 households had patients
with similar strains but the corresponding cultures for
10 index cases were missing. Eight households had
similar MTB strains for both index and contact cases,
indicating possible transmission (see Table 2). How-
ever, household transmission could be higher than we
are reporting if the index cases for the other contacts
were available.
Conclusion
MIRU-VNTR genotyping revealed low clustering and
high diversity of MTB strains in Kawempe municipality
and confirmed earlier reports that MTB strain “Uganda
genotype” is the predominant lineage in Kampala,
Uganda. MIRU-VNTR typing with a panel of 15 loci is
applicable in a Ugandan setting, and can unravel clon-
ally complex strains into individual strains. A nation-
wide study to determine the full spectrum of circulating
MTB strains in Uganda will be helpful.
Additional material
Additional file 1: MTB strain types with reference to the MIRU-
VNTRplus database. Highlighted yellow are the households where
transmission was predicted. Patients with mixed infections are
highlighted in grey.aPTID (Patient Identification); index cases end with 1
(e.g., 1-1-01) while contact cases end with a numerical value > 1 (e.g., 1-
1-03).bBlanks refer to unique strains i.e., those without a matching strain
in the database.cThe numerical figures refer to the number of alleles per
PCR-amplified MIRU-VNTR locus [8,17].
Abbreviations
MIRU-VNTR: mycobacterial interspersed repetitive units - variable number of
tandem repeats; HGDI: Hunter Gaston Discriminatory Index.
Acknowledgements and Funding
We thank Dr. I.C. Shamputa and Dr. C. Barry for technical assistance, and Dr.
P. Supply for access to the technical guide for MIRU-VNTR genotyping.
Gratitude to Dr. S. Zalwango; the staff at the TB wards of Mulago Hospital
Complex, Kampala, Uganda; the TB laboratory team at the JCRC, Kampala;
and Ms Geraldine Nalwadda (Molecular Biology laboratory).
This study was supported in part by the Howard Hughes Medical Institute
Student Research Fellowship and the University of Pittsburgh Student Global
Travel Grant to KRD; the AIDS International Training and Research Program
at Case Western Reserve University, Grant #D43-TW000011; the Tuberculosis
Research Unit, established with Federal funds from the United Sates National
Institutes of Allergy and Infectious Diseases & the United States National
Institutes of Health and Human Services, under Contract Nos. NO1-AI-95383
and HHSN266200700022C/NO1-AI-70022; and the National Institutes of
Health Grants (R01 AI075637-01 & SRO1A1075637-4). DPK was supported by
the Fogarty International Center training support (award #U2RTW006879)
through the Clinical Operational & Health Services Research (COHRE)
Training program at the Joint Clinical Research Center, Kampala, Uganda.
Author details
1Department of Medical Microbiology, School of Biomedical Sciences,
Makerere University College of Health Sciences, Kampala, Uganda.
2Epidemiology and Biostatistics, College of Public Health, University of
Georgia, Athens, USA.3Department of Pediatrics, Boston Medical Center,
Boston University, Boston, USA.
Authors’ contributions
LN and KRD carried out the experimental procedures. LN, DPK, FAK and PRO
analyzed the data. DPK wrote the manuscript. KRD, CCW and MLJ conceived
the study, participated in its design and coordination and proofread the
manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 3 May 2011 Accepted: 11 August 2011
Published: 11 August 2011
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