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Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa
Journal Pre-proof Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa
Nolwazi Londiwe Bhembe, Ezekiel Green PII:
S1567-1348(20)30014-9
DOI:
https://doi.org/10.1016/j.meegid.2020.104182
Reference:
MEEGID 104182
To appear in:
Infection, Genetics and Evolution
Received date:
12 June 2019
Revised date:
31 December 2019
Accepted date:
6 January 2020
Please cite this article as: N.L. Bhembe and E. Green, Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa, Infection, Genetics and Evolution(2019), https://doi.org/10.1016/ j.meegid.2020.104182
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© 2019 Published by Elsevier.
Journal Pre-proof
Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa
Nolwazi Londiwe Bhembe 1* and Ezekiel Green1
Department of Biotechnology and Food Technology, Faculty of Science, University of
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1
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*To whom correspondence should be addressed;
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Johannesburg, Doornfontein, 2028, South Africa
Dr Nolwazi Londiwe Bhembe, Department of Biotechnology and Food Technology, Faculty
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of Science, University of Johannesburg, Doornfontein, 2028, South Africa.
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Tel: +276032441675.
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E-mail: [email protected]
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Journal Pre-proof ABSTRACT Drug-resistant tuberculosis prevalence is still a global challenge. Making it imperative to examine the molecular epidemiology of drug resistant tuberculosis. Molecular epidemiology methods can evaluate transmission patterns and risk factors, ascertain transmission cases of multidrug-resistant tuberculosis (MDR-TB) and furthermore determine transmission patterns in a human populace. This work focuses on MDR-TB isolates in distinguishing them into several species and genotyping the MDR-TB isolates, mainly for epidemiological studies
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using the genomic regions of difference and the spoligotyping techniques. A total of 184 deoxyribonucleic acid isolated from sputum samples that showed resistance against the two
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major first-line anti-tuberculosis drugs (Rifampicin and Isoniazid) were examined. The
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deoxyribonucleic acid samples were amplified with primers specific for each flanking region
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of the genomic regions of difference for the identification of different MTBC species. Isolates were further characterized into different lineages using the spoligotyping commercial kit. The
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M. tuberculosis species was detected in 83.7% (154/184) of the deoxyribonucleic acid
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isolates, followed by the M. caprae in 8.7% (16/184) and the least detected species was the
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M. africanum in 2.2% (4/184). Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. The predominant family (spoligotype) was the Beijing with 67.4% (124/184) strains. This study gives a general overview of drug resistant strains and the circulating strains in the Eastern Cape, South Africa and it shows that the common Mycobacteria in the province is the Beijing strain.
Keywords: tuberculosis; resistant strains; genetic variability; spoligotyping; drug-resistance
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Journal Pre-proof 1. Introduction Tuberculosis (TB) is still an encumbrance global and South Africa is one of the countries that has the highest burden (TB Statistics South Africa, 2019). Approximately 322,000 cases of active TB have been reported in South Africa in 2017 (World Health Organization, 2018a). A diminution was observed in reported TB cases, from 400,000 to 300,000 in 2014 (World Health Organization, 2015). However, there has been a noticeable increase of multidrugresistant TB (MDR-TB) leading to South Africa being part of the countries with the highest
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MDR-TB incidences globally (World Health Organization, 2015). The highest burden of rifampicin-resistant TB (RR-TB) has been reported in South Africa, as well as MDR-TB
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(World Health Organization, 2016). Multidrug-resistant TB is tuberculosis resistant to the
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two principal first line anti-TB drugs (isoniazid and rifampicin) (World Health Organization,
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2018). Approximately 490,000 people in 2016 became ill with MDR-TB globally, and about 110,000 people were resistant to rifampicin (World Health Organization, 2017). Moreover, it
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is anticipated that about 6.2% cases of the 110,000 reported as MDR-TB were XDR-TB cases
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(World Health Organization, 2018b).
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In 2014, about 35,000 multidrug-resistant TB cases were recorded in South Africa (Eyewitness News, 2017). Yet again, there were 19,000 incidences of MDR-TB reported in 2016 from South Africa (World Health Organization, 2017). Tuberculosis in general is still one of the leading, vague health problems in this country, including the Eastern Cape Province with the situation worsening irrespective of the implemented TB control programs such as the direct observed treatment strategy (DOTS) and the vaccine Bacille CalmetteGuerin (BCG) (Karim et al, 2009). The province has the fourth highest TB prevalence with approximately
662/100,000
population
infected
Organization, 2015). 3
with
TB
annually
(World
Health
Journal Pre-proof Different members of the Mycobacterium tuberculosis complex (MTBC) (Forrellad et al, 2013) instigate tuberculosis. The MTBC members are M. tuberculosis, M. africanum, M. bovis, M. bovis BCG vaccine strain, M. canettii, M. caprae, M. pinnipedii, M. microti and M. mungi infecting animals as well as humans (Smith et al, 2006; Young et al, 2008; Alexander et al, 2010). Of these entire complex members, the M. tuberculosis is the most infamous member of the MTBC, causing TB in humans, resulting in an annual report of about 1.5 million deaths (Raviglione et al, 2016). The MTBC encompasses of closely interrelated
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subspecies that have 99.9% genome sequence identity (Comas et al, 2013). The genomic region of difference is reported to be involved in host interaction and pathogenicity, this
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determines the host spectrum of individual MTBC species (Ru et al, 2017). Laboratory diagnosis of human TB relies on acid-fast staining, culture and microscopy
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mainly (Warren et al, 2006). These traditional diagnostic methods have limitations that include sensitivity, specificity and they are time consuming (Parkash et al, 2009). The latest
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tool used in South Africa (GeneXpert/RIF) can diagnose TB from sputum specimens
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identifying the susceptibility or rifampicin resistance in two hours (Rachow et al, 2011).
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However, this tool cannot differentiate between the different MTBC member and it cannot delineate the strain lineages. This is where the genotyping techniques come into play because of their ability to map disseminating strains in a specific region. Studies on molecular epidemiology of MDR-TB are vital because they offer cutting-edge methods to study spreading dynamic forces and evolutionary genetics of the pathogen with supremacy on TB control actions (Kamerbeek et al, 1997). There are several molecular methods that are used to review the molecular epidemiology of TB. These methods include Insertion Sequence 6110 (IS6110) restriction fragment length polymorphism (RFLP) typing,
spoligotyping and
mycobacterial interspersed repetitive unit variable number of tandem DNA repeats (MIRU4
Journal Pre-proof VNTR) (Gupta et al, 2014). In this study there are two tools used, the genomic regions of difference and spoligotyping. We consider these tools imperative for accurate diagnosis of TB and public health surveillance, especially in the Eastern Cape Province, where data on molecular epidemiology of MDR-TB is still scanty. Materials and Methods 2.1.
Study setting and Ethics authorization
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The Eastern Cape Province has the third highest population, with approximately 7 million people (Census, 2011) and it has the second worst burden of about 49% of the population
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living below the poverty mark in rural areas (Bradshaw et al, 2000). This province is
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bordered by Lesotho and the Free State in the north, KwaZulu Natal in the northeast and the
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Northern and Western Cape in the west (Bradshaw et al, 2000). The University of Fort Hare Research Ethics Committee (UREC) permitted this current study (issued ethical clearance
Sample collection
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2.2.
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certificate: REC-270710-028-RA Level 01).
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Samples were collected as earlier described in our previous publication (Bhembe et al, 2014); three thousand eight hundred and ten sputum specimens were collected from patients with tuberculosis symptoms in different infirmaries and clinics in the Eastern Cape Province over a period of 24 months (January 2012 to December 2013). Succinctly, 4.8% (184/3810) DNA samples were used in this present study. A written informed consent form of participants was attained and the confidentiality of the patient’s identities was secured. The Biodata of the patients, such as the gender and age group (0-14, 15-29, 30-44, 45-59, 60 and above) was also collected. The patients were summarized according to age groups of 15 year intervals into 5 groups (0-14 young people, 15-29 youth, 30-44 adults, 45-59 middle age, 60 and above 5
Journal Pre-proof which is the elderly). This grouping was achieved through the use of pivot table by age range, using the Microsoft office excel to perform the analysis and conception of data and information. The samples were transported to the Microbiology laboratory at the University of Fort Hare for further analyses. The personal identifiers of all patients used in this study are imperceptible to safeguard the identity of the patients. 2.3.
Mycobacterial culture
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In the Molecular Pathogenicity and Molecular Epidemiology Research Group (MPMERG)
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laboratory, samples were decontaminated and inoculated on Lowenstein-Jensen (LJ) slants
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that were incubated for approximately eight weeks at 37°C. Mycobacteria isolation and
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identification for each isolate was carried out by the hospitals and clinics using the acid-fast
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staining (AFB) technique.
Antibiotic susceptibility testing
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An antibiotic susceptibility test was executed in the MPMERG laboratory at the University of
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Johannesburg. Briefly, Mycobacterium colony growth from the LJ slants was transferred into
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0.85% normal saline that was in screw-cap tubes. This method was executed using the agardilution proportion method as outlined by Balows et al. (1991) for isoniazid and rifampicin with some modifications as mentioned earlier in our published work (Bhembe et al, 2014). With all sets of experimentations, the M. tuberculosis (H37Rv) strain was used as a positive control. 2.5.
Deoxyribonucleic acid (DNA) isolation
To isolate DNA, the AFB positive isolates were heat-killed by incubating the cells in a water bath for an hour at the temperature of 80o C following the protocol defined by Berg et al.
6
Journal Pre-proof (2009). A volume of 200 µl fractions of the supernatant was aliquated into sterile 1.5 ml Eppendorf tube for each isolate and kept in a -80o C freezer. 2.6.
Molecular detection of MDR-TB
One hundred and eighty-four (184) DNA isolates were confirmed to be MTBC members with the Seeplex® MTB Nested Ace detection assay (Seegene Inc, Korea) following the manufacturer’s instructions. The constituted polymerase chain reaction (PCR) mixture was
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amplified using the MyCyclerT M thermal cycle (Bio-Rad, Cape Town, South Africa) and the
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amplification products were viewed with the Alliance 4.7 transilluminator (UVITEC Limited,
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Cambridge, UK). The DNA isolates were further amplified through a multiplex PCR with
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four primer sets per reaction. For this study, 3 primers per primer set (RD1, RD4, RD9 and RD12) as described by Warren et al. (2006) with some modifications were used. Briefly, each
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PCR reaction contained 2 μl DNA template, 5 μl Q-buffer, 2.5 μl of 10X buffer, 2 μl of 25
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mM MgCl2 , 4 μl of the 10 mM dNTPs, 0.5 μl of each primer (50 pmol/μl) 0.125 μl HotStarTaq DNA polymerase (Fermentas, Thermo Scientific, US) and sterile nuclease free
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H2 O was added to a total volume of 25 μl. The H37Rv (ATCC 27294), strain was used as a
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positive control and sterile H2 O was used as a negative control. Amplification was attained using the MyCyclerT M thermal cycler (BioRad, Cape Town, South Africa). The cycling conditions comprised of 1 cycle at 95o C for 10 minutes (Taq activation cycle), followed by 1 cycle denaturation at 95o C for 15 minutes, 45 cycles of denaturation at 94 o C for 1 minute, primer annealing at 62o C for 1 minute and extension at 72o C for 1 minute followed by 1 cycle of the final extension at 72o C for 10 minutes. The amplified products were electrophoretically segregated on 3.0% agarose gel containing 5 μl Ethidium bromide at 70V for 4 hours. The size of amplicons was estimated by comparison with a 100 base pair (bp) DNA ladder (Thermo Scientific). Images were visualized with a 4.7 7
Journal Pre-proof Alliance
transilluminator
(UVITEC
Limited,
Cambridge,
UK) and
the
results were
interpreted as described by Warren et al. (2006). 2.7.
Sub-lineage classification of MDR-TB isolates and data analysis
The isolates were further characterized into different strains using the spoligotyping commercial kit (Isogene Life Science B.V., Utrecht, The Netherlands) following the protocol as described by Kamerbeek et al. (1997). The genotypes were articulated in binary code and
the MIRU-VNTRplus (http://www.miru-vntrplus.org) (Weniger et al, 2010) and
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into
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octal formats in Microsoft Excel spreadsheets and all the spoligotyping data was deposited
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SpolDB4.0 database (Brudey et al, 2006). The SPOTCLUST (a combination of models) by
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their spacer oligonucleotide typing patterns. 3. Results
Isolation, identification and susceptibility profiles
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3.1.
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Vitol et al. (2006) was used to determine the strain families of the MDR-TB isolates based on
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The 4.8% (184/3810) of the sputum samples investigated in this present study was resistant to
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both the two major first line anti-TB drugs (rifampicin and isoniazid). Multidrug-resistant TB was identified in all 184 samples that were targeted during sample collection. This study explicates the different MTBC members and the lineages detected in different age groups of the study population. We observed that the M. tuberculosis was the most detected species with most patients within the age group range of 15 to 29 years, 35.3% (65/184) followed by those in the age group 30 to 44 years 31.5% (58/184) (Table 1). 3.2.
Differentiation of MDR-TB isolates using RDs
The different amplification products matched the expected base pairs, which designate the presence and absence of the genomic regions of difference (RD1, RD4, RD9 and RD12). It 8
Journal Pre-proof was possible to differentiate between Mycobacterium tuberculosis, which showed four different band sizes (146 bp, 172 bp, 235 bp and 369 bp) and the M. canettii species showed three different band sizes (146 bp, 172 bp and 235 bp). However, it was again possible to differentiate between the M. caprae, which showed two bands (146 bp and 172 bp) and M. africanum species that showed three bands (146 bp, 172 bp and 369 bp) using the primer sets reported by Warren et al. (2006). However, these species were not the only ones detected;
Delineation of MTBC isolates
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3.3.
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the M. bovis species was also identified in this study as shown in Table 2.
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The results show the possibility of differentiating between the different Mycobacterium
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tuberculosis complex members M. tuberculosis, M. caprae, M. bovis, M. africanum and M. canettii. The M. tuberculosis species were detected in 83.7% (154/184) of the DNA isolates
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followed by M. caprae in 8.7% (16/184) and the least detected species was M. africanum in
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2.2% (4/184) (Table 2). Nevertheless, in this study, we were able to detect species that are known to be primarily hosted by humans (M. tuberculosis, M. africanum and M. canettii) and
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species known to be primarily hosted by animals such as goats (M. caprae) and cattle (M.
3.4.
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bovis) from human sputum specimens (Table 2). Distribution of the different genotypes
Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates, with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. However, the TBinsight SPOTCLUST (SpolDB3-based model) was able to identify the families of the isolates that were not found in the database as well as the orphans. A total of 17 families were identified: with the Beijing 67.4% (124/184) as the most prevalent family and the S, BOV3
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Journal Pre-proof and AFRI were the least identified families with only one sample in each family 0.5% (1/184) (Table 3). 4. Discussion The epidemiology of TB has indicated that TB spread from one person/ animal to the next through the air by nuclei droplets produced when the infected person with either pulmonary or laryngeal TB, sneezes or coughs (National tuberculosis Management Guidelines 2014).
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This mode of transmission is more prevalent in a certain social state of affairs (for instance,
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food insecurity and malnutrition, geographic and cultural hindrances to health maintenance
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access and underprivileged housing and environmental conditions) present in South Africa
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(Hargreaves et al. 2011).
investigation
and
control
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Information on molecular epidemiology of MDR-TB is valuable as it supports TB program
of
countries.
However,
MDR-TB
molecular
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epidemiological data of the Eastern Cape Province is still meagre. For better understanding of
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the moving and intensifying clusters of MDR-TB within the province, we have characterized
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the mycobacteria isolated from TB patients using the genomic region of difference and the spoligotyping tools. In this study, the circulating strains in the province were known and their drug sensitivity patterns defined.
The genomic region of difference using RD1, RD4, RD9 and RD12 designated the studied strains as M. tuberculosis, M. bovis, M. caprae, M. canettii and M. africanum (Table1). The M. tuberculosis species proved to be the most dominant that infected most of the TB patients. However, most of the patients infected with MDR-TB were between the age groups of 30 to 44 years 42.4% (78/184) and 15 to 29 years 38% (70/184). This could be due to the economical activeness of people at this ages. Yet again, in this study more females than males are reported to be infected with M. tuberculosis, which is different from what has been 10
Journal Pre-proof reported by other studies, a higher male ratio as compared to females infected with M. tuberculosis (Murray, 1991; Hudelson, 1996). However, this could also be due to selection bias where more female samples than those of males as reported before (Bhembe et al, 2014) were collected from the clinics and hospitals. We noted that species known to be primary hosted by animals were also detected from the samples gathered from human sputum. This shows the possibility of transmission of MTBC
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from animals to humans and vice versa. Nevertheless, this is not easy to learn using the
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spoligotyping tool; so, it needs further discriminatory techniques, which will be utilized for
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future studies on these isolates. Tuberculosis is endemic in South Africa and identifying the
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circulating strains within populations is essential and is important for understanding the epidemiology of the infection in a region, along with supporting the initiation of better
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control programs that will avoid the expansion of MDR-TB at both local and global level. In 2016, the World Health Organization (World Health Organization, 2016) reported about
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19,613 patients detected with rifampicin-resistant TB in South Africa. This number indicated
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a substantial growth from the 2011 reported prevalence of about 13,000 rifampicin-resistant
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cases in this country (World Health Organization, 2016). It is not surprising to find 83.7% of M. tuberculosis in this study samples because this species is primarily hosted by humans. However, the prevalence of TB by this strain proves to be high, which is a problem in the public health domain. There are several reports in South Africa in addition to other countries of the M. tuberculosis Beijing strain reported amongst cases of drug-resistant MDR-TB (Glynn et al, 2002; Stavrum et al, 2009). Previous studies reported in the Eastern and Western Cape Provinces postulate that there is an association of MDR-TB and the Beijing family strains (Bifani et al, 2002; Johnson et al, 2006; Cliota et al,
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Journal Pre-proof 2012; Klopper et al, 2013). However, we cannot prove the association of the Beijing strain and MDR-TB. Results from the SITVIT database and spoligotyping international typing (SIT) number indicates that the Beijing strain is the most prevalent shared type in this study. The strain diversity detected is generally constant with studies reported from other parts of South Africa (Chiota et al, 2007; Mlambeo et al, 2008; Marais et al, 2013). The LAM genotype is the
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second predominant strain in this study. This is not startling because this genotype has been
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reported to be prevalent in the Eastern and Western Cape Provinces of South Africa (Stavrum
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et al, 2009), which could be one of the reasons of the prevalence of this genotype reported in
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this study. This could suggest a continuing spread of this strain in the province.
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The X lineage was also detected in this study (Table 3); this lineage is prevalent in the Northern and Western Cape Provinces (Stavrum et al, 2009). This could mean the strain has
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been transmitted from these provinces, because these provinces borders the Eastern Cape
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Province; the Western Cape Province border this province from the west and the Northern
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Cape Province border this province in the north-west of the escarpment. This study reports 2.7% (5/184) of the BOV (M. bovis subsp. bovis respectively). The primary host of M. bovis is cattle, but a variety of domestic animals, wild animals along with humans may possibly be infected with this species (Ayele et al, 2004; Gibson et al, 2004). Most human cases of bovine TB are observed in countries where bovine TB is not controlled, mostly affecting young people subsequently from either drinking or handling contaminated milk (Cosivi et al, 1998). Data on human TB instigated by the M. bovis species in both industrialized and unindustrialized countries is deficient. A study by Elsabban et al. (1992) showed the 12
Journal Pre-proof proportion of sputum-positive TB patients infected with M. bovis to be 0.4%, 5.4% and 6.4%, while Nafeh et al. (1992) reported that nine out of twenty indiscriminately chosen patients with TB peritonitis were infected with M. bovis and the rest with M. tuberculosis. The M. bovis subsp. M. caprae species was also found in 1.1% (2/184) of the isolates. The species is a causative agent of TB in caprine herds (Duet et al, 2008). This is not surprising as the Eastern Cape Province has the largest numbers of goats in South Africa (Statistics South
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Africa, 2014). This pathogen was isolated from cattle in Central Europe (Pavlik et al, 2002)
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and pigs (Kongpetchsatit et al, 2006). There are less than 1% reported TB cases caused by
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this species in industrialized countries, but other areas or population might experience a
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higher prevalence (Amafu 2006; Rodwell et al, 2008). A study by Bhembe et al. (2017) also reported on this species isolated from slaughtered cattle lymph nodes in the Eastern Cape
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Province. These results may possibly suggest transmission of this pathogen from livestock to humans. The connection between humans and animals has indicated that the mode of
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contagion, such as the use of unpasteurized milk and the proximity between mammals occurs
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between man and animals (McDaniel et al, 2014).
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This study also reports on the AFRI lineage 2.2% (4/184) belonging to the M. africanum species which was detected from the isolates. This lineage has been reported as a prevailing M. bovis clonal complex (Egbe et al, 2017). The M. africanum (subtype I) has traits that resemble those of M. bovis and is widespread in West Africa (Mostowy et al, 2004). The possible route of transmission of M. africanum may perhaps be through the movement of persons from one region to another. Family 33 is also reported in this study with the prevalence of 2.2% (4/184) reported to be belonging to the Euro-American lineage (MagugaPhasha et al, 2017). It is not the first time this family is reported in South Africa, it has been
13
Journal Pre-proof reported in the Limpopo Province (Maguga-Phasha et al, 2017) and the Eastern Cape Province (Bhembe et al, 2017). The T family has also been reported in different provinces of South Africa, which includes the Free State (van Dijk et al, 2016) and the Eastern Cape (Stavrum et al, 2009); which was described as the most predominant family in the province. This present study reports a prevalence of 1.1% (2/184) of the T family. This could mean there is continuing transmission
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of this strain, although it is at a marginal rate. The least detected family in this study is the S
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family 0.5% (1/184); this is not surprising, because the family has been reported with limited
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dissemination through African countries (Mbugi et al, 2016).
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The SpolDB4 database also identified the Manu 1.1% (2/184) and Canettii 2.7% (5/184)
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families. The Canettii family is associated with the M. canettii species. The M. canettii species is a sporadic, smooth, and a variant of the M. tuberculosis commonly isolated in
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Africa from humans (Fabre et al, 2010). The species has been reported worldwide and the
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5. Conclusions
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majority of reported cases are from patients who have either travelled or lived in Africa.
This study validates that the Beijing strain is the prevalent strain disseminating in the Eastern Cape Province. The results of this study denote a reference point of the population structure in the province and may serve, as a drive for future molecular studies. Genotyping of multidrug-resistant Mycobacterium tuberculosis strains is highly imperative for multidrugresistant tuberculosis control as it allows the detection of alleged occurrences and transmission chains. This study evidences that multidrug-resistant TB is instigated by diverse species of Mycobacterium causing the infection in humans, which together form the Mycobacterium tuberculosis Complex. 14
Journal Pre-proof Ethical considerations The experiment was designed and conducted in agreement with the ethical guidelines highlighted by the National Committee for Research Ethics in Science and Technology of South Africa and all sampling and analytical procedures were followed as documented in the University of Fort Hare’s health and safety guidelines. Acknowledgements
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The authors extend their gratitude to the National Research Foundation (NRF) of South
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Africa (grant number: 76255) for a subvention, the University of Fort Hare and the
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University of Johannesburg for letting us use their amenities.
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Conflict of interest
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No conflict of interest declared. References
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Journal Pre-proof
Tables Table 1 Demographic profile of the study population defined using genomic regions of difference Age (years)
Gender of patients with species detected M. bovis
M. caprae
M. canettii
M. africanum
Male
Female
Male
Female
Male
Total
1
0
0
0
0
5
0
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1
1
0
0
70
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Male
Female
Male
Female
0-14
3
1
0
0
0
15-29
38
27
0
0
3
30-44
36
22
0
3
6
5
2
1
2
1
78
45-59
6
17
1
1
1
0
0
0
1
0
27
60+
3
1
0
0
0
0
0
0
0
4
Total
86
68
1
10
6
3
2
3
1
184
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Female
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f
M. tuberculosis
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0
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4
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Journal Pre-proof Table 2 Segregation of MTBC into species as identified following genomic regions of difference Number of DNA
isolated from humans
isolates
Humans
154
83.7%
M. caprae
Goats
16
8.7%
M. bovis
Cattle
5
2.7%
M. africanum
Humans
M. canettii
Humans
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M. tuberculosis
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Primary host, but
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Total
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Percentage
4
2.2%
5
2.7%
184
100%
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Species identified
Journal Pre-proof Table 3 Prevalence of the two major first line anti-TB drugs of the detected genotypes in the Eastern Cape Province of South Africa Family
East-Asian Euro-American Indo-Oceanic Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Indo-Oceanic Euro-American Euro-American Euro-American Euro-American M. bovis M. bovis M. africanum M. bovis M. canettii Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American
Beijing LAM4 X1 X3 LAM3 T1 S LAM9 LAM4 MANU2 X3 X3 X3-variant1 LAM2 BOV1 BOV3 AFRI Bov_4 Caprea Canettii X3 LAM9 Family33 T1 Family33 X3 Family33 Family33 LAM3 LAM9
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pr
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f
SpolDB4 ID
rn
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Pr
Octal code (spoligotype description) 000000000003771 777777607760731 777777377670371 300076777760771 776177607760771 777777777760771 776377777760771 777777607760771 777767607760731 777777607763771 700076777760771 700076777760671 700036777760771 677717607760771 676773777777600 640013777777600 770777777777671 277773777377600 000000000101000 500046637740661 777567607760771 777357757763771 557347637740661 557347607743661 100046637740771 776147607763771 300076777763771 756177607760771 557347607740661
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Shared International Type (SIT) 1 60 1329 2286 33 53 34 42 1750 1247 92 70 91 2271 482 479 181 647 592 ORPHAN ORPHAN Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Total
Prevalence no (%) 124 (67.4) 11 (6) 1 (0.5) 6 (3.3) 5 (2.7) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 2 (1.1) 2 (1.1) 1 (0.5) 1 (0.5) 1 (0.5) 4 (2.2) 1 (0.5) 4 (2.2) 2 (1.1) 5 (2.7) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 184 (100)
25
Journal Pre-proof Credit author statement
Nolwazi Londiwe Bhembe: Data curation, Investigation, Formal analysis, Validation, Visualization, Writing - Original draft preparation.
Ezekiel Green: Conceptualization,
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Methodology, Software, Resources, Supervision.
26
Journal Pre-proof Highlights
This study gives a general overview of multidrug-resistant strains and circulating strains present in the Eastern Cape, South Africa.
Diverse spoligotype patterns were detected in this study, which delineates a high diversity of Mycobacterium tuberculosis strains disseminating in the Eastern Cape Province.
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The Beijing strain was the predominant strain among the multidrug-resistant
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tuberculosis isolates.
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Nolwazi Londiwe Bhembe, Ezekiel Green PII:
S1567-1348(20)30014-9
DOI:
https://doi.org/10.1016/j.meegid.2020.104182
Reference:
MEEGID 104182
To appear in:
Infection, Genetics and Evolution
Received date:
12 June 2019
Revised date:
31 December 2019
Accepted date:
6 January 2020
Please cite this article as: N.L. Bhembe and E. Green, Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa, Infection, Genetics and Evolution(2019), https://doi.org/10.1016/ j.meegid.2020.104182
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© 2019 Published by Elsevier.
Journal Pre-proof
Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa
Nolwazi Londiwe Bhembe 1* and Ezekiel Green1
Department of Biotechnology and Food Technology, Faculty of Science, University of
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1
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*To whom correspondence should be addressed;
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Johannesburg, Doornfontein, 2028, South Africa
Dr Nolwazi Londiwe Bhembe, Department of Biotechnology and Food Technology, Faculty
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of Science, University of Johannesburg, Doornfontein, 2028, South Africa.
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Tel: +276032441675.
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E-mail: [email protected]
1
Journal Pre-proof ABSTRACT Drug-resistant tuberculosis prevalence is still a global challenge. Making it imperative to examine the molecular epidemiology of drug resistant tuberculosis. Molecular epidemiology methods can evaluate transmission patterns and risk factors, ascertain transmission cases of multidrug-resistant tuberculosis (MDR-TB) and furthermore determine transmission patterns in a human populace. This work focuses on MDR-TB isolates in distinguishing them into several species and genotyping the MDR-TB isolates, mainly for epidemiological studies
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using the genomic regions of difference and the spoligotyping techniques. A total of 184 deoxyribonucleic acid isolated from sputum samples that showed resistance against the two
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major first-line anti-tuberculosis drugs (Rifampicin and Isoniazid) were examined. The
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deoxyribonucleic acid samples were amplified with primers specific for each flanking region
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of the genomic regions of difference for the identification of different MTBC species. Isolates were further characterized into different lineages using the spoligotyping commercial kit. The
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M. tuberculosis species was detected in 83.7% (154/184) of the deoxyribonucleic acid
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isolates, followed by the M. caprae in 8.7% (16/184) and the least detected species was the
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M. africanum in 2.2% (4/184). Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. The predominant family (spoligotype) was the Beijing with 67.4% (124/184) strains. This study gives a general overview of drug resistant strains and the circulating strains in the Eastern Cape, South Africa and it shows that the common Mycobacteria in the province is the Beijing strain.
Keywords: tuberculosis; resistant strains; genetic variability; spoligotyping; drug-resistance
2
Journal Pre-proof 1. Introduction Tuberculosis (TB) is still an encumbrance global and South Africa is one of the countries that has the highest burden (TB Statistics South Africa, 2019). Approximately 322,000 cases of active TB have been reported in South Africa in 2017 (World Health Organization, 2018a). A diminution was observed in reported TB cases, from 400,000 to 300,000 in 2014 (World Health Organization, 2015). However, there has been a noticeable increase of multidrugresistant TB (MDR-TB) leading to South Africa being part of the countries with the highest
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MDR-TB incidences globally (World Health Organization, 2015). The highest burden of rifampicin-resistant TB (RR-TB) has been reported in South Africa, as well as MDR-TB
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(World Health Organization, 2016). Multidrug-resistant TB is tuberculosis resistant to the
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two principal first line anti-TB drugs (isoniazid and rifampicin) (World Health Organization,
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2018). Approximately 490,000 people in 2016 became ill with MDR-TB globally, and about 110,000 people were resistant to rifampicin (World Health Organization, 2017). Moreover, it
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is anticipated that about 6.2% cases of the 110,000 reported as MDR-TB were XDR-TB cases
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(World Health Organization, 2018b).
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In 2014, about 35,000 multidrug-resistant TB cases were recorded in South Africa (Eyewitness News, 2017). Yet again, there were 19,000 incidences of MDR-TB reported in 2016 from South Africa (World Health Organization, 2017). Tuberculosis in general is still one of the leading, vague health problems in this country, including the Eastern Cape Province with the situation worsening irrespective of the implemented TB control programs such as the direct observed treatment strategy (DOTS) and the vaccine Bacille CalmetteGuerin (BCG) (Karim et al, 2009). The province has the fourth highest TB prevalence with approximately
662/100,000
population
infected
Organization, 2015). 3
with
TB
annually
(World
Health
Journal Pre-proof Different members of the Mycobacterium tuberculosis complex (MTBC) (Forrellad et al, 2013) instigate tuberculosis. The MTBC members are M. tuberculosis, M. africanum, M. bovis, M. bovis BCG vaccine strain, M. canettii, M. caprae, M. pinnipedii, M. microti and M. mungi infecting animals as well as humans (Smith et al, 2006; Young et al, 2008; Alexander et al, 2010). Of these entire complex members, the M. tuberculosis is the most infamous member of the MTBC, causing TB in humans, resulting in an annual report of about 1.5 million deaths (Raviglione et al, 2016). The MTBC encompasses of closely interrelated
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subspecies that have 99.9% genome sequence identity (Comas et al, 2013). The genomic region of difference is reported to be involved in host interaction and pathogenicity, this
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determines the host spectrum of individual MTBC species (Ru et al, 2017). Laboratory diagnosis of human TB relies on acid-fast staining, culture and microscopy
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mainly (Warren et al, 2006). These traditional diagnostic methods have limitations that include sensitivity, specificity and they are time consuming (Parkash et al, 2009). The latest
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tool used in South Africa (GeneXpert/RIF) can diagnose TB from sputum specimens
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identifying the susceptibility or rifampicin resistance in two hours (Rachow et al, 2011).
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However, this tool cannot differentiate between the different MTBC member and it cannot delineate the strain lineages. This is where the genotyping techniques come into play because of their ability to map disseminating strains in a specific region. Studies on molecular epidemiology of MDR-TB are vital because they offer cutting-edge methods to study spreading dynamic forces and evolutionary genetics of the pathogen with supremacy on TB control actions (Kamerbeek et al, 1997). There are several molecular methods that are used to review the molecular epidemiology of TB. These methods include Insertion Sequence 6110 (IS6110) restriction fragment length polymorphism (RFLP) typing,
spoligotyping and
mycobacterial interspersed repetitive unit variable number of tandem DNA repeats (MIRU4
Journal Pre-proof VNTR) (Gupta et al, 2014). In this study there are two tools used, the genomic regions of difference and spoligotyping. We consider these tools imperative for accurate diagnosis of TB and public health surveillance, especially in the Eastern Cape Province, where data on molecular epidemiology of MDR-TB is still scanty. Materials and Methods 2.1.
Study setting and Ethics authorization
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The Eastern Cape Province has the third highest population, with approximately 7 million people (Census, 2011) and it has the second worst burden of about 49% of the population
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living below the poverty mark in rural areas (Bradshaw et al, 2000). This province is
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bordered by Lesotho and the Free State in the north, KwaZulu Natal in the northeast and the
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Northern and Western Cape in the west (Bradshaw et al, 2000). The University of Fort Hare Research Ethics Committee (UREC) permitted this current study (issued ethical clearance
Sample collection
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2.2.
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certificate: REC-270710-028-RA Level 01).
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Samples were collected as earlier described in our previous publication (Bhembe et al, 2014); three thousand eight hundred and ten sputum specimens were collected from patients with tuberculosis symptoms in different infirmaries and clinics in the Eastern Cape Province over a period of 24 months (January 2012 to December 2013). Succinctly, 4.8% (184/3810) DNA samples were used in this present study. A written informed consent form of participants was attained and the confidentiality of the patient’s identities was secured. The Biodata of the patients, such as the gender and age group (0-14, 15-29, 30-44, 45-59, 60 and above) was also collected. The patients were summarized according to age groups of 15 year intervals into 5 groups (0-14 young people, 15-29 youth, 30-44 adults, 45-59 middle age, 60 and above 5
Journal Pre-proof which is the elderly). This grouping was achieved through the use of pivot table by age range, using the Microsoft office excel to perform the analysis and conception of data and information. The samples were transported to the Microbiology laboratory at the University of Fort Hare for further analyses. The personal identifiers of all patients used in this study are imperceptible to safeguard the identity of the patients. 2.3.
Mycobacterial culture
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In the Molecular Pathogenicity and Molecular Epidemiology Research Group (MPMERG)
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laboratory, samples were decontaminated and inoculated on Lowenstein-Jensen (LJ) slants
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that were incubated for approximately eight weeks at 37°C. Mycobacteria isolation and
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identification for each isolate was carried out by the hospitals and clinics using the acid-fast
2.4.
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staining (AFB) technique.
Antibiotic susceptibility testing
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An antibiotic susceptibility test was executed in the MPMERG laboratory at the University of
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Johannesburg. Briefly, Mycobacterium colony growth from the LJ slants was transferred into
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0.85% normal saline that was in screw-cap tubes. This method was executed using the agardilution proportion method as outlined by Balows et al. (1991) for isoniazid and rifampicin with some modifications as mentioned earlier in our published work (Bhembe et al, 2014). With all sets of experimentations, the M. tuberculosis (H37Rv) strain was used as a positive control. 2.5.
Deoxyribonucleic acid (DNA) isolation
To isolate DNA, the AFB positive isolates were heat-killed by incubating the cells in a water bath for an hour at the temperature of 80o C following the protocol defined by Berg et al.
6
Journal Pre-proof (2009). A volume of 200 µl fractions of the supernatant was aliquated into sterile 1.5 ml Eppendorf tube for each isolate and kept in a -80o C freezer. 2.6.
Molecular detection of MDR-TB
One hundred and eighty-four (184) DNA isolates were confirmed to be MTBC members with the Seeplex® MTB Nested Ace detection assay (Seegene Inc, Korea) following the manufacturer’s instructions. The constituted polymerase chain reaction (PCR) mixture was
f
amplified using the MyCyclerT M thermal cycle (Bio-Rad, Cape Town, South Africa) and the
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amplification products were viewed with the Alliance 4.7 transilluminator (UVITEC Limited,
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Cambridge, UK). The DNA isolates were further amplified through a multiplex PCR with
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four primer sets per reaction. For this study, 3 primers per primer set (RD1, RD4, RD9 and RD12) as described by Warren et al. (2006) with some modifications were used. Briefly, each
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PCR reaction contained 2 μl DNA template, 5 μl Q-buffer, 2.5 μl of 10X buffer, 2 μl of 25
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mM MgCl2 , 4 μl of the 10 mM dNTPs, 0.5 μl of each primer (50 pmol/μl) 0.125 μl HotStarTaq DNA polymerase (Fermentas, Thermo Scientific, US) and sterile nuclease free
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H2 O was added to a total volume of 25 μl. The H37Rv (ATCC 27294), strain was used as a
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positive control and sterile H2 O was used as a negative control. Amplification was attained using the MyCyclerT M thermal cycler (BioRad, Cape Town, South Africa). The cycling conditions comprised of 1 cycle at 95o C for 10 minutes (Taq activation cycle), followed by 1 cycle denaturation at 95o C for 15 minutes, 45 cycles of denaturation at 94 o C for 1 minute, primer annealing at 62o C for 1 minute and extension at 72o C for 1 minute followed by 1 cycle of the final extension at 72o C for 10 minutes. The amplified products were electrophoretically segregated on 3.0% agarose gel containing 5 μl Ethidium bromide at 70V for 4 hours. The size of amplicons was estimated by comparison with a 100 base pair (bp) DNA ladder (Thermo Scientific). Images were visualized with a 4.7 7
Journal Pre-proof Alliance
transilluminator
(UVITEC
Limited,
Cambridge,
UK) and
the
results were
interpreted as described by Warren et al. (2006). 2.7.
Sub-lineage classification of MDR-TB isolates and data analysis
The isolates were further characterized into different strains using the spoligotyping commercial kit (Isogene Life Science B.V., Utrecht, The Netherlands) following the protocol as described by Kamerbeek et al. (1997). The genotypes were articulated in binary code and
the MIRU-VNTRplus (http://www.miru-vntrplus.org) (Weniger et al, 2010) and
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into
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octal formats in Microsoft Excel spreadsheets and all the spoligotyping data was deposited
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SpolDB4.0 database (Brudey et al, 2006). The SPOTCLUST (a combination of models) by
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their spacer oligonucleotide typing patterns. 3. Results
Isolation, identification and susceptibility profiles
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3.1.
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Vitol et al. (2006) was used to determine the strain families of the MDR-TB isolates based on
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The 4.8% (184/3810) of the sputum samples investigated in this present study was resistant to
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both the two major first line anti-TB drugs (rifampicin and isoniazid). Multidrug-resistant TB was identified in all 184 samples that were targeted during sample collection. This study explicates the different MTBC members and the lineages detected in different age groups of the study population. We observed that the M. tuberculosis was the most detected species with most patients within the age group range of 15 to 29 years, 35.3% (65/184) followed by those in the age group 30 to 44 years 31.5% (58/184) (Table 1). 3.2.
Differentiation of MDR-TB isolates using RDs
The different amplification products matched the expected base pairs, which designate the presence and absence of the genomic regions of difference (RD1, RD4, RD9 and RD12). It 8
Journal Pre-proof was possible to differentiate between Mycobacterium tuberculosis, which showed four different band sizes (146 bp, 172 bp, 235 bp and 369 bp) and the M. canettii species showed three different band sizes (146 bp, 172 bp and 235 bp). However, it was again possible to differentiate between the M. caprae, which showed two bands (146 bp and 172 bp) and M. africanum species that showed three bands (146 bp, 172 bp and 369 bp) using the primer sets reported by Warren et al. (2006). However, these species were not the only ones detected;
Delineation of MTBC isolates
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3.3.
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the M. bovis species was also identified in this study as shown in Table 2.
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The results show the possibility of differentiating between the different Mycobacterium
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tuberculosis complex members M. tuberculosis, M. caprae, M. bovis, M. africanum and M. canettii. The M. tuberculosis species were detected in 83.7% (154/184) of the DNA isolates
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followed by M. caprae in 8.7% (16/184) and the least detected species was M. africanum in
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2.2% (4/184) (Table 2). Nevertheless, in this study, we were able to detect species that are known to be primarily hosted by humans (M. tuberculosis, M. africanum and M. canettii) and
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species known to be primarily hosted by animals such as goats (M. caprae) and cattle (M.
3.4.
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bovis) from human sputum specimens (Table 2). Distribution of the different genotypes
Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates, with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. However, the TBinsight SPOTCLUST (SpolDB3-based model) was able to identify the families of the isolates that were not found in the database as well as the orphans. A total of 17 families were identified: with the Beijing 67.4% (124/184) as the most prevalent family and the S, BOV3
9
Journal Pre-proof and AFRI were the least identified families with only one sample in each family 0.5% (1/184) (Table 3). 4. Discussion The epidemiology of TB has indicated that TB spread from one person/ animal to the next through the air by nuclei droplets produced when the infected person with either pulmonary or laryngeal TB, sneezes or coughs (National tuberculosis Management Guidelines 2014).
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This mode of transmission is more prevalent in a certain social state of affairs (for instance,
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food insecurity and malnutrition, geographic and cultural hindrances to health maintenance
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access and underprivileged housing and environmental conditions) present in South Africa
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(Hargreaves et al. 2011).
investigation
and
control
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Information on molecular epidemiology of MDR-TB is valuable as it supports TB program
of
countries.
However,
MDR-TB
molecular
al
epidemiological data of the Eastern Cape Province is still meagre. For better understanding of
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the moving and intensifying clusters of MDR-TB within the province, we have characterized
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the mycobacteria isolated from TB patients using the genomic region of difference and the spoligotyping tools. In this study, the circulating strains in the province were known and their drug sensitivity patterns defined.
The genomic region of difference using RD1, RD4, RD9 and RD12 designated the studied strains as M. tuberculosis, M. bovis, M. caprae, M. canettii and M. africanum (Table1). The M. tuberculosis species proved to be the most dominant that infected most of the TB patients. However, most of the patients infected with MDR-TB were between the age groups of 30 to 44 years 42.4% (78/184) and 15 to 29 years 38% (70/184). This could be due to the economical activeness of people at this ages. Yet again, in this study more females than males are reported to be infected with M. tuberculosis, which is different from what has been 10
Journal Pre-proof reported by other studies, a higher male ratio as compared to females infected with M. tuberculosis (Murray, 1991; Hudelson, 1996). However, this could also be due to selection bias where more female samples than those of males as reported before (Bhembe et al, 2014) were collected from the clinics and hospitals. We noted that species known to be primary hosted by animals were also detected from the samples gathered from human sputum. This shows the possibility of transmission of MTBC
f
from animals to humans and vice versa. Nevertheless, this is not easy to learn using the
oo
spoligotyping tool; so, it needs further discriminatory techniques, which will be utilized for
pr
future studies on these isolates. Tuberculosis is endemic in South Africa and identifying the
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circulating strains within populations is essential and is important for understanding the epidemiology of the infection in a region, along with supporting the initiation of better
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control programs that will avoid the expansion of MDR-TB at both local and global level. In 2016, the World Health Organization (World Health Organization, 2016) reported about
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19,613 patients detected with rifampicin-resistant TB in South Africa. This number indicated
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a substantial growth from the 2011 reported prevalence of about 13,000 rifampicin-resistant
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cases in this country (World Health Organization, 2016). It is not surprising to find 83.7% of M. tuberculosis in this study samples because this species is primarily hosted by humans. However, the prevalence of TB by this strain proves to be high, which is a problem in the public health domain. There are several reports in South Africa in addition to other countries of the M. tuberculosis Beijing strain reported amongst cases of drug-resistant MDR-TB (Glynn et al, 2002; Stavrum et al, 2009). Previous studies reported in the Eastern and Western Cape Provinces postulate that there is an association of MDR-TB and the Beijing family strains (Bifani et al, 2002; Johnson et al, 2006; Cliota et al,
11
Journal Pre-proof 2012; Klopper et al, 2013). However, we cannot prove the association of the Beijing strain and MDR-TB. Results from the SITVIT database and spoligotyping international typing (SIT) number indicates that the Beijing strain is the most prevalent shared type in this study. The strain diversity detected is generally constant with studies reported from other parts of South Africa (Chiota et al, 2007; Mlambeo et al, 2008; Marais et al, 2013). The LAM genotype is the
f
second predominant strain in this study. This is not startling because this genotype has been
oo
reported to be prevalent in the Eastern and Western Cape Provinces of South Africa (Stavrum
pr
et al, 2009), which could be one of the reasons of the prevalence of this genotype reported in
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this study. This could suggest a continuing spread of this strain in the province.
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The X lineage was also detected in this study (Table 3); this lineage is prevalent in the Northern and Western Cape Provinces (Stavrum et al, 2009). This could mean the strain has
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been transmitted from these provinces, because these provinces borders the Eastern Cape
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Province; the Western Cape Province border this province from the west and the Northern
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Cape Province border this province in the north-west of the escarpment. This study reports 2.7% (5/184) of the BOV (M. bovis subsp. bovis respectively). The primary host of M. bovis is cattle, but a variety of domestic animals, wild animals along with humans may possibly be infected with this species (Ayele et al, 2004; Gibson et al, 2004). Most human cases of bovine TB are observed in countries where bovine TB is not controlled, mostly affecting young people subsequently from either drinking or handling contaminated milk (Cosivi et al, 1998). Data on human TB instigated by the M. bovis species in both industrialized and unindustrialized countries is deficient. A study by Elsabban et al. (1992) showed the 12
Journal Pre-proof proportion of sputum-positive TB patients infected with M. bovis to be 0.4%, 5.4% and 6.4%, while Nafeh et al. (1992) reported that nine out of twenty indiscriminately chosen patients with TB peritonitis were infected with M. bovis and the rest with M. tuberculosis. The M. bovis subsp. M. caprae species was also found in 1.1% (2/184) of the isolates. The species is a causative agent of TB in caprine herds (Duet et al, 2008). This is not surprising as the Eastern Cape Province has the largest numbers of goats in South Africa (Statistics South
f
Africa, 2014). This pathogen was isolated from cattle in Central Europe (Pavlik et al, 2002)
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and pigs (Kongpetchsatit et al, 2006). There are less than 1% reported TB cases caused by
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this species in industrialized countries, but other areas or population might experience a
e-
higher prevalence (Amafu 2006; Rodwell et al, 2008). A study by Bhembe et al. (2017) also reported on this species isolated from slaughtered cattle lymph nodes in the Eastern Cape
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Province. These results may possibly suggest transmission of this pathogen from livestock to humans. The connection between humans and animals has indicated that the mode of
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contagion, such as the use of unpasteurized milk and the proximity between mammals occurs
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between man and animals (McDaniel et al, 2014).
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This study also reports on the AFRI lineage 2.2% (4/184) belonging to the M. africanum species which was detected from the isolates. This lineage has been reported as a prevailing M. bovis clonal complex (Egbe et al, 2017). The M. africanum (subtype I) has traits that resemble those of M. bovis and is widespread in West Africa (Mostowy et al, 2004). The possible route of transmission of M. africanum may perhaps be through the movement of persons from one region to another. Family 33 is also reported in this study with the prevalence of 2.2% (4/184) reported to be belonging to the Euro-American lineage (MagugaPhasha et al, 2017). It is not the first time this family is reported in South Africa, it has been
13
Journal Pre-proof reported in the Limpopo Province (Maguga-Phasha et al, 2017) and the Eastern Cape Province (Bhembe et al, 2017). The T family has also been reported in different provinces of South Africa, which includes the Free State (van Dijk et al, 2016) and the Eastern Cape (Stavrum et al, 2009); which was described as the most predominant family in the province. This present study reports a prevalence of 1.1% (2/184) of the T family. This could mean there is continuing transmission
f
of this strain, although it is at a marginal rate. The least detected family in this study is the S
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family 0.5% (1/184); this is not surprising, because the family has been reported with limited
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dissemination through African countries (Mbugi et al, 2016).
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The SpolDB4 database also identified the Manu 1.1% (2/184) and Canettii 2.7% (5/184)
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families. The Canettii family is associated with the M. canettii species. The M. canettii species is a sporadic, smooth, and a variant of the M. tuberculosis commonly isolated in
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Africa from humans (Fabre et al, 2010). The species has been reported worldwide and the
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5. Conclusions
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majority of reported cases are from patients who have either travelled or lived in Africa.
This study validates that the Beijing strain is the prevalent strain disseminating in the Eastern Cape Province. The results of this study denote a reference point of the population structure in the province and may serve, as a drive for future molecular studies. Genotyping of multidrug-resistant Mycobacterium tuberculosis strains is highly imperative for multidrugresistant tuberculosis control as it allows the detection of alleged occurrences and transmission chains. This study evidences that multidrug-resistant TB is instigated by diverse species of Mycobacterium causing the infection in humans, which together form the Mycobacterium tuberculosis Complex. 14
Journal Pre-proof Ethical considerations The experiment was designed and conducted in agreement with the ethical guidelines highlighted by the National Committee for Research Ethics in Science and Technology of South Africa and all sampling and analytical procedures were followed as documented in the University of Fort Hare’s health and safety guidelines. Acknowledgements
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The authors extend their gratitude to the National Research Foundation (NRF) of South
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Africa (grant number: 76255) for a subvention, the University of Fort Hare and the
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University of Johannesburg for letting us use their amenities.
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Conflict of interest
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No conflict of interest declared. References
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Tables Table 1 Demographic profile of the study population defined using genomic regions of difference Age (years)
Gender of patients with species detected M. bovis
M. caprae
M. canettii
M. africanum
Male
Female
Male
Female
Male
Total
1
0
0
0
0
5
0
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1
1
0
0
70
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Male
Female
Male
Female
0-14
3
1
0
0
0
15-29
38
27
0
0
3
30-44
36
22
0
3
6
5
2
1
2
1
78
45-59
6
17
1
1
1
0
0
0
1
0
27
60+
3
1
0
0
0
0
0
0
0
4
Total
86
68
1
10
6
3
2
3
1
184
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Female
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M. tuberculosis
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0
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4
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Journal Pre-proof Table 2 Segregation of MTBC into species as identified following genomic regions of difference Number of DNA
isolated from humans
isolates
Humans
154
83.7%
M. caprae
Goats
16
8.7%
M. bovis
Cattle
5
2.7%
M. africanum
Humans
M. canettii
Humans
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M. tuberculosis
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Primary host, but
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Total
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Percentage
4
2.2%
5
2.7%
184
100%
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Species identified
Journal Pre-proof Table 3 Prevalence of the two major first line anti-TB drugs of the detected genotypes in the Eastern Cape Province of South Africa Family
East-Asian Euro-American Indo-Oceanic Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Indo-Oceanic Euro-American Euro-American Euro-American Euro-American M. bovis M. bovis M. africanum M. bovis M. canettii Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American Euro-American
Beijing LAM4 X1 X3 LAM3 T1 S LAM9 LAM4 MANU2 X3 X3 X3-variant1 LAM2 BOV1 BOV3 AFRI Bov_4 Caprea Canettii X3 LAM9 Family33 T1 Family33 X3 Family33 Family33 LAM3 LAM9
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pr
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SpolDB4 ID
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al
Pr
Octal code (spoligotype description) 000000000003771 777777607760731 777777377670371 300076777760771 776177607760771 777777777760771 776377777760771 777777607760771 777767607760731 777777607763771 700076777760771 700076777760671 700036777760771 677717607760771 676773777777600 640013777777600 770777777777671 277773777377600 000000000101000 500046637740661 777567607760771 777357757763771 557347637740661 557347607743661 100046637740771 776147607763771 300076777763771 756177607760771 557347607740661
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Shared International Type (SIT) 1 60 1329 2286 33 53 34 42 1750 1247 92 70 91 2271 482 479 181 647 592 ORPHAN ORPHAN Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Not in SITVIT Total
Prevalence no (%) 124 (67.4) 11 (6) 1 (0.5) 6 (3.3) 5 (2.7) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 2 (1.1) 2 (1.1) 1 (0.5) 1 (0.5) 1 (0.5) 4 (2.2) 1 (0.5) 4 (2.2) 2 (1.1) 5 (2.7) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 1 (0.5) 184 (100)
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Journal Pre-proof Credit author statement
Nolwazi Londiwe Bhembe: Data curation, Investigation, Formal analysis, Validation, Visualization, Writing - Original draft preparation.
Ezekiel Green: Conceptualization,
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Methodology, Software, Resources, Supervision.
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Journal Pre-proof Highlights
This study gives a general overview of multidrug-resistant strains and circulating strains present in the Eastern Cape, South Africa.
Diverse spoligotype patterns were detected in this study, which delineates a high diversity of Mycobacterium tuberculosis strains disseminating in the Eastern Cape Province.
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The Beijing strain was the predominant strain among the multidrug-resistant
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tuberculosis isolates.
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