論文の公開元へMolecular characterization of Mycobacterium tuberculosis isolates and their association to multidrug resistance in Lusaka, Zambia
概要
PREFACE
It has been over twenty years after declaring tuberculosis (TB) a disease of public health emergence by WHO in 1993, but TB has continued to inflict mankind. Globally, TB is one of the top 10 causes of death, and the world’s top infectious disease killer (above HIV/AIDS) (1). Geographically, most people who developed TB in 2019 were in the WHO regions of SouthEast Asia (44%), Africa (25%) and the Western Pacific (18%) (Figure 1) (1).
TB claims more than a million lives each year and affects millions more, with enormous impact on families and communities. The disease typically affects the lungs (pulmonary TB) but can also affect other organs of the body (extra pulmonary TB). TB can affect anyone and anywhere, but most people who develop the disease (about 90%) are adults. There are more cases among men than women. Men aged 15 years and above accounted for 56% of the people who developed TB in 2019. Women accounted for 32% while children, aged less than 15 years accounted for 12% (1).
Worldwide TB incidence and deaths are falling. However, the pace is not fast enough to reach global TB targets set by WHO End TB strategy which include 90% and 95% reduction by 2035 in the TB incidence rate and annual TB deaths, respectively (Figure2).
The bacteria that cause TB belong to a group known as Mycobacterium tuberculosis (MTB) complex which comprises of closely related microorganisms. The members of MTBc are as follows; Mycobacterium tuberculosis, Mycobacterium canetti, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprea, Mycobacterium pinnipedii and Mycobacterium microti. The natural host for Mycobacterium tuberculosis is mainly humans and this organism is responsible for the typical human TB disease in most part of the world (2).
TB is curable and preventable. Most people (about 85%) who develop TB disease can be successfully treated with a 6-month drug regimen (1). However, a number of factors have been elucidated as drivers of this disease. These risk factors include HIV infection, drug addition, diabetes, malnutrition, smoking and air pollution (3).
Furthermore, the emergency of drug resistant TB cases (organisms can grow in the presence of one or more anti-TB drugs) is threatening global efforts to control TB and threatens to reverse global progress made in TB control so far (4). While the incidence and mortality of drug susceptible TB is generally on the decline (Figure 2), the incidence of drug resistant TB has been described to be variable by country (1), with increasing incidences observed in some countries across the world (Figure3).
Worldwide in 2019, an estimated 465,000 people developed rifampicin-resistant TB (RRTB), of which 78% had multidrug-resistant TB (MDR-TB) (defined as resistance to rifampicin and isoniazid). An estimated 182,000 deaths resulted from MDR/RR-TB. (10). In rates terms, an estimated 3.3% of new cases and 18% of previously treated cases had MDR/RR-TB globally in 2019 (1).
Factors associated with the emergence of drug resistance in new TB cases have been reported to include poor adherence to treatment, limited effective drugs and inadequate health care systems while primary drug resistance has been attributed to poor infection prevention and control measures leading to increased transmission (5).
In Zambia (a Sub Sahara African country) , despite the implementation of Directly Observed Therapy Short Course (DOTS) program and universal BCG vaccination, TB remains a disease of major public health concern and the country is enlisted among the 30 high TB burden countries by WHO due to its high TB incidence (333/100,000 population) (Figure 4).
Most recently (2021), WHO has reclassified Zambia as one of the high MDR/RR-TB burden countries (6). This is consistent with the in-country trend analyses which have demonstrated increasing threat of MDR-TB burden. For instance, the review of national data by Kapata et al (2013) highlighted four-folds increase in the number of MDR-TB patients notified by National TB control program (NTP) between 2000 and 2011 (7) (Figure 5). By 2019, the number of MDR/RR-TB notified by Zambia National TB control Program had increased to 500 cases per annum (8).
In the region, similar trends have been observed in the neighboring Botswana where the 4th national TB drug resistant survey reported a three-fold increase in MDR-TB rates (among new patients) relative to the preceding surveys (Figure 6) (9). By 2019, the proportion of MDR-TB in new cases in Botswana had risen to 3.6% (8).
Furthermore, high MDR-TB prevalence rates (7.7% in new cases and 33.8% in previously treated patients) have been reported from Swaziland by the national TB drug resistance survey [10]. While HIV/AIDS and social economic determinants have been linked to the high TB burden in Zambia and other countries in the region (11), factors driving the increase in MDRTB burden are still unclear.
Although it is unquestionable that most factors responsible for TB pandemic are related to socioeconomic dynamics and insufficient health care systems among others, factors directly related to the microorganism itself are also significant but they are less studied in the third world countries (12). Molecular understanding of a causative agent (M. tuberculosis) can provide an important study platform to investigate possible association of its strains with clinical and epidemiological characteristics.
Today, several techniques have been developed for molecular epidemiological investigations of M. tuberculosis strain diversity and these include spacer oligonucleotide typing (spoligotyping), insertion sequence 6110-based restriction fragment length polymorphism (IS6110-RFLP) and Mycobacterial Interspersed Repetitive Units – Variable Number Of Tandem Repeats (MIRU-VNTR) (13–15). Additionally, next generation whole genome sequencing (WGS) of M. tuberculosis clinical isolates provides invaluable knowledge on genetic diversity and evolution of drug resistance in the M. tuberculosis genomes in circulation (15). Whole genome sequencing is preferred to other typing techniques due to its robustness and high resolution, however, it does not negate the usefulness of other typing tools dueto limitations experienced in resource limited countries.
Although spoligotyping is less discriminatory compared to IS6110-RFLP and MIRUVNTR, this assay is rapid, inexpensive and robust therefore it is often used as a first-line genotyping method. It is the basis for the differentiation of major genotypes of M. tuberculosis such as Beijing, Euro-American sub-lineages, and Central Asian (CAS) families (13).
Some of the spoligotype families are distinctively distributed in specific geographical regions (17). This could signify that they are probably better adapted to certain human populations in those areas (18). For instance, Beijing spoligofamily is predominantly found in far-east Asia whereas Euro-American sub-lineages are predominant in Africa, Europe and the Americas (19).
The Latin-American Mediterranean (LAM) family has been described as the most prevalent M. tuberculosis lineage globally, accounting for approximately 15% of the global TB burden [19]. However, little is known about its epidemiology, biological behavior and disease patterns [12]. Different M. tuberculosis genotypes have been linked to cause drug resistant diseases and TB outbreaks in various regions (20).
There are strong indications that various lineages of M. tuberculosis have different biological characteristics which may influence the TB epidemiology (21). For instance, the Beijing genotype is suggested to be a possibly resistant clone against BCG vaccination, highly pathogenic, transmissible and prone to becoming drug resistant (22). Despite this growing body of knowledge, most TB control strategies are generic with the supposition that all M. tuberculosis strains are equal in terms of transmission dynamics, virulence and drug resistance (22). In various parts of Africa, diverse M. tuberculosis genotypes are driving the epidemiology of drug resistant TB and varied genotypes have been reported across the continent (Figure 7) (23).
M. tuberculosis is intrinsically resistant to many antibiotics, limiting the number of compounds available for treatment. This intrinsic resistance is due to a number of mechanisms including a thick, waxy, hydrophobic cell wall and the presence of drug degrading and modifying enzymes. However, by employing various modes of drug action, a number of drugs including rifampicin and isoniazid (two most powerful first line TB-drugs) have shown efficacious in the treatment of M. tuberculosis disease (Table 1). Eventually, resistance to those drugs (active against M. tuberculosis) has emerged and is conferred mainly by genetic polymorphism (24). For instance, resistance to rifampicin and isoniazid has been associated to mutations in rpoB and katG genes of M. tuberculosis, respectively (25, 26). These chromosomal mutations may confer drug resistance via modification or over-expression of the drug target, as well as by prevention of prodrug activation (24) (Table1).
Furthermore, studies have reported that the rate of mutations causing drug resistance varies according to the lineage to which the strain belongs. For instance, the Beijing family has demonstrated increased mutation rates in vitro compared to the estimated probabilities for the acquisition of resistance by spontaneous mutation which is approximately 1 in 108 bacilli for rifampicin and 1 in 106 bacilli for isoniazid (27).
Broad understanding of those mutations encoding resistance in a specific geographic setting is valuable knowledge for the development and application of new vaccines, drugs and molecular diagnostic tools and understanding the epidemiology of drug resistant TB (28).
Although the overwhelming burden of TB is in developing countries, molecular characteristics of M. tuberculosis have been studied more in industrialized countries than in non-industrialized nations. For instance, in the United States of America nearly each newly identified culture-positive case of tuberculosis is genotyped whereas in the third world countries, where the burden of TB and drug resistant TB is relatively high, genotyping of identified M. tuberculosis strain is not routinely done.
In Zambia, M. tuberculosis genotyping has locally been conducted by two studies, namely; Mulenga et al (2010) in Ndola district and Malama et al (2014) in Namwala district (29, 30). Both studies reported LAM family as the predominant M. tuberculosis genotype circulating in the studied districts. However, neither of those two studies analyzed detailed information relating to the Spoligotype International Types (SIT) of the identified M. tuberculosis families nor did they contextualize identified genotypes in relation to drug resistance. Furthermore, the M. tuberculosis genotypes reported by Mulenga et al and Malama et al were specific for the studied districts. To my knowledge, the M. tuberculosis genotypes isolated in Lusaka (Zambia’s capital city) and their association to multi-drug resistance has not been analyzed. Besides, M. tuberculosis mutational patterns and frequencies encoding drug resistance to rifampicin and isoniazid were un-investigated in Zambia before my study.
For my PhD project, I have utilized spoligotyping to genotype M. tuberculosis cultures isolated from TB patients mainly residing in Lusaka city and stored at the University Teaching Hospital, TB laboratory in Lusaka. Furthermore, I sequenced M. tuberculosis genes associated with drug resistance encoding mutations to rifampicin and isoniazid among MDR-TB isolates from Lusaka and compared the identified mutational frequencies and patterns to those reported in the African region.
Lusaka has the population of 3.3 million people and is both a commercial and administrative town. In addition, it is a getaway connecting the country's four main highways to the neighboring countries on the north, south, east and western part of the continent and hosts the main international airport connecting the country to the globe (Figure 8).
During the 2013 –2014 national TB prevalence survey, Lusaka reported a high prevalence of bacteriologically confirmed TB of 932/100,000 population [31]. Furthermore, Lusaka recorded the highest proportion of MDR-TB patients in the country notified by the national TB program in 2019 (Ministry of Health, 2020).
In chapter I of this thesis, I have described M. tuberculosis sub-lineages and documented their correlation with MDR-TB. Furthermore, I have illustrated gene mutations in rpoB and katG genes and inhA operon conferring resistance to rifampicin and isoniazid, respectively, in chapter II. Additionally, I have shown comparisons in the frequencies of specific mutations identified among M. tuberculosis isolates from Lusaka with those reported by others in the region.
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