Genetic Identification and Mapping of Multidrug-Resistant Tuberculosis in Kenya
BETTY KHAMALA
A Research Proposal Submitted in Partial Fulfillment for the Requirements for the Degree of Master of Science in Molecular Medicine of Jomo Kenyatta University of Agriculture and Technology
2020
DECLARATION
This research proposal is my original work and has not been submitted for any award in any other university or college.
Signature: ……………………… Date: …………………………………
Betty Khamala (TM-305-0874/2017)
This research proposal is submitted for examination with the approval of the following as supervisors:
Dr Raphael Lihana
Signature……………………… Date.………………………………….
Kenya Medical Research Institute
Dr. Joel Bargul
Signature…………………… Date………………………………
Jomo Kenyatta University of Agriculture and Technology
Table of Contents
2.1 Biology of Mycobacterium tuberculosis 5
2.2 Transmission of Mycobacterium tuberculosis 5
2.3 Pathogenesis of Mycobacterium tuberculosis 6
2.4 Clinical manifestation of Mycobacterium tuberculosis 6
2.5. Development of multi-drug resistant tuberculosis (MDR-TB) 7
2.6 Occurrence of MDR-TB strains 10
2.7 Drug-resistant TB in Kenya 11
2.8 Detection techniques of Mycobacterium species 11
2.8.1 Region of Deletion (RD) analysis 11
2.8.2 Nucleic Acid Amplification Tests 12
2.9 Geographical information systems 13
Drug susceptible mycobacteria sample 15
3.5.2 GenoTypeMTBDRplus Drug Sensitivity Testing 17
3.5.8 Georeferencing of MDR-TB to Chest Clinics 17
3.5.11 Ethical considerations 18
3.5.12 Expected Application of the Results 18
List of Abbreviations AND ACRONYMS
AIDS Acquired Immune Deficiency Syndrome
ATCC American Type Cell Culture
ERC Ethical Review Committee
GPS Geographical positioning system
HIV Human Immunodeficiency Virus
INH Isoniazid
inhA It is an enzyme enoyl-ACPreductase.
KNH Kenyatta National Hospital
katG Gene coding for catalase-peroxidase KatG
LJ Lowenstein Jensen
LPA Line Probe Assay
MDR-TB Multi-Drug Resistant Tuberculosis Mycobacterium
MGIT Mycobacterium Growth Indicator Tube
MUT Mutant
MEGA 6 Molecular evolution genetic analysis version 6
NASCOP National Aids and Sexually Transmitted Diseases Control Program
PANTA Polymyxin B, Amphotericin B, Nalidixic Acid, Azlocillin
RIF Rifampicin
RR Rifampicin Resistant
poB gene Agene which rifampicin inhibits the RNA polymerase at the level of the beta subunit encoded on mycobacterium tuberculosis.
SLID Second Line Injectable Drug
SPSS Statistical Packages for Social Scientists
TB Tuberculosis
TNF Tumor Necrosis Factor
WHO World Health Organization
WT Wild type
Abstract
Tuberculosis (TB) has been in existence for years and remains a significant global health challenge. The mycobacterium TB is responsible for the cause of disease to approximately 9 million individuals per year, which is among the highest cause of death globally. The foundation of Drug-Resistant Tuberculosis to various drugs is the biggest coercions towards the intervention of TB. Multi-drug resistance TB (MDR) is referred to as a process of fighting rifampin (RIF) as well as isoniazid (INH). Multidrug-resistant Tuberculosis is not evenly distributed across Kenya; therefore, mapping out areas of high risks of MDR TB will help government prioritize resources and allow interventions and deployment of resources.
The main objective of the study is to genetically identify and map out the distribution of multidrug-resistant tuberculosis strains in clinical isolates received at the National TB reference laboratory. The other objective is to generate information from characterized TB samples at the “National Tuberculosis Reference Laboratory (NTRL)” on how frequent the mutual mutation occurring in the katG, inhA, and rpoB aim gene regions related to INH and RIF resistance of M. tuberculosis complex in Kenya. Further, we hope to obtain the geographical dispersal of MDR-TB in Kenya
The study design will be a cross-sectional and the sample size will be 156. The samples will be collected and transported to the National tuberculosis reference laboratory (NTRL), Nairobi. The bacteria will be detected by culture using Mycobacterium Growth Indicator Tubes (MGIT) used to culture mycobacterium tuberculosis. The genotyping of resistant genes will be done using molecular assay technique of GenoType® MTBDRplus assay version 2.0 to detect RIF- and INH-resistant mutations. Using PCR, the genes answerable for the drug resistance, including rpoB, katG, and inhA, will be augmented and subsequent biotin-labelled amplicons will be crossbred to DNA probes found on the membrane within the strip. For every gene, the assay will be conducted to examine the availability of wild-type (WT) as well as mutant (MUT) probes. Also, the assay will include the following steps: amplification reverse hybridization, extraction of DNA and multiplex PCR. Sequencing will be done on the specific resistance genes.
Data analysis will include SPSS software for analysis of sociodemographic data, MEGA 6 will be used for phylogenetic analysis and Global Positioning System (GPS) will be used for MDR-TB hotspots mapping. The Ethical clearance will be sought from KNH/UON Ethics Review Committee (KNH/ERC). This study will help identify the species and strains in MDR-Tb samples, including their epidemiological links using molecular assays. This, in turn, will help in identifying the type of resistance in Kenya and will guide on appropriate measures by the tuberculosis control program. We expect a higher prevalence of MDRTB since it is a major problem in Kenya.
Chapter One
1.0 INTRODUCTION
1.1 Background information
For centuries, TB has proved to be among the primary cause of public health distresses. It affects approximately 10 million people, close to 1.4 million die annually, 610,000 develop MDR-TB while 8% of Tuberculosis incidents are co-infected with HIV (WHO, 2017). For the last five years, Tuberculosis has been the main cause of mortality among single communicable agent, ranking greater than HIV/AIDS (WHO, 2015). Moreover, ninety-five percent of these Tuberculosis incidents happen in developing countries, where one in 14 new episodes occur in people who are infected with HIV (WHO, 2017). The organism remains a significant source of increased death within individuals with AIDS. Tuberculosis was detected in 374,000 HIV positive individuals in the year 2016 (WHO, 2017). According to WHO, “The portion of TB people co-infected with HIV is greatest in most regions of African countries. Before the emergence of HIV and AIDS, tuberculosis was considered controlled, the infections has been on the higher rise since then, with sub-Saharan Africa taking the lions share. The infections in Kenya are ranked 13th in the world and 5th in Africa.” (WHO, 2017).
Drug-resistant Tuberculosis is a form of TB infection with strains of mycobacterium tuberculosis that is unaffected by at least one of primary anti-Tuberculosis medicine, INH with or without resistance to other Tuberculosis drugs. As per WHO, “Previously treated TB is the strongest risk factor for the progress of multidrug-resistant Mycobacterium tuberculosis (MDR-TB). TB transmission through contacts is increasing due to rising figures of MDR-TB (Sharma et al., 2011). Roughly 3.9% of newfangled diagnosed as well as 20% of retreatment incidents globally have been MDR-TB with visible geographical disparities in occurrence” (WHO, 2013). Similarly, anti-tuberculosis medicines, isoniazid, streptomycin, as well as para-aminosalicylic acid gradually become ineffective in some clinical isolates of TB after their introduction (Pozniak et al., 2011).
Furthermore, Sam asserted that the major rifampin-resistant mycobacterium tuberculosis composite strains that have mutations occurring at 81-base-pair area of rpoB gene which encrypt RNA polymerase β-subunit. These targets are perfect targets for molecular tests for the resistance of rifampicin. Nucleic amplification test amplifies the specific regions of the rpoB gene that is explored with molecular beacons to identify presence of the rifampicin resistance-examining mutations (Sam et al. 2006). Spontaneous mutations responsible for Isoniazid resistance are never focused on one gene. The Drug resistance mutations belonging to katG gene can lead to the reduction of aptitude of catalase to stimulate the prodrug of isoniazid (Zhang et al.., 1992). Mutations occurring in the inhA gene or even its promoter can change a triggered isoniazid binding site resulting in isoniazid resistance (CDC 2009).
Kenya belongs to the nations with high TB and MDR –TB burden in the world (WHO 2017). The incidence of tuberculosis in 2016 stood at 348 per 100,000 of the population, the death rates due to TB was estimated to be 60 per 100,000 of the population without HIV + and 50 per 100,000 of the population with HIV+, these is based to WHO report. The same account presented that 1.3% of newly detected TB individuals as well as 9.4% of formerly treated individuals that had MDR-TB. This data warrants the documentation of drug-resistant strains and observing their spread in the community to contain their spread” (WHO 2017).
This study aims to generate information from characterized TB samples at the “National Tuberculosis Reference Laboratory” (NTRL) on how frequent the mutual mutation occurring in the katG, inhA, and rpoB aim gene regions related to INH and RIF resistance of M. tuberculosis complex in Kenya. Further, we hope to obtain the geographical distribution of MDR-TB in Kenya using samples received at National Tuberculosis Reference Laboratory which could provide a baseline in policy formulation, especially with regards to resource distribution and awareness.
1.2 Statement of the problem
TB is a global health concern with Kenya being listed among the high burden countries. Treatment of tuberculosis is quite expensive, and this has been exacerbated by the advent of drug-resistant strains among the population. Fighting of mycobacterium tuberculosis in known first-line regimens has been on a sharp increase. Currently, many chest clinics worldwide have reported resistance of the pathogen to rifampicin and isoniazid that are considered to be most potent leading line drugs. the Resistance to rifampicin as well as isoniazid leads to an advent of Multidrug-resistant Tuberculosis. Intervention and control of multidrug-resistant TB is resource-intensive and therefore needs a well laid down plan in terms of resource allocation and raising awareness. Currently, no data shows the geographical distribution of multidrug resistance TB strains in Kenya, making resource allocation meant to restrain the spread of secondary MDR-TB a challenge. Absence of such information makes treatment of MDR-TB a fishing expedition that is not guided by scientific facts. This project intends to lay bare such information that will lead to better management technique for control as well as prevention of MDR TB.
1.3 Justification of the studyhttps://essaygroom.com/medication-administration-tuberculosis/
Identifying MDR TB areas through Mapping will help in prioritization of resource allocation and enable efficient deployment of interventions that are often limited to locations where they are most urgently needed. This will help develop site-specific intervention plans against MDR-Tb. This will also help in reducing mortalities and transmission. Molecular diagnostic algorithms are put in place with the overall goal of substantially increasing DR-TB diagnosis, thus proper patient management and cure rates. This study is aimed at identifying the species and strains in MDR-Tb samples, including their epidemiological links using molecular assays. This, in turn, will help in identifying the type of resistance in Kenya and will guide on appropriate measures by the tuberculosis control program.
1.4 Research Questions
1 What is the spatial pattern and distribution of rifampicin and isoniazid drug resistance tuberculosis in Kenya?
- What is the distribution of wild types and point mutations of inhA, rpoB, and katGamong samples obtained at NTRL?
- What is the relationship of point mutations of katG, inhA, and rpoBgenes in different geographical locations in Kenya?
1.5 General Objective
To genetically identify and map out circulation of multidrug-resistant tuberculosis strains in clinical isolates received at National TB reference laboratory
1.5.1 Specific Objectives
- To regulate the spatial pattern and distribution for rifampicin and isoniazid drug resistance tuberculosis in Kenya.
- To determine the distribution of wild types and point mutations of inhA, rpoB, and katGamong samples obtained at NTRL.
- To determine the relationship of point mutations of katG, inhA, and rpoBgenes in different geographical locations in Kenya.
Chapter Two
2.0 Literature Review
2.1 Biology of Mycobacterium tuberculosis
Mycobacterium. Tuberculosis is an etiologic agent in person, and it is also non-motile, non-encapsulated, non-spore developing Gram-positive, rod-shaped bacterium (Ducati et al. 2006). Tortoli noted that “Mycobacterium tuberculosis is characterized by a thick, sophisticated, lipid-rich, acid-fast cell wall which is commonly associated with disease instigation and considerably slow growth. Today, there are more than 150 officially recognized Non-Tuberculous Mycobacterium (NTM) present in the environment, and only two or three dozen are familiar to clinicians, most of them being saprophytic soil bacteria (Tortoli, 2014). Mycobacterium Tuberculosis Complex (MTC) consist of minority members of these species that are pathogenic to humans and animals, causing mycobacterial disease; they include M. Tuberculosis, M. terrae, M. avium, M. simiae, M. Bovis, M. africanum and M. leprae (Tortoli, 2014).
More so, a cell wall of the M. tuberculosis is related to the gram-positive organisms but have a higher lipid content (Alderwick, Harrison, Lloyd, & Birch, 2015). The lipids are long-chain fatty acids that present that require heat to achieve staining. This exclusive thick cell wall of Mycobacteria is also responsible for its resistance to the lethal effects of acids, alkalis, and detergents. This characteristic stands out during the isolation of the organism from other bacteria for culturing (Alderwick et al., 2015). M. tuberculosis grows best at a temperature range of 35 – 37oC (Cole, 2010).
2.2 Transmission of Mycobacterium tuberculosis
Person to person interaction is the most common mode of tuberculosis transmission and is by inhalation of aerosols dispersed in the air after a cough from an infected person. The aerosolization happens at a quicker rate, especially during coughing (Yates, 2016). Bovines are also a reservoir for tuberculosis, and there are reports of cow-human transmission (Ibrahim et al., 2016).
2.3 Pathogenesis of Mycobacterium tuberculosis
Immediately after taking in M. TB, natural immune reactions comprising of alveolar macrophages as well as granulocytes starts to fight infection and in other people, bacilli are killed by the immune system, whereas in others, infection is established (Modlin et al.., 2013). Multiplication of bacilli in the macrophages as well as localized lymph nodes affects lymphatic and hematogenous distribution, with seeding of numerous organs, that ultimately results in another pulmonary disease (tuberculosis outside the lungs). Retention of bacilli in macrophages and extracellular among granulomas confine further duplication and control tissue degrading, causing a dynamic balance among pathogen and host (Modlin et al.., 2013). According to Getahun, there is a period where bacterial reproduction and distribution starts, then immunologic containment of feasible bacilli follows. The outcome of the process is also asymptomatic latent tuberculosis infection, that is well-defined as the condition of immune control, persistent bacterial viability, and lack of evidence of the clinical symptoms of lively tuberculosis (Getahun et al.., 2015).
2.4 Clinical manifestation of Mycobacterium tuberculosis
Mycobacterium tuberculosis can affect various part of body, including bone, central nervous system among other organ systems, but it is chiefly the lung that is the main organ affected (Drobniewski et al., 2011). Pulmonary tuberculosis patients have a sustained loss of weight and persistent cough for at least three weeks. Symptoms like blood in sputum, dyspnea, fever, chest pain, night sweats, and anorexia have also been shown to be widespread among TB patients (Deus et al., 2012; Goyal et al., 2012). Cough is a predominant symptom; initially, the cough might be less severe; however, inflammation and tissue necrosis ensue, and sputum is later produced (Suzuki et al., 2010). Geriatric patients with Tuberculosis may not present typical symptoms of Tuberculosis infection since they may not support a significant immune response (WHO, 2011). Gillespie and Yoon states, “Active TB infection in this age group may manifest as persistent pneumonitis” (Gillespie, 2011; Yoon et al., 2012)
Figure 2 1: The diagram shows the phylogenetic diversity of M. tuberculosis complex gene. The main groups 1, 2 and 3 are indicated at the top of the figure with blue, green and red arrows, respectively. (Donoghue et al., 2004)
2.5. Development of multi-drug resistant tuberculosis (MDR-TB)
Drug resistance is divided into two categories, either primary drug resistance (bacilli isolated from individuals who have never taken drugs) or secondary drug resistance (bacilli isolated from patients who have had TB medication for not less than one month (Weyer et al., 1992). Research asserts that resistance to drugs is related to the spontaneous mutations in the gene encoding for both drug targets and enzymes engagement in drug activation (Somoskovi et al., 2001). In M. tuberculosis, the drug resistance is attributed to nucleotide substitutions, additions, or obliterations in precise resistance- defining sections of the genomic aims or initiating enzymes of anti-TB chemotherapeutic agents. The mutations are exclusively confined to chromosomal DNA and not linked; therefore, the probability of the strain creating the spontaneous mutation to all drugs is extremely little. The development of resistance to drugs can be sketched back to the start of treatment of M. TB in 1944. Concerning this discovery of management as well as clinical application in the year 1943-1945 through one W. Selman. The Streptomycin was extremely efficient in management of all Mycobacteria species and strains. But that lasts for a small period of fruitful application, resistance incidents regarding Streptomycin monotherapy were chronicled within the population. Afterward, p-amino alicyclic acid, Isoniazid, Pyrazinamide, plus Rifampicin were supplementary to the schedule, which significantly decreased emergent incidents. The consequential cross protection for the administration of Isoniazid and Streptomycin completely cured tuberculosis. This was due to mutants that resisted streptomycin were killed by isoniazid. (Iseman, 1994).
The anti TB drugs such as Isoniazid which act by wedges the production of cell-wall mycolic acids, which are chief element of M. tuberculosis cell envelope. The drug targets the ketoacyl-ACP synthase (KasA), enoyl-acyl carrier protein reductase (InhA), fatty-acid, and complex of an acyl carrier protein (AcpM) (Somoskovi et al., 2001). presence of catalase peroxidase in the bacteria stimulates INH which is a pro-drug. (Iseman, 1994; Somoskovi et al. 2001). Enzyme catalase-peroxidase is also programmed for by ‘catalase-peroxidase gene’ (katG). In INH the resistant strains, katG is changed; as such, decreasing the initiation ability of pro-drug INH (Iseman, 1994; Bifani et al., 2008). Notably, the point mutation at the Ser315Thr lessens catalase-peroxidase movement through approximately 50%’; therefore, constructing high-level confrontation to INH (Pfyffer, 2000; Bifani et al., 2008).
According to Pfyffer, and Bifani, rifampicin mode of action is to kill early metabolically active M.TB which produces sterilization action on dominant organism with high metabolic activity. The drug interfere with the RNA synthesis by boring to bacterial RNA polymerase. The bacterial RNA polymerase is fixed by the rpoB gene. Therefore, mutations in rpoB prevents binding of the drug to RNA polymerase because of associated modifications. Pyrazinamide (PZA) mode of action of done through the fusion of short-chain fatty acid precursors. It is also a prod-rug which is triggered to pyrazinoic acid (POA) through bacterial pyrazinamide (PZA)/nicotinamidase. It brings about cytoplasmic acidification and hinder cellular metabolic activities. The changes in the base pair in gene for the bacterial pyrazinamidase (pZase)/nicotinamidase brings about amino acid substitutions and nucleotide insertions or deletions.
Furthermore, the transmutation leads to non-sense mutations in pncA organizational gene or putative promoter area resulting in flashy pZase activity. Both M. Bovis as well as M. bovis BCG, resists PZA due to the exclusive C to G point of mutation in codon 169 of the pncA. Ethambutol (EMB) is also an antTB drug which act by inhibiting the biosynthesis of arabinogalactan, is the major polysaccharide for Mycobacterial cell wall. Additionally, drug interferes with the building up of cell wall arabinan of arabinogalactan, lipoarabinomannan and then induces the pile of β-D-arabinofuranosyl-P-decaprenol, an in between in arabinan biosynthesis. Ethambutol focus on an enzyme arabinosyl-transferase. The Mutations in codon 306 of embB, embCAB operon, in amino acid residues Asp328, and Gly406 and Glu497 are alleged to account for the resilient phenotype” (Pfyffer, 2000; Bifani et al., 2008).
According to Iseman, Pfyffer, and Bifani, Streptomycin perform at ribosomal protein S12 as well as 16S rRNA of the 30S sub-unit of the ribosome leading to bacterial protein synthesis intrusion, destruction of the cell membrane, and RNA synthesis production consisting of mis-reading, mis-coding of genetic code and inhibition of respiration. Mutations in rpsL gene that encodes ribosomal protein S12 as well as rrs gene which encrypts 16S rRNA restricts the accomplishment of the drug “(Iseman, 1994; Pfyffer, 2000; Bifani et al., 2008).
2.6 MDR-TB strains Occurrence
Strains of M. tuberculosis causing MDR-TB disease are distributed worldwide (Amor et al., 2008; Keshavjee et al., 2008). Several species and strain lineages of M. tuberculosis are associated with specific geographical localities. Freeman, Filliol, and Glynn state, “The strains belongs to different families as well as sub-families which consists of six key phylogenetic lineages that are also sub-structured geographically. The phylogenetic lineages are Indo-Oceanic (IO), West- African 2, West-African 1, East Asian (EA), East-African-Indian (EAI) and Euro-American. The Beijing family of East Asia lineage comprises of large clusters and often linked with illness outbreaks universally” (Freeman et al., 2005; Filliol et al., 2006; Glynn et al., 2008).
All the six lineages are represented on the African continent. So far, records indicate that MDR-TB strains belonging to Euro-American family are predominant in Africa (Gagneux et al., 2005). Other lineages are reported to occur frequently in patients within specific countries. The two West African 1 and two lineages are prevalent in the West African countries (Homolka et al., 2008) while East-African-Indian lineage frequently occur in Central African countries (Niobe-Eyangoh et al., 2004). Three lineages comprises of Indo-oceanic, Euro-American and East-African-Indian, are prevalent in East Africa. In South African countries, the East Asian descent is prevalent among MDR-TB patients (Gagneux et al., 2005).
MDR tuberculosis scenarios have been reported in all nations found in the East African region. Some of the countries with high prevalence include the Democratic Republic of Congo (DRC) on the western side, where the MDR-TB prevalence was noted as 2.3 percent (WHO, 2006). A prevalence of 1.2 percent was also documented in Rwanda in the southwestern part, with a possibility of drug resistance amplification effect (Umubyeyi et al., 2007). Tanzania, to the South, was estimated at 1.1%, with predominant strains being of Central Asian Sub-family, Latin American Mediterranean as well as East-African-Indian families (Eldholm et al., 2006). Predominant families in Kenya were CAS- Kili and LAM11-ZWE, CAS1-Dehli, EAI, LAM9, and T family. Beijing strains were also reported (Githui et al., 2004).
2.7 Drug resistant TB in Kenya
In the coastal part of Kenya, monoresistance was most common with isoniazid at (16.0%), Rifampicin (2.1%), Streptomycin (18.3%) and Ethambutol (10.0%) in a freshly diagnosed individuals while within the formerly cured patients, and resistance to streptomycin was at 58%, Ethambutol 53%, isoniazid 41% and rifampicin at 23%. Prevalence of MDR-Tuberculosis, referred to as resistant to either isoniazid or rifampicin, was 10 (4.8%) among the recent and formerly treated individuals (Yonge et al., 2017).
In another research, it was reported that 37 of 30.2 percent of the iso;lates were resistanttothe isoniazid; the four of 1.4 percent to rifampin and the 30 to of 10.4 prcent to the pyrazinamide. Additionally, the double resistance was seen in the 4 of 1.4 percent isolates that were resistant to pyrazinamide as well as isoniazid; the four of the 1.4 percent to the isoniazid as well as streptomycin and 1 of 0.3 percent to streptomycin as well as rifampin . The two remaining isolates of 0.7 percent were also multidrug-resistant and then 1 triple resistant with the additional to Ethambutol.
2.8 Detection techniques of Mycobacterium species
Effective management and control of tuberculosis through means of vaccination as well as treatment, primarily for MDR-TB, needs detailed information concerning pathogen species or strains (Asiimwe et al. 2008). Furthermore, analysis related to phenotypic growth and biochemical features have restricted biased and repeatability influence to type associates of the MTC. As such, precise capturing of MTC team can be attained by engaging molecular typing techniques that are quick and extremely biased.
2.8.1 Region of Deletion (RD) analysis
Region of deletion or difference analysis (RD) is a PCR based genotyping tool that analyses specific deleted areas in the genome of MTC. Amplification success or failure of a given part within the genome precisely differentiates members of the MTC. In the MTC, unidirectional chromosomal region deletions are occurring in the genome over generations forming separate species (Gagneux et al., 2005). The deletions at different loci in the genome were exploited to develop a fast, simple, and consistent PCR based typing technique for MTC (Huard et al., 2003). The panel of typing composed of several chromosomal regions: Rv3120, Rv1970, IS1561, Rv1510, Rv3877/8, Rv0577 and 16S rRNA were then developed. The PCR products (amplicons) pattern of the panel, specified by failure or success, differentiates members of the MTC and segregates them from Mycobacteria other than tuberculosis (MOTTS).
2.8.2 Nucleic Acid Amplification Tests
Several rapid molecular tools have been accessible over many years now to help in the speedy diagnosis of disease. In 2008, WHO suggested the application of the molecular line probe assays (LPAs) for increased identification of multidrug-resistant (MDR) Tuberculosis known as Tuberculosis resistant to the first-line drugs isoniazid [INH] as well as rifampin [RIF]). After two years, World Health Organization permitted the application of Gene- Xpert MTB/RIF assay for the quick recognition of MDR Tuberculosis openly in the sputum samples. Ling states that, “the Genotype MTBDRplus (Germany, Hain Life science GmbH, Nehren,) is an example of molecular line probe assay and consists of the probes particular for the M. tuberculosis complex, and for collective rifampin (RIF) resistance-conferring mutations and even the subset of mutations convening resistance to the isoniazid (INH). As per the issue of MTBDRplus assay, the meta-analysis showed the joint specificity of 98.4% as well as specificity of 98.9%, for the aim of uncovering the if the RIF resistance of 99.2 and 88.7 percent respectively for showing INH resistant; however, almost all of research involved utilized the cultured isolates as well as smear-positive respiratory specimens (Ling 2008). The original validation research indicated both MTBDRplus as the proper procedure for the exposure of resistance for smear-positive respiratory specimens, as well as the assay most efficient if used for smear-negative respiratory samples which had M. tuberculosis in culture; 16/20 of 80% delivered interpretable outcome for the RIF. Similarly, 14/19 of 74% showed interpretable result for INH (Barnerd 2008).
Xpert MTB/RIF is a programmed, heminested actual PCR which detects MTB and rifampicin sensitivity through the application of molecular beacons (Boehme et al. 2010). Shama asserts that “results for Gxpert are out within 2 hours as compared to conventional which takes between 8 to 10 weeks. This is the cartridge founded on nucleic acid amplification test (CBNAAT) which is user friendly and require less technical training. Gene Xpert reagent kills the bacilli thus reduces the biosafety risks (Shama et al. 2015).
2.9 Geographical Information Systems
Geographical information systems (GIS) is a programmed system for display, storage analysis, capture, and retrieval of spatial information (Vant et al. 1995; Boelaert et al. 1998). GIS delivers ultimate platforms for merging of disease-specific data and their examination in relation to the surrounding social, population settlements, and health services as well as natural environment (WHO, 2006). They are appropriate for evaluating epidemiological information, exploring trends, and interrelationships that are hard to determine in tabular format. More so, GIS authorizes policymakers to effortlessly envision issues regarding the existing health and social services, natural surroundings and efficiently focus on the resources (WHO, 2006). The data kept in GIS is connected to the geographic points. All the points are associated to one another through the application of typical coordinate system.
Previous studies have been conducted to connect the used geographic scrutiny within the isolates molecularly characterized through IS6110-based RFLP examination to classify undistinguishable Tuberculosis scenarios in Fort Worth-Dallas region found in Texas. There was the identification of 2 spatial clusters matching 2 zip codes in downtown site. The case gives an instance of how geographic units affect the explanation of results; as such, these can influence decisions concerning resource distribution as well as the early discovery of outbreaks of tuberculosis disease (Moonan et al. 2004). Another study was conducted in Benin by the application of molecular tools and GIS to characterize a potential epidemic of MTB Beijing Strain at the Cotonou (Affolabi et al. 2009). This study showed isolates of streptomycin-resistant MTB strains in many individuals living as well as working in the same region and going to the similar home pub.
Furthermore, results from this research gave direction to NTP to assume operative actions in curbing tuberculosis at local as well as individual level. Conducted spatial analysis of RFLP Tuberculosis incidents classify geographic indicators as well as socio-demographic liable for circulation of Tuberculosis within Hong Kong are through the help of GIS. Factors such as, poverty, education level as well as the elderly age, were essential elements of the rate of spread of tuberculosis in various regions of Hong Kong. Likewise, there was absence of socio-demographic factors associated to spread of tuberculosis (Chan Yeung et al. 2005). Bishai also led the geographic as well as molecular examination of tuberculosis incidents in town of Baltimore then summarized that citizens with cases of similar MTB strains were spatially gathered and as such, linked to the low socio-economic status and increased rate of drug use (Bisha et al. 1998).
Chapter Three
3.0 MATERIALS AND METHODS
3.1 Study area
The study will take place at “National Tuberculosis Reference Laboratory” situated in Nairobi County (Kibra Constituency). The laboratory collects samples from patients all over the nation as it is a reference laboratory. A mean of 12000 samples are received annually. The Laboratory serves TB patients countrywide, and screens for resistance patterns in Tuberculosis isolates.
3.2 Study design
This will be the cross-sectional laboratory study. The samples received at National tuberculosis reference laboratory Nairobi during the study period will be isolated and tested for Multidrug resistance. The sampling will be done equally from all counties in Kenya to give a true representation of circulation of the MDR TB.
3.3 Study population
The research population will encompass all samples received at the National Tuberculosis Reference Lab.
3.3.1 Inclusion criteria
Drug-resistant mycobacteria positive sample
Samples from different counties in Kenya.
3.3.2 Exclusion criteria
Drug susceptible mycobacteria sample
3.4 Sample Size
The sample size required will be calculated by application of statistical Fisher’s exact test, and 95% confidence interval will be used.
n = Z2 1-α/2 P (1-P)
δ2
n = minimum sample size
Z 1-α/2= Z statistic for a 95% level of confidence (1.96)
P = Proportion of target population estimated to be MDR resistant (0.094) (WHO, 2017)
δ = Precision with a 95% confidence interval that offers a margin of error of 0.05.
Substituting for the variables:
n= (1.96)2 x 0.094(1-0.094)1 = 130
(0.05)2
20% to increase the power of the study
20 x 130 = 156
100
Final sample size 156
3.4.1 Sampling procedures
The sampling procedure will be a census of all samples coming in from different counties until the sample size is reached. The samples received at NTRL during the study period will be enrolled into the study until the required sample size is achieved.
3.5 Laboratory methods
3.5.1 Culture
The ABBL MGIT tube from the “Becton Dickinson” comprising 7 mL improved middle brook 7H9 broth will be applied, where an upgrading supplement and concoction of antibiotics containing nalidixic acid, polymyxin B, trimethoprim, azlocillin, and amphotericin B, will be added. Further, tubes will be nurtured at the 37°C. Evaluations will be done every day for approximately three weeks after which it will be done once each week subsequently for culture positivity till termination of 6 weeks with the application of BBL Micro MGIT System. Later, positive tube will be established again through use of ZN staining as well as sub culturing on the blood agar plate. The period to reveal (TTD) of Mycobacteria will be founded on the time of the initial instrumental signal of positivity (Hasegawa et al. 2002).
3.5.2 Geno Type MTBDR plus Drug Sensitivity Testing
“The molecular technic that is used to detect mutations and identification for both rifampicin and isoniazid. Genotype MTBDR plus version 2.0 is the assay of choice and is done following manufacturer’s instructions. It is a rapid method of identifying MDR-TB and PCR process. Amplicons shall be used that are biotin labelled and through hybridization they bind to DNA probes bound on membrane on the strips. At the end of hybridization, visible bands will be detected. The test is done using GT Blot, which is automated. The strips is permitted to dry and then read by a genoscan.
For each gene, the test will look for missing world types and mutant probes (MUT). The test is done to detect the availability of wild-type (WT) as well as mutant probes. The strip have 27 reaction zones comprising of six controls including (amplification, conjugate , MTBC, inhA controls, katG, rpoB), four MUT (MUT His526Asp ,rpoB MUT Asp516Val, rpoB, rpoB MUT Ser531Leu and rpoB MUT His526Tyr), four MUT probes (inhA MUT1 Cys15Thr, inhA MUT2 Ala16Gly, inhA MUT3A Thr8Cys, and inhA MUT3B Thr8Ala), two MUT probes (katG MUT Ser315Thr1 and katG MUT Ser315Thr2), eight rpoB WT (WT-1 to WT-8), one katG WT (WT-315) and two inhA WT (WT-15/-16 and WT-8) . This assay will include will comprise of the following step: reverse hybridization ,DNA extraction, and multiplex PCR amplification (Hillemann et al. 2007).
3.5.8 Geo referencing of MDR-TB to Chest Clinics
From the analyzed samples chest clinics where samples have been received from will be identified and mapping of MDR-TB hotspots done. Mapping of MDR-TB hotspots will be mapped using Global Position System (GPS) (W.H.O. 2016). Using a hand-held GPS machine, coordinates of all identified hotspots will be outlined (Sharma et al. 2011).
3.5.9. Data Management
And Data Analysis
The collection of data will be keyed in to MS-Access spreadsheets, where data can be retrieved quickly and reliably. The database management system access will be restricted to authorized personnel only. Data will be stored on password protected flash disks and computer’s hard drive as well as in another computer for safety purposes in case of system breakdown involving the main equipment.
Data cleaning and validation will be executed to realize the desired dataset to export into a Statistical Package Format (IBM SPSS) for analysis. The phylogenetic data will be analyzed with MEGA. The differences between wild type and mutant genes will be analyzed using chi square. The GPS will be used to map the MDR TB to specific clinics.
3.5.11 Ethical considerations
Ethical clearance for the study will be acquired from “Kenyatta National Hospital Ethical Research Committee and the University of Nairobi.” Confidentiality of sample details will be keenly observed when de-identifying the samples. The samples will then be processed for normal routine culture and drug resistance testing. The study is a laboratory-based study which uses clinically isolated culture from primary specimen. The isolates are not collected directly from the patient. The isolates will not be linked to a particular patient’s identification but will be linked to county and regions where the samples came from.
3.5.12 Expected Application of the Results
This study is aimed at identifying the species and strains in MDR-Tb samples, including their epidemiological links using molecular assays. Identifying MDR Tb areas through Mapping will help in prioritization of resources allocation by national tuberculosis program and enable efficient deployment of interventions that are often limited to locations where they are most urgently needed.
3.5.13 Study limitations and solutions.
The study limitations will include obtaining quality specimens and transportation of the samples from the counties. The challenge will be overcome by conducting regular training of patients on sample collection. The transportation of samples will be based on the existing mechanism of sample transportation that employ cold chain.
Time frame of activities
| ACTIVITY | JAN-APRIL | APRIL-MAY | JUNE-AUGUST | SEPT | OCT | NOV | DEC | JAN- FEB | MAR-JUN | JULY |
| Proposal development and presentation | ||||||||||
| Approval of proposal by ERC | ||||||||||
| Implementation of approved proposal and data collection | ||||||||||
| Submission of the draft thesis to supervisors | ||||||||||
| Letter of intent to Submit Thesis | ||||||||||
| Preparation and submission of Thesis | ||||||||||
| Thesis defense | ||||||||||
| Submission of quarterly progressive reports | ||||||||||
| Graduation |
Budget
| Item | Quantity | Unit Price | Cost (KES) |
| MGIT Tubes | Two sets | 36000 | 72000 |
| PANTA | 2sets | 10000 | 20000 |
| Forward & Reverse primers synthesis | 12 sets | 2000 | 24,000 |
| Pipette tips (250µl) | Three packs | 3,000 | 9000 |
| Pipette tips(1ml) | Four packs | 4,000 | 16,000 |
| MTB DR Plus Kit | 2 | 100,000 | 200,000 |
| Data analysis | 1 | 15,000 | 15,000 |
| Total | 356,000 | ||
Budget Justification
The project will be nested in an ongoing surveillance program. The capital expenditure has been taken care of and the expenses will be only the consumables & reagents.
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