Theses & Dissertations

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    Open Access
    Development of Loop-mediated Isothermal Amplification (lamp) for detection of macrolide-resistant genes in Streptococcus Pneumoniae
    (Kamuzu University of Health Sciences, 2020-12-01) Msefula, Jacquline
    Antibiotic resistance is fast becoming a major health problem, which makes it difficult to treat several infections, including Streptococcus pneumoniae infection. Increasing access to antibiotics, presumptive treatment especially in low-resource settings, and self-medication are some of the factors contributing to the rise in antibiotic resistant pathogens. Therefore, a robust surveillance platform for emergence of drug-resistant pathogens is a public health imperative. Diagnostic tools that could help with early detection, monitoring of resistance patterns and determining the prevalence of pathogens and pathogen resistance are urgently needed. In this study we have addressed the latter need by developing a method for the detection of macrolide resistant Streptococcus pneumoniae using loop-mediated isothermal amplification (LAMP) assay. The LAMP assay has increased specificity and sensitivity with a reduced time frame of detection. Rational: There is a growing need to develop diagnostic tools in early detection of antimicrobial resistance and real time surveillance. We aimed to develop a LAMP method for the rapid detection of antimicrobial resistant S. pneumoniae that could help in monitoring macrolide resistance in resource limited areas. Methods: A laboratory method, development study was conducted to develop a LAMP assay for the detection of macrolide resistance genes in S. pneumoniae from Blantyre, Malawi. S. pneumoniae isolates were obtained from an on-going community-based pneumococcal carriage surveillance project. Nasopharyngeal swabs were collected from children below the age of five in urban Blantyre. Isolates’ resistance profiles including erythromycin and azithromycin resistance were determined using phenotypic methods at Malawi-Liverpool-Wellcome Trust (MLW) laboratories. Whole genome sequencing (WGS) was done at the University of Oxford, United Kingdom. LAMP primers for the detection of macrolide resistance genes mefA and ermB were designed from two sequenced S. pneumoniae isolates genomes using Premier Biosoft software. The cut-off for amplification and the sensitivity and specificity of the LAMP primers was determined using the Receiver operating characteristic curve (ROC) Antimicrobial resistance phenotyping on culture was used as the gold standard test. Resistance results turn-around time was calculated for both culture and the LAMP assay. Results: 79 S. pneumoniae isolates were analyzed on the LAMP assay and results were compared to conventional culture method. Primers designed to detect azithromycin, mefA had sensitivity = 97.1% and specificity = 100%, at 95% confidence interval 0.952 to 1.000; while for erythromycin, ermB had sensitivity = 97.1% and specificity = 95.8%, at 95% confidence interval 0.452 to 0.701. LAMP assay amplified both azithromycin and erythromycin resistance genes within 30 minutes of the run. Furthermore, detection time for both ermB and mefA primers including DNA isolation and LAMP assay took <3 hours versus the conventional culture methods antimicrobial sensitivity testing (AST) which ranges from 24 to 48 hours to generate results. Conclusion: The study successfully developed and evaluated the performance of the LAMP assay for mefA and ermB gene in S. pneumoniae isolates. LAMP assay sensitivity and specificity performance for detection of mefA was excellent being a classifier of 0.98 the area under the Receiver Operating Curve (ROC). While for ermB it was an average classier with 0.57 area under ROC when both compared to conventional AST methods. The turnaround time for LAMP assay from pure isolate was less than 3 hours including nucleic acid isolation and detection compared to culture AST, which ranges from 24 to 48 hours. The study developed LAMP assay that can be used in the detection of bacterial resistance and provide results in timely fashion. Further evaluation of performance of the LAMP assays is recommended.
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    Open Access
    Healthcare-associated urinary tract infection in the Surgery Department at Queen Elizabeth Central Hospital: Deciphering risk factors and antimicrobial resistance patterns of isolated bacteria
    (Kamuzu University of Health Sciences, 2021-12-01) Gabriel, Kambale Bunduki
    In this dissertation, I investigated the risk factors associated with healthcare-associated infections among patients admitted in surgical wards of the Surgery department of the Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi. A particular focus was on healthcare-associated urinary tract infection (UTI). This dissertation is a result of three studies. The first one was a systematic review and meta-analysis of uropathogenic Escherichia coli (UPEC) and specifically their antimicrobial resistance and virulence profiles. The second study was a point-prevalence survey on healthcare-associated infections (HAI) and antimicrobial use in the surgery department at QECH. The third study was a cross-sectional study investigating risk factors associated with UTI and catheter-associated UTI. In addition, the study determined the antimicrobial resistance patterns of isolated bacteria from urine samples from patient suspected with hospital-acquired UTI. From the systematic review and meta-analysis, 1,888 UPEC isolates were included in the analysis. High antimicrobial resistance rates were observed among the antibiotic class of tetracycline in 69.1% (498/721), followed by sulphonamides in 59.3% (1119/1888), quinolones in 49.4% (1956/3956), and beta-lactams in 36.9% (4410/11964). Meanwhile, virulence factors with highest prevalence were immune suppressors (54.1%) followed by adhesins (45.9%). The point prevalence of HAI was 11.4% (n=12/105) (95% CI: 6.0%-19.1%), including 4 surgical site infections, 4 urinary tract infections, 3 bloodstream infections and 1 bone/joint infection. We identified the following risk factors for HAI; length-of-stay between 8 and 14 days (OR=14.4, 95% CI: 1.65-124.7, p=0.0143), presence of indwelling urinary catheter (OR=8.3, 95% CI: 2.24-30.70, p=0.003) and the history of surgery in the vii past 30 days (OR=5.11, 95% CI: 1.46-17.83, p=0.011). 29/105 patients (27.6%) were prescribed antimicrobials, most commonly the 3rd-generation cephalosporin, ceftriaxone (n=15). The prevalence of confirmed HA-UTI was 53.1% (179/337, 95% CI: 47.8-58.4). The CAUTI was observed in 53.9% (28/52, 95% CI: 40.0-67.1). Risk factors associated with HA-UTI and CAUTI were the age of patients, patients who are not married, low educational level (none or primary school), prostatic diseases, patients presenting UTI symptoms, hospital length of stay (>7 days). The most frequent isolated bacteria from patient with confirmed HA-UTI were E. coli in 46.4% (83/179), Klebsiella spp in 11.7% (21/179), Citrobacter spp in 9.5% (17/179), S. aureus in 5.9% (16/179), Enterobacter spp in 5.5% (10/179), Acinetobacter spp in 5% (9/179), Pseudomonas spp in 3.4% (6/179) and Enterococcus spp in 2.8% (5/179). Other emerging bacteria with potential of causing wide ranges of infections were also observed. These included Raoultella spp in 2.2% (4/179), Kluyvera ascorbata in 1.7% (3/179), Morganella morganii in 0.6% (1/179) and Proteus vulgaris in 0.6% (1/179). Resistance rates observed were 2.3% for carbapenems (meropenem and imipenem) (4/171 for each), 10.5% (18/171) for amikacin, 21.6% (36/167) for fosfomycin, 36.0% (58/161) for chloramphenicol, 50.1% (84/165) for nitrofurantoin, 53.9% (69/128) for amoxicillin-clavulanate and 54.0% (95/176) for ciprofloxacin. Healthcare-associated infections constitute a relatively high burden in the surgical ward of QECH. Reinforcing infection prevention and control measures will help in reducing their prevalence and hence reduce antimicrobial resistance. Empiric antibiotic therapy for UTI in the Surgery Department should be revised based on the antimicrobial resistance patterns of isolated bacteria.
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    Open Access
    Determinants of decisions between clinician and patient to prescribe antimicrobials: A clinician’s perspective
    (Kamuzu University of Health Sciences, 2021-10-19) Chalusa, Morris
    Antimicrobial resistance is a great public health challenge which has been accelerated by the inappropriate use of antimicrobials. The inappropriate use of antimicrobials is associated with the increased risks of prolonged hospital stay, self-medication of self-limiting conditions and more admissions to hospitals. Some of the factors that increase antimicrobial resistance appear to be modifiable and are determine by clinician-patient communication. Increased antimicrobial resistance was the cause of severe infections in the year 2012 in Malawi. Out of a total of 100,000 recorded deaths, 70,200 (70.1%) were attributable to infectious diseases. A qualitative study was used to find out what factors determine the decision between clinician and patient to prescribe antimicrobials when not necessitated in Mulanje, Malawi, through the use of unstructured questionnaires, semi-structured interviews and focus group discussions. Clinicians’ knowledge of antimicrobial resistance as well as their communication skills was also sought. Interview and open ended survey responses were translated, transcribed and coded for themes. Participants pointed out that patient preferences, patient belief and clinician lack of education were among the factors that contribute to the decisions to prescribe antimicrobials. Most clinicians showed lack of knowledge on the definition of antibiotic and antimicrobial resistance. Inappropriate use of antimicrobials is facilitated by prescribing decisions made by clinicians who are greatly influenced by their patients. Improving antimicrobial prescription requires educational interventions for both clinicians and patients.
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    Open Access
    Assessing antibiotic prescribing patterns and utilization of microbiological tests results for common bacterial infections in under five inpatients at Ntchisi District Hospital, Malawi
    (Kamuzu University of Health Sciences, 2021-04-01) Kondowe, Davie
    Antibiotics have remained useful to humans and animals in the treatment of serious infectious diseases for decades now. Unfortunately, irrational antibiotic use has resulted in the emergency of antimicrobial resistance. The aim of this study was to assess antibiotic prescribing patterns and utilization of microbiological test results in common bacterial infections, and enhance existing interventions for the improvement of rational antibiotic use. This was a hospital based study, designed as prospective cross-sectional, which was conducted at Ntchisi district hospital, Malawi. The study used a structured questionnaire which involved prospectively reviewing and recording information from files of under-five patients admitted in peaditric ward. Convenience sampling method was used to enroll participants in the study. The study enrolled 373 participants who were prescribed antibiotics. The outcomes of the study were to assess antibiotic prescribing patterns in under five inpatients, evaluate utilization of microbiological test results of common bacterial infection in under five patients and find out the correlation or association between antibiotic prescribing pattern and utilization of microbiological test results in under five inpatients. According to Malawi Standard Treatment Guidelines, among the 373 recruited participant 76.68 % were appropriately prescribed antibiotics and 23.32 % were inappropriately prescribed antibiotics (p=<0.001). The most prescribed antibiotics were a combination of Benzyl penicillin and gentamycin 276(74.0%), followed by ceftriaxone 87(23.2%) and metronidazole 10(2.7%) (p=<0.001), 318(85.25%) had antibiotic prescription without a request for microbiological test and only 55(14.75%) had antibiotic prescription with a requested microbiological test (p=<0.001). Among the 55(14.75%) cerebral spinal fluid (CSF) samples sent to the vii laboratory, 46/55 CSF samples were analyzed and all the samples had negative result. Culture and sensitivity was not performed because of lack of laboratory resources. There was no utilization of these microbiological test results to maintain, change or discontinue treatment by the prescribers. It was observed that antibiotic prescriptions were made empirically. The study found that the correlation between antibiotic prescribing pattern and utilization of microbiological test results was not statistically significant (p=0.288).
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    Open Access
    Comparison of malaria parasite clearance times during Quinine and Artesunate administration for cerebral malaria in Blantyre, Malawi
    (Kamuzu University of Health Sciences, 2021-07-01) Saidi, Alexuse Mustaph
    Malaria which is caused by Plasmodium species is one of the most important human parasitic diseases. In recent years, preceding 2014, the most severe form of the disease, cerebral malaria, was recommended by World Health Organization (WHO) to be treated with quinine, but due to its increased side effects over the recently discovered drug, artemisinin derivatives, WHO substituted quinine as first line treatment with artemisinin derivatives in combination with a long-acting drug such as lumefantrine, piperaquine, amodiaquine, mefloquine, pyronaridine or sulfadoxine-pyrimethamine. Delay in parasite clearance time in treated patients is the main characteristic of parasite resistance to a particular antimalarial drug. Malaria parasites have already shown resistance to the currently recommended artemisinin derivatives in South East Asia, a development that prompted WHO to recommend periodic monitoring of the drug's effectiveness in endemic countries. Malawian children admitted with cerebral malaria (CM) between 2010 and 2019 in a long-standing pathogenesis study at Blantyre’s main referral hospital, Queen Elizabeth Central Hospital (QECH) were retrospectively sampled at admission and every six hours until two consecutive malaria smears were negative. This was done to monitor parasite clearance times across those years. Yearly average clearance time was compared to 2014, the year when artesunate replaced quinine as first-line therapy for CM in Malawian hospitals. Parasite clearance time was shown to be slower during the quinine era compared to the artesunate, an indication that artesunate is superior to quinine. There was no increase in clearance times from the onset of artesunate as first-line therapy, showing that the current recommended anti-malarial drug is still effective.