Laboratories, space and equipment: Immunology
Over the years, the Department of Medical microbiology has built capacity for research and training in immunology. The department has the first and only fully fledged immunology training laboratory in the University. The laboratory collaborates with the Makerere University Walter Reed, Medical Research Council and Join Clinical Research Centre Laboratories. The laboratory can perform and teach all T-cell and immunoglobulin based immunological techniques.

Equipment available in the immunology lab: Water baths, centrifuges, pipette aides; ELISA reader; ELISA washer; ELISA spot; Refrigerators and freezers; Liquid nitrogen tanks.

Clinical Microbiology
The Department started as a clinical microbiology department for the University and has been on the fore front of promoting clinical microbiology for the University and country. Below is a summary of the recent developments in the department:

  • Clinical Microbiology Laboratory: The Department of Medical Microbiology has the oldest teaching clinical microbiology laboratory in the country that has embarked on an international (ISO) accreditation process. This laboratory has taught well qualified and successful 10 students in Master of Medicine in Microbiology as well as 3 doctoral students.

Equipment available in the Clinical Microbiology lab: Microscopes; Centrifuges; Freezers; Refrigerators; Incubators; Fluorescent Microscope; Autoclave; weighing scale.

  • BSL-3 Mycobacteriology laboratory:The Department has just completed construction of a state of the art BSL-3 laboratory that can enable it handle highly infectious agents. It is the only one in the University. This will enable the Department to practically teach on wide range infectious agents that no other University in the country can.

Equipment available in the BSL-3 laboratory: A high containment area, well equipped P3 facility; MGIT machine; 2 Level two bio-safety cabinets CO2 incubators; inverted microscopes; refrigeration facilities.

  • Mycology laboratory:The Department of Medical Microbiology has the only clinical mycology laboratory in the country. It has been instrumental in detecting existing and new fungal infections in the era of HIV/AIDS. This will be a powerful resource to disseminate knowledge on diagnosis of fungal diseases. 
  • Virology: Currently the Department collaborates with Uganda Virus Research Institute to teach practical virology. This collaboration will continue as we aim to build our own capacity.

Molecular biology laboratory

  • The Molecular Biology Laboratoryis the main research and training laboratory in the department. Research scope is wide, spanning from basic molecular studies to apply and operational research in infectious diseases that cause most of the disease burden in Uganda. Other studies focus on TB Molecular Epidemiology, Drug resistance studies and Molecular Diagnostics services offered to the teaching hospitals and other research laboratories in Uganda. 
  • Equipment available in the molecular Diagnostic laboratory: Two Thermocyclers; Real time PCR machine; Gel documentation systems (UV illuminators and Stratalinker, networked gel Bioimaging system); Basic equipment for manipulation of DNA/RNA (Incubators, spectrophotometers, electrophoretic apparatus (vertical and horizontal), etc), air and water incubators, hybridization oven, Gene quants, Vacuum blotters, pH meter; Ice making machine, autoclave, water distiller, Refrigerators and freezers, equipped dark room.
  • Molecular Diagnostic laboratory:The Department has acquired funds to construct a clinical diagnostic molecular laboratory. It will be the first one in the region. This will tremendously increase the capacity of the Department to teach and introduce molecular techniques in the diagnosis of ailments in the University and country at large.

Lecture Space of the department of Medical Microbiology

The Department has nine spacious office laboratories, one of which is being used as a computer room for post-graduate students. There are also two study rooms reserved for MSc and PhD students. The department has a big teaching laboratory, accommodating up to 400 students. The College of Health Sciences has provided funds for extensive renovation of this teaching laboratory: the learning environment has never been better for student in the College of Health Sciences.

Computer laboratory

The Department has a computer laboratory (6 x 8 meters) for post-graduate students, equipped with 5 desktop computers, each fully connected to internet.

Personnel

The department of Medical Microbiology has a total number of 15 academic staff members. Two are full Professors, two associate Professors, two senior lecturers, four full lecturers, three assistant lecturers and two teaching assistants. Five of the academic staff members are doing doctoral studies and they are in the advanced stage of their studies. 90% of the support staff members have first degrees and 50% have master’s degrees.

  •  Clinical microbiologists:The Department has 7 well trained clinical microbiologistsand 5 technologists.
  • Immunologists:The department is working with five dedicated immunologists and many visiting immunologists with keen interest to develop the proposed program.

Library:-

The College of Health Sciences has a well equipped library containing current editions of text books of immunology and clinical microbiology which are available to students.  The library also has access to the leading journals and electronic books in immunology and clinical microbiology.  Graduate students are also provided passwords to access medical literature electronically using the WHO/HINARI initiative (World Health Organization / Health InterNetwork Access to Research Initiative). In addition, the department also has a fully equipped book bank. Page Break 

Graduate studies

The department has two running graduate programmes:

  • MMED Microbiology
  • Doctoral program: The department has gone ahead and started a doctoral program in immunology with 5 fully sponsored PhD immunology students. The doctoral program will also serve as an avenue for the masters graduates. Although it is new, the doctoral program has been very favourably reviewed by Welcome Trust, Karolinska institute and Case Western Reserve Universities that are well recognized immunology teaching institutions.  With capacity to handle a doctoral program, it should be feasible to start a master’s program and later an undergraduate one.

Researchers wishing to utilize the departmental research facilities are welcome.  

Download the departmental Research Policy (pdf)

Download application for utilizing the departmental research facilities (pdf)

The department of Medical Microbiology strongly believes that microbiological research will play a leading role in solving Uganda’s medical ailments. In order to address the need for continued research and training in infectious diseases, the department has rapidly transformed into a vibrant research and training center within the College of Health Sciences. Research in our department is integrated, spanning from basic research to applied research and operation/clinical research. We believe in this integration since basic research provides a firm foundation for applied and operational research. Undergraduate and graduate (masters and PhD) students from a wide range of the university’s programs are actively involved in our research programs. Upon finishing, the graduates pursue careers in research or health care or the academia.

Research in the department can be an individual grant support to a departmental faculty, or a collaborative study between the faculty and any other scientist or study group.

The department’s research focus is mainly in the following areas, mirrored by the laboratories where various research activities are localized:

  • Molecular biology of infectious diseases: based at the Molecular Biology and diagnostics laboratories
    • Strategic basic research on the biology of pathogens aiming at drug/vaccine discovery and development of diagnostic tests and molecular epidemiological tools for screening of infectious diseases
    • Molecular epidemiology of infectious diseases
    • Molecular mechanisms of drug resistance
    • Routine Molecular diagnostics
  • Immunological research: based at the Immunology laboratory
    • Immunology of infectious diseases
    • Pregnancy immunology
  • Mycobacteriology: based at the BSL3 Mycobacteriology laboratory

TB Vaccine study clinical trials

  • Clinical/Operational research: based at the clinical Microbiology and mycology laboratories

1) Current Research projects in the Molecular Biology Laboratory

Cell to Cell signaling in Mycobacteria- this project is funded by NIH grant #1R01A175637-01. This project has is supporting three graduate students, one doing a PhD in Molecular Microbiology and the other two are doing Masters degree in Molecular Biology. All the the students are in their advanced years of study. PI: Dr Moses L. Joloba

Molecular Biology of Mycobacterium tuberculosis: Molecular Characterization of MTB complex in Kampala- this project is funded by Sida-Sarec. The project supported a PhD student through Makerere University-Karolinska Institute collaboration, and a Masters student who studied the Molecular Epidemiology of Mycobacterium bovis in Kampala. These students accomplished their studies successfully and their work was published in referral journals. PI: Dr Moses L. Joloba

Molecular Biology of Mycobacterium tuberculosis: Evaluation of Rapid Methods for Diagnosis of Multi-Drug Resistant TB in Kampala”This is another Sida-Sarec supported project. The project is funding one PhD student and one Msc student, now in their final years of study. A manuscript from this work was published by BMC Infectious diseases, and it is one of the most highly accessed papers this year. PI: Dr Moses L. Joloba

Molecular Biology of Mycobacterium tuberculosis: Phenotypic and Microevolution of MTC of Uganda Genotype: this study belongs to the Mycobacteria tuberculosis Strain working groupof the Tuberculosis Research Union (TBRU).This project is aiming at investigating the role of different genotypes of MTC in transmission, treatment response, clinical and radiological presentation of MTC. A PhD student and an international student are supported by this project. PI: Prof. Henry Boom; CO-PI: Dr Moses L. Joloba

Molecular Biology of Streptococcus pneumoniae: Transformation Efficiencies of Drug susceptible and Drug Resistant Pneumococci- This study was completed last year and the data revealed that there were no differences in transformation efficiencies among drug resistant and drug susceptible serotypes of Streptococcus pneumoniae. The study funded a Tanzanian Masters student. A manuscript is ready for submission for publication.
PI: Dr Moses L. Joloba

Prevalence of infection with multiple strains of Mycobacterium tuberculosis among patients with pulmonary tuberculosis in Kampala, Uganda: This study was done with Howard Hughes funding, aiming at evaluating the presence of multiple strain infections in a high TB burden country using the Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) molecular typing technique in our laboratory.
This study was pioneered by an international Medical student from the University of Pittsburg, USA, with initial support from AITRIP and the Forgarty scholarship program. The study also supports a local Msc student. PI: Kate Dickmanns, CO-PI: Dr Moses L. Joloba

Tuberculosis Drug resistance survey in Kampala:This study is funded by the European Union, and it has supported two Master’s students: one in Public Health and another in Molecular biology. PI: Dr Moses L. Joloba

Characterization of Extended spectrum B-lactamases elaborated in Uganda: this is a doctoral study funded by Makerere University School of Graduate studies and belongs to Dr. F.C. Najjuka, a lecturer in the department. The study has so far supported up to seven undergraduate students. PI: Dr F.C. Najjuka

A multi-centre comparative trial of efficacy and safety of sodium stibogluconate (SSG) versus paromomycin (PM) versus combination of SSG and PM as the first line treatment for visceral leishmaniasis in Ethiopia, Kenya, Sudan and Uganda. PI: Dr. E. Ssentongo & Prof J.Olobo

Malaria Vaccine Studies in Uganda: Site Preparation, Infrastructure Development, And Capacity Building For Clinical Trials. PI: Prof. J. Olobo

The efficacy of artemether- lumefantrine therapy and assessment of possible molecular markers of resistance in Uganda. PI: Dr. Hakim Sendagire

Comparison of the development of thymidine analogue mutations with CD4 monitoring alone versus CD4 monitoring plus viral load monitoring in naïve HIV-1 individuals on first-line antiretroviral therapy in Africa. PI: Dr. Hakim Sendagire

The phylogeography of Kaposi’s sarcoma-associated herpesvirus 8 in Uganda. CO-PI: Dr Henry Kajumbula

Services in the new Molecular Diagnostic Laboratory

Increased emphasis on diagnosis of human diseases by molecular genetic analysis has occurred in the recent years. Clinicians have become increasingly aware of the tremendous power of molecular-based tests for the diagnosis of human diseases. Molecular diagnostic tests, originally developed in a research setting, have been commercially packaged into a myriad of formats that are sufficiently quick and simple for effective use in clinical diagnosis of human diseases. The use of these diagnostic assays is becoming increasingly common in many clinical settings in Uganda, and is likely to extend to major referral hospitals in the near future. Thus, as the molecular basis of more diseases is elucidated, these technologies will continue to be methods utilized in clinical settings in the near future. This surge of new molecular-based tests has created the need for individuals trained in the theory and practice of performing or developing these tests. Funded by Sida-Sarec, this laboratory is constructed to meet this need. Upon completion, this laboratory will be fully equipped with facilities to detect all human pathogens and genetic diseases.

In 2006, the molecular biology laboratory introduced PCR for identification of MTC infections as a routine for the first time in Uganda. Since then, approx 5000 samples (culture and sputum) from the Joint Clinical Research Center have been speedily and accurately worked on. In addition, A number research programmes also bring samples to the laboratory for identification of MTC. Occasionally, clinicians and health workers bring suspect specimen to rule out MTC infection from their patients. So we deal with a diverse source of specimen and samples.  Rigorous steps and utmost care are taken to ensure quality of results disseminated.

Ugandan clinicians, health workers and researchers: We look forward to processing your samples using robust and reliable molecular techniques

Download PCR protocol for identification of M. tuberculosis

Download IS6110-RFLP Protocol for fingerprinting of M. tuberculosis

Completed projects in the Molecular Biology laboratory

Evaluation of various methods for rapid detection of multi-drug resistant tuberculosis:This was a WHO/TDR grant support to Moses Joloba.In this study four new methods (one molecular and other 3 culture based) were compared with the conventional indirect Bactec method for rapid detection of rifampicin resistance. The methods werecompared for turnaround time, cost and technical ease. Year of completion: 2007. Manuscript published in the open access journal, BMC Infectoius diseases where it is highly accessed. PI: Dr. Moses Joloba
Role of Cell-Cell signaling homologues in Mycobacteria Signaling: This was an RO3 NIH grant support to Moses Joloba. A genetic approach was used to identify and characterize mycobacteria genes that are required for production or sensing extracelluar signals. In addition, biochemical methods were used to purify signals. The grant was upgraded to RO1 (see cell to cell signaling in mycobacteria). PI: Dr. Moses Joloba

2) Current Research Projects in the Immunology Laboratory

This is the first and only fully fledged teaching immunology teaching laboratory in the department and Makerere University. The laboratory collaborates with the Makerere University Walter Reed, Medical Research Council, Uganda Virus Research Institute and the Joint Clinical Research Centre Laboratories. The laboratory is fully equipped performs and teaches all T-cell and immunoglobulin based immunological techniques. The department started a doctoral program in immunology with 5 fully sponsored PhD immunology students, based in this laboratory. The doctoral program also serves as an avenue for the training of masters graduates. Although it is new, the doctoral program has been favorably reviewed by Welcome Trust, Karolinska institute and Case Western Reserve Universities that are well recognized immunology teaching institutions.

Collaborative Research studies

The University of Washington PIP (Partners in prevention study) Study:  This completed study was about Randomized Placebo Controlled Trial of HSV-2 Suppression to prevent HIV transmission among HIV discordant couples. The serology and immunological screening on patient samples were done in the immunology laboratory.

The University of Washington COS (Couples Observational Study): This is a study among HIV discordant couples. This study explores the role of HSV-2 suppression in prevention of HIV transmission in discordant couples. Unlike previous studies, this study includes all participants irrespective of their level of immune suppression or being on ARVs

University of Washington PrEP Study:This study is about Parallel comparison of Tenofovir and Emtricitabine/Tenofovir  Pre- Exposure Prophylaxis to prevent HIV-1 Acquisition with in HIV-1 Discordant couples.

The immunological tests done in the laboratory:

  • HIV-ELISA
  • HSV-2 ELISA
  • Hepatitis B  ELISA
  • RPR  + TPHA
  • Agglutination tests
  • Western blotting/Immunoblotting

3) Current Research Projects in the BSL3 Mycobacteriology Laboratory

The AERAS Mycobacteriology BSL2 laboratory
Aeras TB vaccine study supported construction of the BSL2 Laboratory in Makerere University. This laboratory is the first of its kind in the University and it is used for liquid and solid culture of mycobacteria, Molecular tests as well as Drug susceptibility testing.

Epidemiological Studies towards Phase III TB Vaccine Trials in Uganda
Many TB cases and deaths occur in resource-limited settings in Africa where access to health services is often limited. Efforts are currently underway for developing and testing of more effective TB vaccines. Testing of these vaccines in areas where they are most needed has advantages in spite of the limited capacity to conduct such trials in these settings. Capacity building activities in these settings including TB epidemiological studies are therefore necessary. In Uganda, TB vaccine activities to build capacity for future phase III TB vaccine trials are being trained. Two prospective cohorts of 2500 BCG-vaccinated infants and 7000 adolescents aged 12-18 years will be under TB surveillance for two years. The studies are conducted in the Iganga/Mayuge Demographic Surveillance Site (DSS) located in a rural/peri-urban setting in Eastern Uganda.

The Biosafety level III Mycobacterialogy Laboratory
The Department of Medical Microbiology College of Health Sciences Makerere University provided space and other technical support, and AERAS global TB Vaccine foundation funded the construction and staffing of Bio-safety Level 3 laboratory for the TB vaccine study. The laboratory has a Director, a Manager, a supervisor, a field Manager, two technologists, one data Clark, two support staffs and two drivers.

4) Research and services in the Clinical Microbiology laboratory

This is the clinical diagnostic branch of the department of Medical Microbiology. The clinical laboratory offers affordable and quality diagnostic services to a plethora of specimens from patients suspected to have infectious diseases.  We offer bacterial, parasitic, fungal, and viral diagnosis detection in the specimens. The laboratory is strategically located centrally in Kampala, surrounded by a number of hospitals and clinics within the city. The clinical laboratory has increasingly become the nations referral centre for the public and private hospitals/clinics within the Kampala area, and countrywide.

The Clinical Laboratory receives specimens from a number of sources. These include Mulago Hospital, Infectious Diseases Institute, Paediatric Infectious Diseases Institute, Makerere University-Johns Hopkins University Clinic, Private Clinics and Private not-for-profit health facilities in Kampala City and upcountry, as well as from walk in individual patients.

Specimens of any type are processed here. Modern techniques raging from biochemical, serological, culture to molecular biology techniques are employed to identify disease causing Microorganisms. This work is executed by dedicated personnelcomposed of Specialist Clinical Microbiologists, technologists and support staff.
During 2008, the laboratory processed about 7000 specimens.

The major clients for the clinical microbiology laboratory include:

  • The Infectious Disease Clinic of The Infectious Disease Institute (IDI)- idi.mak.ac.ug
  • The Baylor Uganda, Paediatric HIV Clinic
  • The Makerere University- Johns Hopkins Collaboration
  • A number of Graduate students (Masters and PhD) doing Research Projects for their dissertations

Collaborative Research
A joint grant application ‘The Environmental Transmission of the AIDS associated Pathogen Cryptococcus neoformans in Africa’ has been submitted in collaboration with a group from the University of Minnesota for funding.
Through this grant, a number of equipment will be bought for the lab.

Accreditation:
The Clinical Microbiology laboratory has embarked on the process of accreditation.

Specimens Tests Performed

  • Biopsy Tissues e.g. Pleura, Bone marrow etc
  • Blood
  • Corneal scrapping
  • CSF (Cerebrospinal fluid)
  • Fluid aspirates (Pleural, Peritoneal, Joint, Pericardial, Cysts, Hydrocele)
  • Genital Specimens e.g urethral swab, High vaginal swab
  • Hair, Skin, & Nails for Fungal diagnosis  
  • Nasopharyngeal and oro-pharyngeal swabs
  • Pus
  • Semen
  • Skin snips for Microfilaria
  • Sputum, Induced sputum, Bronchoalveolar Lavage, Tracheal aspirates
  • Stool for bacterial pathogens
  • Stool for parasites
  • Swabs
  • Urine

5) The Mycology laboratory

The Department of Medical Microbiology has the only clinical mycology laboratory in the country. It is instrumental in detecting existing and new fungal infections in the era of HIV/AIDS. The lab is a powerful resource to disseminate knowledge on diagnosis of fungal diseases in Uganda.

Masters student’s research

The laboratories of the department of medical microbiology provide a vibrant research and training environment for both undergraduate and graduate students. It is these laboratories (Clinical Microbiology, Molecular Biology, Molecular Diagnostics, Immunology, Mycobacteriology and Mycology laboratories) that support students’ research and/or training. We serve health professionals, research scientists and students’ interests spanning from high school vacationists, undergraduates, Masters, and PhD candidates. Whether you are a student, health professional or high caliber Researcher (local or International), the department of Medical Microbiology warmly welcomes you to visit her research facilities for a friendly discussion.

Below is a list of Master’s students who successfully completed their graduate research in the department of Medical Microbiology. Except where noted, students’ research was totally funded by the department.


2006:

1) Factors associated with multi-drug resistant tuberculosis among retreatment patients attending mulago tuberculosis clinic. By Alice Asiimwe Rwego, MbChB, MSC. (Clinical Epidemiology)-Mak.

Supervisors: Moses L. Joloba, PhD, & Alphonse Okwera, MMED

THESIS ABSTRACT

Tuberculosis (TB) drug resistant tuberculosis (MDR-TB) in particular is emerging as an increasing important cause of morbidity and death. Drug-resistant tuberculosis is a significant threat to tuberculosis control because only a few effective drugs are available against m. tuberculosis. Even the best available treatment is often unsuccessful. MDR-TB among re-treatment patients attending the Mulago TB clinic has been found to be 13.3% and is increasing. The factors associated with MDR-TB are not well known in Uganda.
Objective: to determine the factors associated with multi- drug resistant tuberculosis among re-treatment patients attending Mulago TB clinic with the aim of informing strategy on preventing further spread and development of MDR-TB.

Methods: A case control design, the cases being patients with MDR-TB and the controls being patients with susceptible TB. A study setting was Mulago TB clinic in Kampala with participants enrolled from the welcome trust study.
Study participants: all TB patients with MDR-TB who fulfilled the eligibility criteria were recruited as cases and all TB patients with susceptible TB who fulfilled the eligibility criteria were recruited as controls.
Sampling: cases and controls were recruited and enrolled consecutively until the number was realized. Data collection, management and analysis: Data was collected using a semi-structured questionnaire, entered into Epi-info version 6.04 and exported to SPSS for analysis.

Results: 58.7% of the cases were male with median age of 33 years compared to 66.4% of the controls with a median age of 36years. Adherence level to anti-TB drugs was low among cases (55.6%) and controls (56.3%).Having worked in the hospital was the only socio-demographic factor that was negatively associated with multi-drug resistant tuberculosis (or=0.14; 95%CI 0.03-0.76). A participant who had worked in the hospital was almost 7 times less likely to have MDR-TB than one who had never.None of the clinical factors (adherence to treatment, HIV status and admission to hospital) was found to be significantly associated with MDR-TB.Participants with MDR-TB were 18 times more likely to have heard about MDR-TB than those who did not (or=17.85, 95% CI 6.45-49.30). Receiving drugs from private clinics/pharmacies and duration after diagnosis of TB before start of treatment were not significantly associated with MDR-TB

Conclusion: adherence to TB drugs was low among both cases and controls. Participants who had MDR-TB had a greater chance of hearing about MDR-TB while working in hospital seemed to be protective from having MDR-TB.


2) Reliability of multiplex PCR method in detection of methicilin resistance and panton-valentine leukocidin genes in Staphylococcus aureus. By Freddie Bwanga, MbChB, MMED (Microbiology)-Mak.

Supervisors: Moses L. Joloba, PhD, & Deogratious H. Kaddu-Mulindwa, PhD

THESIS ABSTRACT

There has been a recent increase in community-acquired methicillin resistant Staphylococcus aureus infections associated with strains possessing lukS and lukF genes that code for a highly virulent bi-component cytotoxin, the Panton-Valentine Leukocidin (PVL). Thus detection of PVL genes in a methicillin resistant S.aureus isolate is a genetic marker of community acquired infection and could be used to characterize the source of MRSA infection. Methicillin resistance in S. aureus is predominantly associated with production of penicillin binding protein 2a, which is encoded by the mecA gene. Currently the mecA and the lukS-lukF genes can be detected in S. aureus using PCR runs and it is regarded as the reference method. To detect all the three genes, two separate PCR runs are done. However it is possible to detect the presence of the three genes in one PCR run which could be cost effective. The aim of this study was to assess the reliability of the multiplex PCR test compared to the reference individual tests in simultaneous detection of the mecA and lukS-lukF genes in S.aureus. Individual and multiplex PCR tests for the mecA and lukS-lukF genes were performed on 220frozen stocks of S. aureus isolated from various clinical specimens, between 200 and 2006. The sensitivity and specificity of the multiplex test in detecting the mecA gene were 85.1% and 94.8% respectively. For the PVL genes, the corresponding values were 55.8% and 98.3% respectively. This study has shown that multiplex PCR is a sensitive and specific method of detecting the mecA gene but has low sensitivity in detecting the PVL-encoding genes lukS-lukF in S. aureus. The values obtained in this study were lower than those in two studies in Canada and Cleveland where the sensitivity and specificity were 100% for mecA and 98% and 100% for PVL. The multiplex method should be further evaluated before it is recommended for routine co-detection of the mecA and lukS-lukF genes


 
3) The distribution of mycobacterium tuberculosis complex species among PTB patients presenting at the national TB treatment center at Mulago hospital, Kampala, Uganda, by PCR method. By Candin Godfrey Mawa, MbChB, MMED (Microbiology)-Mak.

Supervisors: Moses L. Joloba, PhD, & William Worodria, MMED.

THESIS ABSTRACT

Tuberculosis (TB), caused by the bacterium mycobacterium tuberculosis complex (MTC) is one of the deadliest pathogens in human history. Tuberculosis control today relies on case identification, treatment (case holding) until cure and in certain instances prophylaxis with drugs such as in case of recent infection without clinical or radiological manifestation in non- BCG vaccinated children less than 5 years of age, living in close contact with patients and presenting with a strong positive reaction to tuberculin. The gold standard of TB diagnosis and speciation is based on the demonstration of AAF bacilli in clinical specimens, culture and setting biochemical tests. However classification based on physical and biochemical properties are tedious, interruption subjective and therefore can give ambiguous results. Molecular typing techniques particularly based on amplification or lack of specific DNA sequences has been found to give superior results. Despite high prevalence of TB in Uganda species identification of MTC is still a remote practice, particularly speciation based on molecular techniques, although there have been attempts to classify these organisms mainly on basis of biochemical tests. Strain typing allows the tracing of epidemiologically related cases including virulent or MDR strains, identification of nosocomial infections, differentiations of new cases from relapses and identification of laboratory contaminants.
This study describes molecular typing of 300 M. tuberculosis complex isolates cultured from sputum of patients described in the literature based on genomic deletion sequences namely: RD9, TbD1, RD4, RD1, RD12. The distribution of the molecular type pattern suggests that the population of M. tuberculosis complex strains isolated from PTB patients attending nation TB treatment centre at Mulago was predominantly m. tuberculosis constituting 89.3% contrary to the previous study a decade ago which showed that M. africanum was the predominant species in Kampala constituting 67%. This time M. africanum constituted only 2.7% of the isolates,22 (7.3%)were identified as M. tuberculosis ancient strain, and 2 (0.7%) were identified as M. canettii. the study concludes that M.tuberclosis is the predominant MTC species causing TB among patients aged twelve years and above presenting at the national TB treatment centre at Mulago hospital, Kampala, Uganda. M. africanum constitutes a small percentage of only 2.7% and M. canettii 0.7%. From the above findings, the study safely recommends that there is urgent need to carry out similar speciation studies up- country. Such studies should in addition search for possible accentuating factors for TB infection such as HIV. The study further recommends that molecular methods of diagnosis and speciation be imported for routine use in all microbiology laboratories in this country, as this would promote good TB management and control.                         


4) Campylobacter spp isolates and their sensitivity pattern from children with acute diarrhohea at Mulago hosipital complex Kampala, Uganda. By Mshana Eliatosha, MD, MMED (Microbiology)- Mak.

Supervisors: Moses L. Joloba, PhD, Deogratious H. Kaddu-Mulindwa, PhD & Kakooza Mwesigwa Angelina, MMED.

THESIS ABSTRACT

Campylobacter species are a frequent cause of entries and less often of extra intestinal infection in humans. Infection is usually transmitted through contaminated water and food with animal’s excreta especially from poultry. Infection rate in broilers in Uganda is 87% but there is no data on these organisms in human infection in Uganda. In other developing countries infection rate has been found to be between 5-20%. This study aimed at finding the prevalence of campylobacter spp among children with acute diarrhea attending Mulago hospital, Kampala, Uganda.

Main objective: The aim of the study was to determine the proportional of campylobacter spp infection and the antibacterial sensitivity of these organisms among children with acute diarrhea at Mulago hospital, Kampala, Uganda.

Materials and methods: A cross sectional study was conducted on 226 children with acute diarrhea attending Mulago hospital from June to October 2005. Serial sampling method was used to obtain the sample size. Stool specimens were  obtained, examined macroscopically and microscopically for white blood cells (WBC) and gram stain; and culture was done in micro-aerophilic environment using blood free campylobacter media (containing cefoperazone, charcoal, and deoxycholate). Identification was done using gram stain, catalase, oxidase and susceptibility to nalidixic acid, cephlothin and sodium hippurate hydrolysis. Disc susceptibility tests for erythromycin (15µg), ampicillin (10µg) and ciprofloxine (5 µg) were done.

Results: Study population was made up of 226 children with acute diarrhea, the majority were from Kampala district 138 (61.1%), the mean age was 16 months, and 42.5% were infants. A total of 68 (30.5%) had used antibiotics before stool culture. While blood cells were seen in 56.2% of stool specimen, 78% of the study population didn’t keep any animal at home.Campylobacter spp were isolated in 21 (9.8%) from 226 stool specimens cultured:
Campylobacter jejuni 17 (80.9%), campylobacter coli 1 (4.8%) campylobacter lari 2 (9.5%) and campylobacter jeji/coli (4.8%). There was association between presence of white blood cells and culture results (p=0.001), also there was association between use of antibiotics and low culture rate of campylobacter spp (p=0.031). There was no association between keeping animals at home and isolation rate of campylobacter spp (p=0.617) also being an infant did not predispose to infection with campylobacter spp (p=0.176). All campylobacter isolates were sensitive to erythromycin using disc susceptibility test, 20% were resistant to ampicilin and only 1 (5%) was resistant to ciprofloxin. This isolate was identified as campylobacter lari. The sensitivity and specificity of gram stain in diagnosing campylobacter infection was 76% and 99.5%, respectively.

Conclusion: Campylobacter infection is prevalent in Ugandan’s in other developing countries. There is strong association between the presence of white blood cells in stool and positive culture of campylobacter. Use of antibiotics affects the culture of campylobacter spp and the gram stain is specific for diagnosing campylobacter infection where facilities are limited


.2007:

1) Aetiology and antimicrobial susceptibility pattern of pyoderma in children presenting to Mulago hospital. By Joyce N. Balagadde, MBChB, MMED., Mak.

Supervisors: Philippa Musoke, PhD., Fred Kambugu, MMED & Moses L. Joloba, PhD

THESIS ABSTRACT

Introduction: P. yoderma is well-recognizes cause of morbidity in children in the under-developed tropical environment. It is curable if diagnosed early and appropriately treated, otherwise can potentially result in life-threatening complication. Treatment is empiric and relies on knowledge of the likely etiologic bacteria and their local antibacterial susceptibility pattern.

Objective: To determine the etiology and antibacterial susceptibility pattern of pyoderma in children presenting to skin clinic and assessment center of Mulago hospital.

Methods: Childrenaged 2 month to 12 years who fulfilled the eligibilitycriteria were enrolled. Clinical diagnostic was followed by specimen collection for bacteria culture and sensitivity testing HIV testing was done.

Results: We enrolled 398 children. The prevalence of HIV infection was 3.8%. Primary and secondary pyoderma was diagnosed in 34% and 56% of children respectively.
Ecthyma, folliculitis and furunculosis were the most common primary pyoderma while infected eczema, tinea capitis, popular urticaria and scabies were the most common secondary pyoderma. S .aureus and P. pyogenes were recovered from 70% and 43% of children respectively. Gram negative bacteria were recovered from 8%. Sensitivity of S. aureus to vancomyicin and oxacillin was 99.6% and 98% respectively. Resistance of S. aureus to penicillin was 97%. Resistance of S. aureus and S. pyogenes to erythromyicin was 36% and 42% respectively.

Conclusions: S. aureus and S. pyogenes were the predominant etiologic agents of pyoderma. The prevalence of methicillin resistant S. aureus was very low (2%). Resistance of S. aureus and S. pyogenes of erythromycin was high.


2) Prevalence of methicillin resistant Staphylococcus aureus among isolates from surgical site infections in Mulago hospital, Kampala, Uganda. By Ojulong Julius, MbChB, MMED (Microbiology)- Mak.

Supervisors: Moses L. Joloba, PhD, & Deogratious H. Kaddu-Mulindwa, PhD

THESIS ABSTRACT

Background: Methicillin resistant Staphylococcus aureus( MRSA) is a worldwide health problem. MRSA isolates are resistant to penicillins and all other B-lactam antibiotics. Nosocomial MRSA is also resistant to variety of other antibodies classes. MRSA infections are associated with a high morbidity and mortality particularly in developing countries where more expensive drugs like vancomycin are affordable. The objective of this study was to determine the prevalence of MRSA among S. aureus isolates from surgical sites infections in Mulago hospital, Kampala, Uganda.

Methods: One hundred and eight pus swabs were collected from patients with surgical site infections. Swabs were introduced for culture at microbiology laboratory faculty of medicine, Makerere University. S. aurues was identified biochemically. All S. aurues isolates were subjected to oxacllin agar screen and then tested with a polymerase chain reaction (PCR) assay for detection of the mecA gene which codes for oxacillin resistance.

Results: Out of the 188 specimen cultures, 54(28.7%) grew S. aurues. Seventeen (31.5) of the 54 isolates were confirmed as MRSA by PCR.

Conclusion: This study shows a high pr


evalence of MRSA in surgical site infections in Mulago hospital.

3) Clinical profile and antimicrobial susceptibility of pnuemococcal bacteria among febrile patients admitted to the emergency medical ward at Mulago hospital. By Grace Namayanja, MbChB, MMED., Mak.

Supervisors: Alice Namale, MMED., Moses L. Joloba, PhD, Robert A. Salata, MD & Harriet Mayanja Kizza, MD, MSC.

THESIS ABSTRACT

Introduction: Streptococcus penumoniae is major cause of morbidity and mortality worldwide more especially in the immuno-compromised individuals. An estimated 50-60% of in-patients on the medical wards of Mulago hospital are immuno-compromised due to HIV infection. Increasing resistance of S. pneumonia bacteraemia to available antimicrobial agents may worsen clinical outcome in resources constrained settings, there are limited data on prevalence, clinical profile, and antimicrobial susceptibility of S. pneumonia among hospitalized patients in Uganda.

Objectives: To determine the prevalence, clinical profile and antimicrobial susceptibility patterns of S. pneumonia bacteraemia among febrile patients admitted to the emergency medical ward at Mulago hospital.

Methods: Descriptive cross sectional study with follow up of patients with confirmed S. pneumonia bacteraemia on blood culture. Febrile patients with an oral temperature of greater than or equal to 37.8°C were sampled consecutively until the sample size was achieved. Using a standardized questionnaire, data on socio-demographics clinical features and outcome were collected. Blood was drawn for complete blood count, serum chemistry, bacterial culture and sensitivity. Data was analyzed using SPSS version 12.0.
Study setting: Emergency medical ward, Mulago hospital Kampala, Uganda.
Study participants: A total number of 386 febrile patients aged 13-81 years, who were admitted from November 2006 to March 2007 in the emergency medical ward were enrolled.

Results: The prevalence of S. pneumonia bacteraemia was 9.8% (38/386). Of these, 68% (26/38)were HIV infected. The mean oral temperature was 38.6°C and mean duration of fever was 3 weeks. Cough was reported by 78.9 %( 30/38) and headache by 376.8 %( 14/38) with a mean duration of 3 weeks. Cigarette smoking was reported by 15.8%.
Multilobar consolidation on chest x-ray was noted in 58% (11/19). The mean neutrophil percentage was 77.4= 12.6% with a neutrophilia of greater than or equal to 75% present in 68% (26/38). Impaired renal function with creatinine of greater than or equal to1.3mg/id was found in 68% (26/38). Cough (p=0.014), chest signs (p=0.048), meningeal signs (p=0.001), neutrophil percentage (p=0.004), multilobar consolidation (p=0.001) were significantly associated with S. pneumonia bacteraemia, but cigarette smoking (p=0775) was not. All S. pneumonia bacteraemia isolates were resistant to contromoxazole, but all were susceptible to cenftriaxone and ethromycin while only 21.1% were susceptible to penicillin. On follow up of the patients, the mean hospital stay was 8.6 days; 34.2% developed septicaemia, 28.9% pneumonia while 13.0% developed meningitis. Complete recovery was noted in 78.9% (30/38), and mortality in 7.9% (3/386), Staphylococcus aureus 1.6 %( 6/386), Pseudomonas areruginosa 5, E. coli 5, Klebisella pneumonia 3, Haemophilus influenza 2, A cinetobacter 2 and Norcadia 1.

ConclusionS. pneumoniae bacteraemia is common among febrile patients admitted on the medical emergence ward at Mulago hospital. Presentation is characterized by fever, cough, and headache. The isolates are resistant to contrimoxazole and penicillin which are the commonly available antibiotics. Mortality is more likely in patients with leucopenia, anemia, HIV infection, meningitis and dehydration.


4) Accuracy of sputum polymerase chain reaction in the diagnosis of tuberculosis among sputum smear negative adult PTB suspects in Mulago hospital. By Lydia Nakiyingi, MbChB, MMED (Internal Medicine)-Mak.

Supervisors:Roy Mugerwa, Harriet Mayanja & Moses Joloba, PhD.

THESIS ABSTRACT

Introduction and rationale: Accurate and early diagnosis of TB is crucial for effective patient management and TB control. The sensitivity of the available diagnosis method, sputum smear microscopy is very low; ranging from 30to 70% and it is even lower in TB/HIV co-infected patients. This has resulted into an increased number of SSN PTB suspects, yet sputum culture for confirmation of TB is not readily available and not routinely done. Therefore a need for rapid and accurate tests for the diagnosis of SSN TB, particularly those using molecular techniques like sputum PCR. Sputum PCR is now available in Uganda in research settings and no study has been done to evaluate its accuracy in the diagnosis of TB among SSN adult PTB suspects.

Objective: To evaluate the accuracy of in house sputum PCR as compared to sputum culture in the diagnosis of TB among AFB sputum smear negative adult PTB suspects in Mulago hospital and to also describe the clinical characteristics associated with positive sputum PCR.                           

Methods: This cross sectional study was conducted from September 2007 to February 2008 on adult patients admitted on the emergency medical wards of Mulago hospital complex.  A pre-tested and standardized questionnaire was administed to consenting PTB suspects who were then asked to provide 2 early morning sputum samples and a proportion of each sample was subjected to smear microscopy using ZN staining. SSN PTB suspects meeting the inclusion criteria were recruited consecutively into the study until a sample size of 205 patients was attained. The remaining portions were each subjected to sputum culture using LJ media and a proportion of the second sputum was subjected to sputum PCR after processing
Data was collected using a coded questionnaire, entered using EPI-INFO 6.04 and analyzed using STATA version 10.0. using LJ sputum culture as the “gold standard”, we analyzed for the diagnostic accuracy  of sputum PCR by computing Sensitivity, specificity, Positive and negative values as well as diagnostic likelihood ratios. Bivariate analytical methods were conducted to describe the factors associated with positive sputum PCR.

Results: of the 320 consenting PTB suspects screened, 115 patients were AFB smear positive and were started on anti-tuberculosis treatment, 205 were AFB smear negative and the inclusion criteria. Compared to LJ culture, the sensitivity and specificity of the in-house sputum PCR were 75% and 35% respectively and the positive and negative predictive values were 39% and 72.4% respectively.
Body temperature below 37.5° (or 0.423(0.22-0.82), p- value 0.0009) was significantly associated with positive sputum PCR among AFB smear negative PTB suspects.
Conclusion: the diagnostic accuracy of the available in-house sputum PCR is low. Body temperature below 37.5°C is associated with positive PCR in AFB smear negative PTB suspects

Recommendations: the available in- house sputum PCR cannot be adopted as a rapid and accurate alternative to LJ culture detection of TB in AFB smear negative PTB suspects. The diagnostic accuracy of this test, therefore, has improved in order for it to be worthwhile and beneficial. the in-house PCR should be compared to a more sensitive “gold standard” like mycobacteria growth indicator tube (MIGIT) system and a study should be done to compare other possible in-house PCR methods in our setting to the “gold standard”.


Evaluation of PCR for direct detection of Mycobacterium tuberculosis complex from sputum samples. By Alarakol Simon Peter.

Supervisors: Moses L. Joloba, PhD & Nakavuma Jessica, PhD.

THESIS ABSTRACT

BACKGROUND: Tuberculosis (TB) is a public health problem causing up to 3 million deaths worldwide. In Uganda TB is the third top killer disease especially in the HIV patients.
Routine diagnosis of TB in the laboratory uses ZN microscopy which has low sensitivity.
Objective: this study evaluated PCR for direct detection of MTB complex from sputum samples.

Method: A total of 180 sputum samples were collected from 60 suspected TB patients.
Thirty ZN negative and 30ZN positive were included in the study. Three specimens were collected from each patient. Specimens were digested in N- Acetyl L- Cysteine (NALC)-4 NaOH. DNA extracted from sputum samples was used for PCR amplification of 1S6110 sequence which is specific for M.tb complex.

Results: The proportion of samples detected by PCR for ZN negative and ZN positive smears were 46 (51%) and 73 (81.1%) respectively. Using three samples from each patient, PCR detected M.tb in all (100%) of ZN positive samples and 80% of ZN negative samples.
Conclusion/recommendation: PCR is a highly sensitive diagnostic tool and therefore can be used in the detection of mycobacterium tuberculosis complex in ZN negative patients.


2008:

1) Prevalence and factors associated with risk of HIV occupational exposure and PEP utilization among healthcare workers in Mwanza referral hospitals-tanzania. By Samuel Sumba James, MbChB, MMED., Mak.

Supervisors: Moses L. Joloba, PhD, Sarah Nabwire Ssali, PhD & B. Gumodoka, MMED.

THESIS ABSTRACT
Introduction: Its is estimated that about 40 million people are living with HIV/AIDS globally, and of those two thirds are sub Saharan Africa. In Tanzania the prevalence of HIV/AIDS among adults is 7%. The increasing number of persons being treated for HIV associated illness makes it likely that more health care workers will encounter patients infected with HIV and therefore they are at a high risk of occupational exposure, more so in developing countries with high incidence of blood borne viruses and increased risk of occupational injuries. The use of post exposure prophylaxis (PEP) for HIV reduces the chance of infection.

Objective: Theobjective of this study was to study prevalence and factors associated with risk of HIV occupational exposure and PEP utilization among healthcare workers in Mwanza referral hospital.

Methods A cross sectional study was conducted between January and march 2008 in Mwanza referral hospital(North west of Tanzania mainland). For quantitative data, a total of 363 health care workers were initially selected by sampling proportionate to size of each hospital and by occupational category in the respective hospitals. Consecutive sampling was done within the different categories (units). Qualitative data were collected from a total of six key informants, three from each hospital selected purposively.

Results: The overall prevalence of risk of HIV occupational exposure was 33.9% (95% CI=29.0-38.8). Risk of HIV occupational exposure was high among healthcare workers in Mwanza regional hospital (Sekou-Toure), (OR 2.44, 95% CI-1.54-3.85). Those working in the department of surgery and obstetrics were more likely to experience, (OR 1.92, 95% CI= 1.19-3.13) and (OR 1.45, 95% CI=0.84-2.50) respectively. The rate of utilization was higher among those who worked for forty hours or more per week (OR 5.44,95% CI 1.04-28.59). Health care workers who knew the procedures for PEP were more likely to utilization the services, ( OR 5.88, 95% CI 1.64-20.00).Some of the possible key barriers to utilization of PEP services for HIV among other were lack of knowledge and information on PEP services for HIV among healthcare workers, stigma and professional discrimination.

Conclusion: T his study demonstrates that healthcare workers in Mwanza referral hospital are at an increased risk of HIV occupational exposure, and the utilization of PEP services for HIV is suboptimal.


2) Assessment of smear microscopy in a TB programme setting in Kampala, Uganda: combining blinded re-checking, culture and polymerase chain reaction. By Sande Obondo James, MbChB, MMED (Microbiology)- Mak.

Supervisors: Moses L. Joloba, PhD., & William Worodria, MbChB, MMED.

THESIS ABSTRACT
Background:  In developing countries TB case detection by quality-assured bacteriology using Acid- Fast direct smear microscopy is one of the DOTS components of the stop TB strategy in an era of increasing HIV- related TB. Uganda’s TB case detection rate is low may be due to poor performance of AFB smear microscopy at peripheral laboratories and failure to use new rapid diagnostic tools such as PCR. Blinded rechecking of smears, the EQA method of AFB smear microscopy has not been performed against mycobacterial culture in assessing performance of AFB direct smear microscopy at peripheral laboratories, and the accuracy of per in TB case detection is not known in our setting.

Objective: This study aimed at determining the magnitude of missed smear-positive cases at peripheral laboratories in Kampala by performing blinded rechecking of smears against LJ culture as the gold- standard, and also determines the accuracy of PCR using Lowenstein Jensen (LJ) culture as the gold- standard.

Methods: This was a cross-sectional study in four health units in Kampala, from February 2008 to may 2008. ZN smears from 296 spot sputum samples of new TB suspects were prepared and read by technologists at the peripheral laboratories and the re-read (blinded rechecking)by technologists at reference laboratory. LJ culture and IS6110 PCR were performed on NaOH/NALC – processed sputum from which the original smears were prepared. HIV status of participants was determined.

Results: Sixty-eight percent of the TB suspects were HIV- positive, 23% HIV negative and 90% unknown status. The magnitiutude of missed smear-positive TB cases at peripherical laboratories was 19.2% (9 missed smear- positive cases) when compared to culture, and 5.7% (3 missed smear-positive cases) when compared to blinded rechecking. There was 91.8% observed agreement between blinded rechecking and culture, 98.7% observed agreement between reading of smears at peripherical laboratory and re- reading at the reference laboratory (Kaapa=0.930). PCR showed sensitivity, specificity, positive and negative values of 98.0%, 84.6% and 99.4% respectively in all suspects. Of the 19 culture cases (94.7%). Of the 111 culture- negative smear-negative HIV- positive TB suspects, negative predictive values of PCR in smear- negative HIV- positive TB suspects were 94.7%, 97.3%,85.7% and 99%, respectively.

Conclusion:
Blinded rechecking is a satisfactory EQA method of smear microscopy in our setting. PCR is very sentive and highly specific in detecting TB cases in smear-negative HIBV-Positive TB suspects. The magnitude of missed smear- positive cases was worse at peripheral laboratories than at reference laboratory, but not statically different.


3) Molecular characterization of Mycobacterium bovis isolates from selected slaughter houses in Kampala. By Jeniffer Asiimwe, BVM, MSC (Molecular Biology)-Mak.

Supervisors: Moses L. Joloba, PhD & Nakavuma Jessica, PhD.

THESIS ABSTRACT

In order to gain an insight into the genetic diversity and geographical sub-structuring of M. bovis strains in Uganda,170 samples were collected from the major slaughter houses in Kampala, and cultured for  mycobacteria isolation. A total of 21 mycobacteria isolates (12.4% of the samples collected) were obtained from the study. PCR based identification revealed that 48% (n=10) 0f the mycobacteria other than tuberculosis (MOTTS). Spoligotyping revealed that three of the M. bovis isolates had spoligopatterns that had previously been reported in Uganda while the seven were new. In this study, one of the M. bovis had a spoligo pattern identical to that observed in a TB-HIV co-infected individual from a parallel study in Rubaga division, Kampala (unpublished observations). The M. tuberclosis isolates lack spacer 40 which is characteristic to most of the isolates previously isolated from humans in Uganda. IS6110 RFLP analysis of nine of the M. bovis isolates obtained in this study revealed that most of these strains (except two)were high copy number strains with more than five copies of IS6110. Molecular typing of the M. bovis and M. tuberculosis isolates revealed a high degree of heterogeneity among the strains and a high level of strain dissemination in a country where cattle movements are not controlled. Although a few clusters were identified on analysis of the spoligotypes and RFLP patterns, no geographical sub-structuring was observed. This study also highlights the fact that in regions with high prevalence of HIV positivity, a cycle of cattle to human to human and human to cattle could be easily established especially in a country where many communities are economically dependent upon cattle and are in frequent close contact with them. It is therefore recommended that there should be a grater degree of co-operation between veterinary and medical policies that will provide adequate data for the formulation of sustainable control strategies for TB in Uganda.

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4) Analysis of HIV-1 subtypes among blood donars in Uganda using a multi-region hybridization assay. By Bagaya Ssentalo, BBLT, MSC (Molecular Biology)-Mak.

Supervisors: Moses L. Joloba, PhD., Fred Wabwire-Mangen, PhD., and Miguel A. Arroyo, PhD.

THESIS ABSTRACT

Background: Uganda has been a focus of HIV/AIDS intervention efforts, including vaccine clinical trials. HIV -1 genetic diversity poses challenges for design of efficacious vaccines.
Data on HIV-1 genetic diversity is crucial for design of an effective vaccine in Uganda. Some previous studies have reported   discrepant results probably due to varied sensitivity of different sub-typing methods and different study populations. Also no study sampled HIV-1 across the entire country.
Study objectives: (i) to determine the HIV-1 prevalence and (ii) HIV-1 subtype distribution among blood donors attending donation centers located in five different regions of Nakasero/ Kampala (central), Mbale (eastern), Fortportal (wersten), Mbarara (sourthen), and Gulu (northern region).

Methodology: 6,192 samples were collected from anonymous blood donors in five regional blood banks Uganda. All samples were tested for HIV-1  using MUWRP laboratory/FIDA approved algorithm. HIV-1 viral load and HIV-1 sub typing was performed on HIV-1 positive samples using Roche Amplicator HIV-1 monitor Test v1.5 and MHAacd respectively.

Results: HIV-1 prevalence among blood donors was 1.3% but was highest in Kampala at 1.8% and Gulu at 1.5% and was the lowest in Fortportal and Mbarara at0.9% and 1.0% respectively. Prevalence increased with increased age and was 3.2% among the 34-39 and 3.4% in the 50+ age groups. HIV-1 subtypes A accounted for 50% of cases, 25% subtype D, 2% subtype C and recombinants AD made up 20% and 3% for AC. Subtype A was dominant in 4 out of 5 regional blood banks while subtype D predominated in fort portal.  Subtype distribution was compared across gender but the HIV-1 prevalence was higher in female (1.6%) blood donors than in males (1.3%)

Conclusions: this study adds information to the HIV-1 subtype distribution in Uganda and informs vaccine design and clinical trials. HIV- 1 pure sub type A was the most predominant sub type among blood donors in Uganda and the proportion of pure sub type C was very low in this study. Recombinant HIV being responsible for almost a quarter of cases among blood; a low risk population further depicts the increasing role of recombinants and dynamic nature of the HIV/AIDS pandemic HIV -1 subtype distribution was comparable with respect to gender. This study demonstrates development of the capacity to genotype HIV-1 using the real-time PCR based MHA acid technique for the time in Uganda.


5) Bacterial aetiology and antimicrobial susceptibility of chronic suppurative otitis media in HIV-Infected children attending the paediatric infectious disease clinic in Mulago hosipital. By Jimmy Sekitoleko, MbChB, MMED., Mak.

Supervisors: Turitwenka Edward, MSc & Moses L. Joloba, PhD

THESIS ABSTRACT

The HIV/AIDS pandemic is one of the most devastating ever seen. Sub-Saharan Africa is one of the worst hit, although it is home to 10% of the world’s population, 60% of people with HIV/AIDS live in it. HIV destroys the body’s immune system leading to development of multiple pathogenic conditions, chronic suppurative otitis media  among them. Approximately 15% of children infected with HIV present with chronic suppurative otitis media. Poorly managed chronic suppurative otitis media can result into complications, among these are; hearing loss due to tympanic membrane perforation, septicaemia, mastoiditis, facial nerve palsy, extra and intracranial infections and some of the are fatal. Many antimicrobial agents are on the Ugandan market; however their efficacy on bacterial agents of C.S.O.M in HIV infected children is unknown.
Objective: The aim of the study was to determine the bacterial agents of chronic suppurative otitis media and their antimicrobial susceptibility in HIV infected children.

Study design: This was a case- series study
Study setting: The study was conducted in the pediatric infectious disease clinic located in Mulago hospital.
Study population: This involved 41 HIV positive children aged between 0-12 years attending pediatric infectious diseases clinic who met the inclusion criteria.
Outcome measures: The objective was to determine the types of bacterial agent’s of C.S.O.M in HIV infected children and their antimicrobial susceptibilities to commonly available antimicrobial agents.
Results: During the study period between October and December 2007, 41 children were assessed.Bacterial agents isolated in order of percentage frequently included: Proteus mirabilis (37%), Pseudomonas aeruginosa (21.7%), Klebsiella pneumonae (10.8%), Escherichia coli and Staphylococcus aureus each contributed (8.7%), Enterobacta (6.5%), Morganella morgani (4.4%) and finally Streptococcus pneumonae with (2.2%).
Ciprofloxacin was 78% effective on all isolates, followed by gentamycin with effectivity of 65%, ceftriaxone with 63%, augmentin with 26% and chloramphenicol with 24%.
Chloramphenicol which is commonly used in form of ear drops was found to be effective on only 24% of all isolates.

Utility of the study results: The findings were recorded on pre-tested data collection sheets and analyzed. It is hoped that results of this study will contribute into the knowledge base of bacterial etiology of chronic suppurative otitis media and antimicrobial susceptibility in HIV infected children.


2009:

1) Prevalence of toxoplasma gondi infection among adult HIV patients in mualgo hospital, Kampala, Uganda. By Erima Bernard, MbChB, MMED (Microbiology)- Mak.

Supervisors: D.H. Kaddu-Mulindwa, PhD., Fred M. Kironde, PhD., & Edward Ddumba, MMED.

THESIS ABSTRACT

Introduction: Toxoplasma gondii is a major cause of neurological morbidity and mortality among patients with advanced acquired immunodeficiency syndrome (AIDS). There are very few published studies on human toxoplasmosis in Uganda. The magnitude of the problem among the HIV/AIDS patients in Uganda is not known. The relationship between circulating T- cells and the infection with Toxoplasma gondii has not been adequately investigated.
Objective: the goal of this study was to determine the prevalence of Toxoplasama gondii infection and describe its manifestation using the laboratory tests among adult HIV positive patients attending Mulago Hosipital.
Design: across sectional and descriptive study.

Methods: Three hundred (300) adult HIV infected patients receiving health care on the medical wards and outpatients clinics at Mulago hospital were enrolled. Three (3) ml of whole blood was collected for analysis. The circulating CD4+ T-Cell count was determined using FACS caliber (Becton Dickinson) flow cytometry system. Anti-T. gondii IgG antibodies were screened by an agglutination technique using Toxoscreen DA kit (bio-merieux); and the presence of T. gondii specific DNA  was examined for using nested PCR in the patients’ blood.

Results: A hundred thirty one (131) males and a hundred sixty nine (169) females were enrolled in the study. The participants’ age ranged from 18 years, with mean age of 34 years. The mean CD4+ T cells count was 175 cells/ µL (0.0 to 1361 cells/ µL).
Seventy percent (70%) of samples had CD4+ T-cell count less than 200 cell/ µL.
The ser0- prevalence of T. gondii infection in this study population was 59.7%. out of the 300 samples, 62.0% had T.gondii specific DNA (T. gondii B1 gene). Therefore, 23.3% had acute toxoplasmosis, 38.7% had reactivated toxoplasmosis, 20% had latent toxoplasmosis, and only 18% participants in the study were not infected with T. gondii. The proportion of patients with reactivated toxoplasimosis did not have a lower mean CD4+ T cell count compared to those with latent toxoplasmosis.

Conclusion: The sero- prevalence of toxoplasmosis among HIV/HIV patients attending Mulago hosipital is very high with significant proportions having acute, latent, or reactivated toxoplasmosis. Levels of CD4+ T cell count was not related to presence or type of toxoplasma infection.


2) Benson Kidenya, MD, MSC (Molecular Biology)-Mak.

Supervisors:Moses L. Joloba, PhD., & Nakavuma Jessica, PhD.

THESIS ABSTRACT

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3) Mycobacterium tuberculosis genetic diversity in Mbarara, South Western Uganda. By Joel Bazira. MbChB, MMED-Mbarara Univ.

Supervisors: Moses L. Joloba, PhD.

Comparison of transformation frequencies of commonly drug resistant and susceptible serotype of streptococcus pneumonia

By: Benson Kidenya, MD, MSC (Molecular Biology)-Mak

Supervisors:Moses L. Joloba, PhD., & Nakavuma Jessica, PhD.

Abstract

Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. There are at least 90 serotypes of pneumococcus but it has long been observed that drug resistance is found only in a limited number of pneumococcal serotypes, 6, 9, 14, 19 and 23. There is no compelling mechanism to explain this restriction. Its ability to develop natural competence for genetic transformation is thought to be a major mechanism for horizontal acquisition and initial development of resistance. It still remains to be elucidated why only few serotypes of S. pneumoniae are drug resistant. During this study, it was hypothesized that the serotypes that are commonly associated with drug resistance have higher transformation frequency than the ones that are frequently susceptible. Thus, an in vitro investigation of genetic transformation frequency of these serotypes was compared with the other serotypes (1, 3, 18, 29, 33 and 35) of S. pneumoniae under the influence of the synthetic competence-stimulating peptidesto assess their transformation frequency. The tranforming DNA was the genomic DNA carrying Tn916-like transposon with the mefE gene that confers erythromycin resistance. The findings suggest that the serotypes-serogroups 6, 9, 14, 19 and 23 that are highly associated with the drug resistance phenotypes do not exhibit a higher degree of transformation efficiency than other serotypes. The serotypes (6, 9, 14, 19 and 23) with highest frequency of resistance are carried in children for a long period of time. Children are more likely to take antibiotics than any other age group. Thus, the serotype association with drug resistance in general is probably due to prolonged exposure to transforming DNA resulting from longer carriage and to a greater selective pressure from antimicrobials.

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Evaluation of PCR for direct detection of Mycobacterium tuberculosis Complex in sputum samples in Kampala, Uganda

By Simon P Alarakol
Supervisors: Moses L. Joloba, PhD & Nakavuma Jessica, PhD.
Thesis Abstract
BACKGROUND:  Correct diagnosis of tuberculosis (TB) remains a challenge particularly in developing countries where the incidence is alarming due to the strong association with Human Immunodeficiency Virus (HIV). Polymerase Chain Reaction (PCR) as a Rapid diagnostic technique can potentially provide correct and timely results, reducing the transmission of TB in communities. We evaluated PCR for direct detection of Mycobacterium tuberculosis complex (MTC) in sputum samples using Ziehl-Neelsen (ZN) as the gold standard. Two approaches were used in the detection of mycobacterium tuberculosis complex in ZN negative TB; Direct PCR-amplification on sputum sediments as templates and extraction of genomic DNA from sputum.
METHODS: Sixty patients, 30 ZN-negative and 30 ZN-positive, were included in the study. Three sputum specimens were collected from each patient and treated with N-acetylL-cycteine (NALC)-4% NaOH. Chromosomal DNA was extracted from sputum and used as template for PCR amplification of IS6110 which is specific for MTC species.
RESULTS: The proportion of samples detected by PCR in the ZN negative and ZN positive smears were 46 of 90 (51%) and 73 of 90 (81%), respectively.  Using the three samples from each patient, PCR detected MTC bacilli in all the 90 (100%) ZN positive samples and 80% of ZN negative samples.
CONCLUSION: PCR is a highly sensitive diagnostic tool and therefore can be used in the detection of mycobacterium tuberculosis complex in ZN negative TB.

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2010:
Lydia Nabyonga
Sylvia Wanzala
Katabazi Ashaba Freddie
Olia Alex
Eugene
Muyombya William
Gafirita James
Tusubira Evans

The laboratories of the department of medical microbiology provide a vibrant research and training environment for both undergraduate and graduate students. It is these laboratories (Clinical Microbiology, Molecular Biology, Molecular Diagnostics, Immunology, Mycobacteriology and Mycology laboratories) that support students’ research and/or training. We serve health professionals, research scientists and students’ interests spanning from high school vacationists, undergraduates, Masters, and PhD candidates. Whether you are a student, health professional or high caliber Researcher (local or International), the department of Medical Microbiology warmly welcomes you to visit her research facilities for a friendly discussion.

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Below is a list of doctoral students who successfully completed their graduate research in the department of Medical Microbiology. Except where noted, students’ research was totally funded by the department.

2008:

1) Molecular characterization of mycobacterium tuberculosis complex in Kampala, Uganda. By Benon B. Asiimwe

Supervisors: Gunilla Kallenius, PhD., Moses L. Joloba, PhD., & Tuija Koivula, PhD.

THESIS ABSTRACT
Uganda is one of the countries with the highest burden of tuberculosis (TB) in sub-saharan Africa, ranked 16th among the 22 highest-burden countries in the world. Poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated most in the increase of TB. Different species, strain families and lineages of the Mycobacterium tuberculosis Complex (MTC) are now known to have differences in virulence, clinical presentation as well as transmission potential. This study determined the predominant species as well as strain lineages that cause TB in Rubaga division, Kampala; analyzed TB transmission in HIV-seropositive and HIV-seronegative TB patients and the prevalence of resistance to key anti-tuberculosis drugs. Furthermore, the study characterized cattle-derived isolates of M. bovis from slaughter-cattle at a peri-urban city abattoir so as to set up a database of M. bovis strains for comparison with infections in humans in future studies.
To achieve this, 386 consecutive newly presenting sputum smear positive patients resident in and attending TB clinics in Rubaga division were enrolled. Infecting species for 344 cultures were determined by a solely PCR-based typing panel that determined presence or absence of regions of difference (RDs) in the MTC; strain types and families were determined by spoligotyping, and dynamics of TB spread in HIV co-infected vis-à-vis HIV-seronegative TB patients by standard IS6110-RFLP fingerprinting methodology. All but one of the 344 isolates in the study were M. tuberculosis, the other being M. bovis. Spoligotyping revealed predominance of the T2 family, which was in turn predominated by a previously described   “Ugandan genotype” group of strains. Further characterization of 139 Uganda genotype strains revealed an internal deletion in the RD724 locus, a polymorphism that defines one major sub-lineages of M. tuberculosis commonly seen in the central African human host population. Resistance to isoniazid was found in 8.1% of 344 strains, while all 15 (4.4%) strains resistant to rifampicin were also multi-drug resistant. IS6110-RFLP analysis of isolates from 80 HIV-seronegative patients revealed no difference in the level of diversity of DNA fingerprints observed in the two serogroups (P = 0.615), patients aged <40 years (P = 0.100), and sex (P = 0.715). However, 54% (99/183) of the patients shared fingerprints (average cluster size of 2.9), suggesting a high transmission rate in this community. There was no association between any starin types in the sample with either drug resistance or HIV sero-status of the patients. Eleven M. bovis and six non tuberculous mycobacteria were isolated from tissue samples of 87 carcasses. Worryingly, six carcasses showing obvious and multiple sites of infection were not condemned as unfit for human consumption, creating a potential for spread of M. bovis in the food chain and to humans through consumption of contaminated meat, a very important health concern in a resource-poor high disease-burden setting.
The study has shown that M. tuberculosis is the predominant species of the MTC in Kampala, and the spoligotype-specific and RD724-deleted “Uganda genotype” the predominant strain type. The TB epidemic in Kampala is localized, mainly caused by the closely knit T2 spoligotype family of strains, and strain types common in neighboring countries were minimal. Additionally, strain types were neither associated with drug resistance, nor HIV sero-status. The study further showed evidence of a high rate of recent transmission of TB in Rubaga with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristic, including HIV sero-status.

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2009:


2) Characterization of in vitro anti-malarial sensitivity and evaluation of genetic polymorphisms in P. falciparum associated with resistance in Uganda. By Nsobya Samuel, BBLT, MSC (Molecular Biology), PhD thesis submitted.



2010:

Bwanga, Freddie
RAPID TESTS FOR MULTIDRUG RESISTANT TUBERCULOSIS IN LOW INCOME SETTINGS

Fredagen den 24 september 2010, kl. 09.00.
Swedish Institute for Infectious Diseases Control Solna, Sweden in Gard-Aulan hall.

 

ISBN: 978-91-7457-034-2  

 


Supervisors:

  1. Moses L. Joloba, PhD, Makerere University College of Health Sciences, Kampala, Uganda
  2. Hoffner S, Karolinska Institute, Stockholm, Sweden

Abstract:
Tuberculosis (TB) is at epidemic levels in the resource-limited settings (RLSs) due to HIV/AIDS, poverty and insufficient TB control programmes. These factors are also contributing to TB drug resistance. Patients with multidrug drug resistant tuberculosis (MDR-TB) do not respond to first line drugs. These patients require unique drug regimens, making it necessary to routinely screen for MDR-TB. Screening for MDR-TB with the Lowenstein-Jensen proportion method (LJPM), which is common in the RLSs is a very slow process – taking 2-3 months. More rapid tests suitable for RLSs are urgently needed. In this thesis, a comparison of the technical and operational performance of several rapid tests for MDR-TB was done, and the most optimal tests for RLSs are proposed.
In paper I, a meta-analysis of rapid tests for direct detection of MDR-TB was conducted. The direct nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) and Genotype® MTBDRplus (GT-DRplus) were highly sensitive and specific, and far more rapid than the conventional indirect drug susceptibility testing (DST).
In paper II, the NRA, MODS, Mycobacterium Growth Indicator Tube (MGIT 960), GT-DRplus, Alamar blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and resazurin assays were compared head-to-head for indirect detection of MDR-TB at the National Tuberculosis Reference Laboratory (NTRL) Kampala. The NRA, MGIT 960, GT-DRplus and MODS were the most sensitive and specific tests, with significantly shorter time to results compared to the LJPM.
In paper III, the direct NRA and MODS assays were compared at the NTRL on sputum specimens from consecutive re-treatment TB patients. Interpretable results were obtained in over 90% of the samples with both assays. The median days to results were 10 with the NRA and 7 with MODS. The direct NRA was more sensitive and specific, and was cheaper.
In paper IV, the sensitivity, specificity, time to results (TTR) and reproducibility of the direct GTDRplus against the MGIT 960 was assessed. Sensitivity and specificity were 100% and 96% for detection of rifampicin resistance; 81%, and 100% for isoniazid resistance; and 92%, and 96%, for MDR-TB, respectively. The TTR was 1-3 days, and concordance of results between the Molecular Laboratory at Makerere University and the FIND Diagnostics Laboratory was 98%.
In paper V, we applied spoligotyping to study the clustering rate and predominant genotypic strains of 99 MDR-TB strains isolated from patients in Kampala. Eighty-three percent of the strains occurred in clusters, and the T2 lineage was the largest single cluster.
Conclusion. The direct NRA and the GT-DRplus appear to be the most appropriate tests for MDR-TB in RLSs. The NRA being the cheapest test can be applied where resources are extremely limited, while the ultra rapid but commercially available GT-DRplus can be used where resources permit.


List of papers from the dissertation

 

Direct susceptibility testing for multi drug resistant tuberculosis: a meta-analysis
Bwanga F, Hoffner S, Haile M, Joloba ML.
BMC Infect Dis, 2009; 9: 67

Evaluation of seven tests for the rapid detection of multidrug-resistant tuberculosis in Uganda.
Bwanga F, Joloba ML, Haile M, Hoffner S.
Int J Tuberc Lung Dis, 2010; 14: 890-895

Direct Nitrate Reductase Assay versus Microscopic Observation Drug Susceptibility for rapid detection of MDR-TB in Uganda.
Bwanga Freddie, Melles Haile, Sven Hoffner, Emmanuel Ochom, Moses L. Joloba.
Manuscript

Rapid screening of MDR-TB using molecular Line Probe Assay is feasible in Uganda.
Albert H, Bwanga F, Mukkada S, Nyesiga B, Ademun JP, Lukyamuzi G, Haile M, Hoffner S, Joloba M, OBrien R.
BMC Infect Dis, 2010; 10: 41

High clustering of MDR-TB strains in Kampala, Uganda: Predominance of the T2 lineage.
Bwanga Freddie, William George Muyombya, Sven Hoffner, Melles Haile, Benon Asiimwe, David Kateete, Fred Katabazi, Jennifer Asiimwe, Maria Wijkander, Moses L Joloba.
Manuscript

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GENETIC POLYMORPHISMS IN PLASMODIUM  FALCIPARUM ASSOCIATED WITH RESISTANCE IN ARTEMISININ COMBINATION THERAPY: COMPLEXITY OF INFECTION DURING SHORT-TERM CULTURE AND CHARACTERIZATION OF IN VITRO   SENSITIVITY IN KAMPALA UGANDA
ABSTRACT

BY
NSOBYA SAMUEL LUBWAMA, BLT, DipMLT, Msc.Mol.Bio.
2006/HD11/4547U
Supervisors:

  • Prof. Moses L. Joloba, Makerere University College of Healthy Sciences, Kampala, Uganda.
  • Prof. Rosenthal, University of California (SF), USA.

Background
Artemisinin-based combination therapy (ACT) is currently advocated in Africa as a means of improving treatment efficacy and slowing the development of drug resistance. However, the selection of resistant parasites, particularly to artemisinin partner drugs, remains a concern. In addition, evaluations of in vitro drug sensitivity of fresh clinical isolates have been limited for components of artemisinin-based combination therapies, and associations between in vitro measures, clinical outcomes, and genotypes are uncertain. We also know that P.falciparum infections are commonly polyclonal in many areas, and, since not all parasites successfully grow in culture, it is unclear how well in vitro culture represents the complexity of clinical infections.
Objectives      
In this work, the following objectives were addressed: i) assessed changes in occurrence of various polymorphisms of a drug resistant allele’s of pfmdr-1 and pfcrt between baseline and new infections during therapy with artesunate-amodiaquine (AS/AQ). ii) evaluate changes in complexity of infection(COI) during culture. iii) assess the impact of various amodiaquine containing regimens on the sensitivity of recurrent P. falciparum isolates. iv) determine in vitro  senstivity patterns of P. falciparum isolates in Uganda against various antimalarial drugs. v) evaluate associations between parasite genetic polymorphisms, in vitro  drug sensitivity, and clinical outcomes after antimalarial combination therapy.
Methods
The following methods were used to address the above objectives: i) polymorphisms of the pfmdr-1 and pfcrt genes known to play a role in altered drug sensitivity to some antimalarials were analysed by PCR and restriction fragment length polymorphism. We compared results between pretreatment isolates (n=201) and in new infections (n=132) in (AS/AQ) identified by genotyping over 28 days of follow-up from samples collected in a randomized trial for the treatment of uncomplicated malaria in Tororo, Uganda. We also evaluated pfcrt haplotypes at positions 72-76 in 90 randomly selected pretreatment and 90 randomly selected new infection ii) to study COI, we placed into culture 211 samples from children with uncomplicated malaria in Kampala.Uganda. Of these samples, 98(46%) were successfully cultured for at least 9 days. We determined COI daily based on msp-2 polymorphisms. In 25 samples initially mixed which lost some of strains, known gene polymorphisms of dhps, dhfr and pfmdr-1 were anlysed using restriction fragment polymorphism. iii) using P. falciparum isolates obtained from a clinical trial comparing efficacy of (AQ/SP andAQ/AS or AL), the impact of AQ-containing therapies on the sensitivity of 61 P. falciparum isolates causing recurrent infections soon after prior therapy and changes in resistance-mediating polymorphisms of pfmdr-1 gene were assessed. iv) for the in vitro  sensitivity studies, IC50s for 241 P. falciparum isolates obtained  between 2006 and 2008 were analysed in Kampala using an HRP-2-based ELISA.
Results
The findings for each objective above were as follows: i) in our study of the selection of pfmdr-1 polymorphisms, AS/AQ arm  selected for the mutant  alleles 86Y,(91%(182/201) to 97%(128/132) (p=0.03) and 1246Y,83%(167/201) to 91%(120/132)(p=0.05):86Yand1246Y, 81.6%(164/201) to 90.2%(119/132) (p=0.04). In contrast, the prevalence of the wild type allele Y184 increased from pretreatment to new infection isolates (171/201, 85.1% to 122/132, 92.4%, p=0.04). Only wild type alleles were seen at positions 1034 and 1042. For pfcrt, the CVIET haplotype at positions 72-76 was seen in all 180 samples analyzed ii) in studies of in vitro  cultures, COI decreased over 9 days of culture from 1.73 to 1.56. New strains appeared after day 0 in 20 (44%) out of 45 cultures. Strains disappeared after day 0 in 25(56%) of 45 cultures that were initially mixed; persisting strains more commonly had wild type dhfr(C59) and dhps(K540), and mutant pfmdr-1(86Y) sequences. iii) for the in vitro  susceptibility studies, parasites from subjects previously treated with AQ/SP or AQ/AS within 12 weeks were less sensitive to AQ(n=18; mean IC50 62.9 nM; range 12.7-158.3 nM) than parasites from those not treated within 12 weeks (n=43; mean IC50 37.5 nM; p=0.022; range 6.3-184.7 nM) or only those in the treatment arm that did not contain AQ (n=20; mean IC50 28.8 nM; p=0.017; range 6.3-121.8 nM).The proportion of strains with polymorphisms expected to mediate diminished response to AQ (pfmdr-1 86Y and 1246Y) increased after prior AQ therapy, although differences were not significant. iv) in vitro  sensitivities (geometric mean IC50 (nM), range) were assessed for: chloroquine(CQ)(101.1, 15.6-767,n=181); monodesethylamodiaquine(MDAQ)(66.4,6.5-312,n=206);quinine(QN)(94.4,15.4-761,n=196); lumefantrine(LUM)(0.51,0.19-29.4,n=200);piperaquine(PIP)(6.1,5-6.8,n=199);and dihydroartemisinin (DHA)(0.55, 0.13-4.8, n=212). Sensitivities were positively correlated between CQ, MDAQ, and QN (r=0.4-0.6; p<0.001 by Pearson’s correlation), but not for other comparisons. Sensitivities to CQ, MDAQ, and QN, but not to the other drugs, decreased over the course of the study. Considering common Pfmdr-1 polymorphisms, parasites highly sensitive to MDAQ, QN, and LM, but not PQ, were more likely to have wild type sequence at allele N86Y; there were no clear associations at Y184F or D1246Y

Conclusion
The following conclusion were made from this sudy: i) we report that although AS/AQ combination therapy is effective in clearing malaria parasites, in re-infections most of strains selected are associated with decreased response to AQ in Uganda. ii) for the first time in Uganda, we have developed a molecular research laboratory to study fresh clinical isolates of P. falciparum by performing culture, in vitro culture and molecular studies. iii) to the best our knowledge, this is the first assessment of complexity of infection during in vitro culture of freshly isolated malaria parasites. In drug efficacy trails, strains missed on day 0 and seen after treatment will be misclassified as new infections, thus understating levels of drug resistance. iv) loss of strains during culture of freshly isolated malaria parasites may be due to diminished fitness of some drug resistant strains. v) these results suggest diminishing efficacy of AQ-containing combination regimens as they are increasingly used in Uganda. vi) the generated baseline in vitro sensitivity data for the first time in Uganda will be used as yard stick to monitor emerging early resistance to ACTs.
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