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