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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