Medical Microbiology

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Light forge : a microfluidic high throughput platform for rapid and affordable detection of drug resistant strains of tuberculosis.
(2015) Mbano, Ian Maheti.; Balagaddé, Frederick.
Tuberculosis is one of the most deadly infectious diseases currently plaguing the global community. Unfortunately, lack of accessible, reliable and affordable diagnostic tools in the high disease burden, and resource poor regions such as Sub-Saharan Africa has hampered efforts to eradicate the epidemic. This study documents the development of a microfluidic platform called Light Forge, which is capable of detecting genetic drug resistance signatures in M.tuberculosis DNA. The first phase of this study involved a molecular drug susceptibility assay on 7 strains of M.tuberculosis using the high resolution melt analysis at the rpoB, katG, mab-inhA and gyrA loci with the Light Cycler96 . These findings compared with phenotypic drug susceptibility testing and Sanger sequencing. The results from the preliminary tests showed that the commercial system could detect positive strains at sensitivity estimates of 86%, 17% , 0% and 100% for rpoB, katG, mab-inhA and gyrA respectively. Detection of non-synonymous mutation in gyrA region for all test strains halted further testing. The rpoB gene was selected for on chip profiling with the Light Forge system due to the higher sensitivity. The results from the Light Forge showed that the system was capable of detecting test strains with 100% sensitivity, with modest reproducibility and correspondence with the phenotypic drug susceptibility profiles and the sequencing results. A microfluidic TB assay based on the Light Forge system is on the horizon based on the findings of the study. However, more work is required to incorporate other genes and ultimately design the best-equipped device for the clinical setting.
Microchemostat technologies for characterization of efflux pumps associated with multidrug resistance in mycobacterium tuberculosis.
(2016) Mackenzie, Jared Stuart.; Balagaddé, Frederick.
Abstract available in PDF file.
Microfluidic technologies for capturing and concentrating human immunodeficiency virus-1 (HIV-1) particles.
(2016) McArthur, Chanelle Crystal.; Balagaddé, Frederick.
HIV-1 RNA assays are routinely used in developed countries to monitor the effectiveness of antiretroviral therapy (ART). These assays require well-trained operators, expensive equipment and reagents, and established laboratory infrastructure. These requirements limit their usefulness in resource-limited settings where people are most afflicted by the HIV-1 epidemic. Recent advances in microfluidics and nanotechnology offer new approaches for rapid, low-cost, robust and simple HIV-1 viral load monitoring systems. Here we describe an approach within a microfluidic device to directly detect HIV-1 virus particles using an immune sandwich assay that includes anti-gp120 antibodies -conjugated to polystyrene microspheres and fluorescently labelled goat anti-HIV-1 FITC detection antibodies. The anti-gp120 antibody-conjugated microspheres were employed to capture and concentrate HIV-1 particles, whereas the FITC detection antibodies were used to generate fluorescent signal that represented the number of captured viruses. In the presence of HIV-1 particles, addition of microspheres and FITC detection antibody led to the formation of a microsphere/HIV-1 particle/FITC detection antibody complex. This complex was measured by analysing the fluorescence intensity produced by the FITC detection antibody bound to the HIV-1 particle within the complex. We demonstrated the utility of an in-house microfluidic device and assay in detecting 1x106 virus particles/μl with a significance of (p≤0.01). This assay was completed within 3.8 hours, without any pre- or post- treatment of reagents.
Microfluidic technologies for genomic interrogation of mycobacterium tuberculosis clinical isolates using the polymerase chain reaction (PCR) and high resolution melting analysis (HRMA).
(2015) Mandizvo, Tawanda.; Balagaddé, Frederick.
Background: A number of Mycobacterium tuberculosis (Mtb) genes have been shown to be under positive selection pressure in the presence of anti-TB therapy. This results in the selection of drug resistant phenotypes associated with genetic changes—which can be point mutations, deletions and/or insertions. Some mutations from multiple genes have been documented to be associated with reduced susceptibility to anti-TB drugs such as rifampicin, ethambutol, carpreomycin and fluoroquinolones. The list is continuously updated as new mutations are discovered and validated. In principle therefore, there is an urgent need to design robust molecular diagnostics and more efficacious therapeutic strategies that are able to indicate diverse genetic mechanisms behind drug resistance in individual isolates Materials and Methods: We used the LightForge system we developed at K-RITH. This LightForge system is a fluorescence detection based, highly scalable microfluidic platform. It interrogates Mycobacterium tuberculosis strains using Real-Time PCR and High Resolution Melt Analysis (HRMA) on a chip. Results and Discussion: We have used this LightForge system to identify clinical Mtb strains resistant to rifampicin—a frontline drug used to treat tuberculosis, relative to a susceptible strain H37RV, based on mutations in the rpoB gene. This system has the potential to contribute towards a low-cost solution to diagnosis of multidrug resistant tuberculosis—a current critical global healthcare challenge. The interrogation of clinical Mtb isolates—including R35, KZN 605 and Tkk 01-062—using the LightForge system has detected mutations linked to rifampicin resistance including single nucleotide polymorphisms (SNPs) in a congruous manner with commercial systems. Conclusions: In preparation for diagnosis of clinical samples, this LightForge approach is now being expanded to incorporate detection of genetic markers linked with resistance to other TB drugs that include fluoroquinolones and isoniazid based on mutations in gyrA, katG and Mab-inhA regions of the Mtb genome. The scalability of LightForge can also be harnessed to conduct digital PCR (dPCR), a critical tool for detecting genetic heterogeneity in Mtb.
Technologies for a user-friendly microfluidic system for portable applications.
(2015) Kunota, Tafara Takunda Remigio.; Balagaddé, Frederick.
In the same way that the HIV virus subdues the human immune system, the HIV/AIDS epidemic has severely overloaded the health service infrastructure in resource limited countries and threatens to systematically suppress societies’ capacity to cope with killer diseases. The epidemic has also directly impacted the health workforce, causing absenteeism, attrition (due to illness and death), and increased demand for provider time and skills. Advanced and miniaturized microfluidic systems can perform complex biotechnological functions such as growing bacteria, sequencing DNA and identifying disease causing pathogens. As a technology, microfluidics offers so many advantages but it also suffers from a variety of technological drawbacks that limit its wide spread practical application in hospitals and patient setting. Microfluidic systems require a lot of time (6 hours to an entire work-day) to set up and the set-up process requires the meticulous attention of highly trained personnel. We proposed the development of an automated, time conservative and user-friendly fluid-transport system (off-chip to on-chip) for Microfluidic Large Scale Integration platform based microfluidic devices. Using multilayer soft-lithography, micro-electric actuators and a LabVIEW graphical user-interface, a user-friendly automated microfluidic fluid transport system was developed. In comparison to the conventional manual loading system, the developed system can save at least 60% of the total chip preparation time required during the off-chip to on-chip fluid loading process. This system can be extended and made compatible with other devices that require complex off-chip to on-chip loading processes in microfluidic large scale integration platform based systems.

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Browsing Medical Microbiology by Author "Balagaddé, Frederick."