Development of a Colorimetric Loop-Mediated Isothermal Amplification Assay for the Detection of Trypanosoma cruzi in Low-Resource Settings.

Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi (T. cruzi). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi. We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

Bacterial inactivation using silver-coated magnetic nanoparticles as functional antimicrobial agents.

The ability for silver nanoparticles to function as an antibacterial agent while being separable from the target fluids is important for bacterial inactivation in biological fluids. This report describes the analysis of the antimicrobial activities of silver-coated magnetic nanoparticles synthesized by wet chemical methods. The bacterial inactivation of several types of bacteria was analyzed, including Gram-positive bacteria ( Staphylococcus aureus and Bacillus cereus ) and Gram-negative bacteria ( Pseudomonas aeruginosa , Enterobacter cloacae , and Escherichia coli ). The results have demonstrated the viability of the silver-coated magnetic nanoparticles for achieving effective bacterial inactivation efficiency comparable to and better than that of silver nanoparticles conventionally used. The bacteria inactivation efficiency of our silver-coated MnZn ferrite (MZF@Ag) nanoparticles was also determined for blood platelets samples, demonstrating the potential of utilization in inactivating bacterial growth in platelets prior to transfusion to ensure blood product safety, which also has important implications for enabling the capability of effective separation, delivery, and targeting of the antibacterial agents.

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics.

The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC's ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC's performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings.