HRP2 and HRP3 cross-reactivity and implications for HRP2-based RDT use in regions with Plasmodium falciparum hrp2 gene deletions.

BACKGROUND: The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs. METHODS: Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2-/hrp3+), HB3 (hrp2+/hrp3-) and 3BD5 (hrp2-/hrp3-)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH. RESULTS: At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density. CONCLUSIONS: Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.

Shear-Thinning and Thermo-Reversible Nanoengineered Inks for 3D Bioprinting.

Three-dimensional (3D) printing is an emerging approach for rapid fabrication of complex tissue structures using cell-loaded bioinks. However, 3D bioprinting has hit a bottleneck in progress because of the lack of suitable bioinks that are printable, have high shape fidelity, and are mechanically resilient. In this study, we introduce a new family of nanoengineered bioinks consisting of kappa-carrageenan (κCA) and two-dimensional (2D) nanosilicates (nSi). κCA is a biocompatible, linear, sulfated polysaccharide derived from red algae and can undergo thermo-reversible and ionic gelation. The shear-thinning characteristics of κCA were tailored by nanosilicates to develop a printable bioink. By tuning κCA-nanosilicate ratios, the thermo-reversible gelation of the bioink can be controlled to obtain high printability and shape retention characteristics. The unique aspect of the nanoengineered κCA-nSi bioink is its ability to print physiologically-relevant-scale tissue constructs without requiring secondary supports. We envision that nanoengineered κCA-nanosilicate bioinks can be used to 3D print complex, large-scale, cell-laden tissue constructs with high structural fidelity and tunable mechanical stiffness for regenerative medicine.

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.

Low-Cost 3D Printers Enable High-Quality and Automated Sample Preparation and Molecular Detection.

Most molecular diagnostic assays require upfront sample preparation steps to isolate the target's nucleic acids, followed by its amplification and detection using various nucleic acid amplification techniques. Because molecular diagnostic methods are generally rather difficult to perform manually without highly trained users, automated and integrated systems are highly desirable but too costly for use at point-of-care or low-resource settings. Here, we showcase the development of a low-cost and rapid nucleic acid isolation and amplification platform by modifying entry-level 3D printers that cost between $400 and $750. Our modifications consisted of replacing the extruder with a tip-comb attachment that houses magnets to conduct magnetic particle-based nucleic acid extraction. We then programmed the 3D printer to conduct motions that can perform high-quality extraction protocols. Up to 12 samples can be processed simultaneously in under 13 minutes and the efficiency of nucleic acid isolation matches well against gold-standard spin-column-based extraction technology. Additionally, we used the 3D printer's heated bed to supply heat to perform water bath-based polymerase chain reactions (PCRs). Using another attachment to hold PCR tubes, the 3D printer was programmed to automate the process of shuttling PCR tubes between water baths. By eliminating the temperature ramping needed in most commercial thermal cyclers, the run time of a 35-cycle PCR protocol was shortened by 33%. This article demonstrates that for applications in resource-limited settings, expensive nucleic acid extraction devices and thermal cyclers that are used in many central laboratories can be potentially replaced by a device modified from inexpensive entry-level 3D printers.

Nuclear, biological, and chemical combined injuries and countermeasures on the battlefield.

The Armed Forces Radiobiological Research Institute (AFRRI) has developed a research program to determine the major health risks from exposure to ionizing radiation in combination with biological and chemical warfare agents and to assess the extent to which exposure to ionizing radiation compromises the effectiveness of protective drugs, vaccines, and other biological and chemical warfare prophylactic and treatment strategies. AFRRI's Defense Technology Objective MD22 supports the development of treatment modalities and studies to assess the mortality rates for combined injuries from exposure to ionizing radiation and Bacillus anthracis, and research to provide data for casualty prediction models that assess the health consequences of combined exposures. In conjunction with the Defense Threat Reduction Agency, our research data are contributing to the development of casualty prediction models that estimate mortality and incapacitation in an environment of radiation exposure plus other weapons of mass destruction. Specifically, the AFFRI research program assesses the effects of ionizing radiation exposure in combination with B. anthracis, Venezuelan equine encephalomyelitis virus, Shigella sonnei, nerve agents, and mustard as well as their associated treatments and vaccines. In addition, the long-term psychological effects of radiation combined with nuclear, biological, and chemical (NBC) injuries are being evaluated. We are also assessing the effectiveness of gamma photons and high-speed neutrons and electrons for neutralizing biological and chemical warfare agents. New protocols based on our NBC bioeffects experiments will enable U.S. armed forces to accomplish military operations in NBC environments while optimizing both survival and military performance. Preserving combatants' health in an NBC environment will improve warfighting operations and mission capabilities.