Targeted Next Generation Sequencing for malaria research in Africa: current status and outlook.

Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed.

Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes.

Cry toxins are primarily a family of insecticidal toxins produced by the bacterium Bacillus thuringiensis (Bt). However, some Cry toxins, called parasporins (PSs), are non-insecticidal and have been shown to differentially kill human cancer cells. Based on amino acid homology, there are currently six different classes of parasporins (PS1-6). It is not known what role parasporins play in nature, nor if certain PSs are associated with Bt found in particular environments. Herein, we present ten parasporin-containing isolates of Bt from the Caribbean island of Trinidad. Genes coding for PS1 and PS6 were found in isolates associated mainly with artificial aquatic environments (e.g., barrels with rain water), while Bt possessing two novel PS5-like genes (ps5-1 and ps5-2), were isolated from manure collected directly from the rectum of cattle. The amino acid sequences inferred from the two PS5-like genes were 51 % homologous to each other, while being only 41 or 45 % similar to PS5Aa1/Cry64Aa, the only reported member of the parasporin five class. The low level of amino acid homology between the two PS5-like genes and PS5Aa1 indicate that the two PS5-like genes may represent a new class of parasporins, or greatly expand the level of diversity within the current parasporin 5 class.

Matched Placental and Circulating Plasmodium falciparum Parasites are Genetically Homologous at the var2csa ID1-DBL2X Locus by Deep Sequencing.

In pregnancy-associated malaria, infected erythrocytes accumulate in the placenta. It is unclear if in polyclonal infections this results in distinct peripheral and placental parasite populations. We used long amplicon deep sequencing of Plasmodium falciparum var2csa ID1-DBL2X from 15 matched peripheral and placental samples collected at delivery from a high transmission area to determine genetic homology. Despite substantial sequence variation and detecting 23 haplotypes, the matched pairs mostly contained the same genetic variants, with 11 pairs sharing 100% of their variants, whereas others showed some heterogeneity. Thus, at delivery, peripheral and placental parasites appear to intermix and placental genotypes can be inferred through peripheral sampling.

Increased risk of low birth weight in women with placental malaria associated with P. falciparum VAR2CSA clade.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA's ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99 g; 95% Confidence Interval [CI]: -466.43 g,-69.55 g) and higher odds of low birthweight (<2500 g) (Odds Ratio [OR] 5.41; 95% CI:0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

Improvements in the hemodynamic stability of combat casualties during en route care.

Three Forward Aeromedical Evacuation platforms operate in Southern Afghanistan: UK Medical Emergency Response Team (MERT), US Air Force Expeditionary Rescue Squadron (PEDRO), and US Army Medical Evacuation Squadrons (DUSTOFF), each with a different clinical capability. Recent evidence suggests that retrieval by a platform with a greater clinical capability (MERT) is associated with improved mortality in critical patients when compared with platforms with less clinical capability (PEDRO and DUSTOFF). It is unclear whether this is due to en route resuscitation or the dispatch procedure. The aim of this study was to compare prehospital Shock Index (SI = heart rate / systolic blood pressure) with admission values as a measure of resuscitation, across these platforms. Patients were identified from the Department of Defense Trauma Registry, who were evacuated between June 2009 and June 2011 in Southern Afghanistan. Data on platform type, physiology, and injury severity was extracted. Overall, 865 patients were identified: 478 MERT, 291 PEDRO, and 96 DUSTOFF patients and groups were compared across three injury severity scoring (ISS) bins: 1 to 9, 10 to 25, and 26 or greater. An improvement in the admission SI was observed across all platforms in the lowest ISS bin. Within the middle bin, both the MERT and PEDRO groups saw improved SI on admission, but not the DUSTOFF group. This trend was continued only in the MERT group for the highest ISS bin (1.39 ± 0.62 vs. 1.09 ± 0.42; P = 0.001), whereas a deterioration was identified in the PEDRO group (0.88 ± 0.37 vs. 1.02 ± 0.43; P = 0.440). The use of a Forward Aeromedical Evacuation platform with a greater clinical capability is associated with an improved hemodynamic status in critical casualties. The ideal prehospital triage should endeavor to match patient need with clinical capability.