In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

Background: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. Methods: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance. Findings: The suite of Plasmodium assays achieved analytical sensitivities ranging from 25-188 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. Interpretation: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance.

Recent identification and structural modeling of Treponema pallidum ’s ( Tp ) repertoire of outer membrane proteins (OMPs) represent a critical breakthrough in the decades long quest for a syphilis vaccine. However, little is known about the antigenic nature of these β-barrel-forming OMPs and, more specifically, their surface exposed regions, the extracellular loops (ECLs).

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Understanding temporal and spatial dynamics of ongoing malaria transmission will be critical to inform effective interventions and elimination strategies in low transmission regions approaching elimination. Parasite genomics are being used as a tool to monitor epidemiologic trends, including assessing residual transmission across seasons or importation of malaria into these regions. Southern Province, Zambia is a low-transmission setting with seasonal malaria. We genotyped 441 Plasmodium falciparum samples using molecular inversion probes at 1,832 positions across the genome, using dried blood spots collected from 2012-2018 from 8 health centers in the catchment area of Macha Hospital in Choma District. We show that highly related parasites persist across multiple seasons, suggesting that the persistence of malaria is at least in part fueled by parasites seeding across the dry season. In addition, we identify clusters of clonal parasites that are dissimilar to the general population, suggesting that introduction of parasites from elsewhere may contribute to the continued malaria burden. We identified signals of population size fluctuation over the course of individual transmission seasons, suggesting a ramp-up of malaria transmission from a seasons beginning. Despite the small spatial scale of the study (2,000 sq km), we identified an inverse relationship between genetic relatedness of parasite pairs and distance between health centers, as well as increased relatedness between specific health centers. These results, leveraging both genomic and epidemiological data, provide a comprehensive picture of fluctuations in parasite populations in this pre-elimination setting of southern Zambia.