Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Early syphilis in Guangzhou, China: presentation, molecular detection of Treponema pallidum , and genomic sequences in clinical specimens and isolates obtained by rabbit infectivity testing.

Background: The global resurgence of syphilis requires novel prevention strategies. Whole genome sequencing (WGS) of Treponema pallidum ( TPA ) using different specimen types is essential for vaccine development. Methods: Patients with primary (PS) and secondary (SS) syphilis were recruited in Guangzhou, China. We collected ulcer exudates and blood from PS participants, and skin biopsies and blood from SS participants for TPA polA polymerase chain reaction (PCR); ulcer exudates and blood were also used to isolate TPA strains by rabbit infectivity testing (RIT). TPA WGS was performed on 52 ulcer exudates and biopsy specimens and 25 matched rabbit isolates. Results: We enrolled 18 PS and 51 SS participants from December 2019 to March 2022. Among PS participants, TPA DNA was detected in 16 (89%) ulcer exudates and three (17%) blood specimens. Among SS participants, TPA DNA was detected in 50 (98%) skin biopsies and 27 (53%) blood specimens. TP A was isolated from 48 rabbits, with a 71% (12/17) success rate from ulcer exudates and 69% (36/52) from SS bloods. Twenty-three matched SS14 clade genomes were virtually identical, while two Nichols clade pairs had discordant tprK sequences. Forty-two of 52 unique TPA genomes clustered in an SS14 East Asia subgroup, while ten fell into two East Asian Nichols subgroups. Conclusions: Our TPA detection rate was high from PS ulcer exudates and SS skin biopsies and over 50% from SS whole blood, with RIT isolation in over two-thirds of samples. Our results support the use of WGS from rabbit isolates to inform vaccine development. Summary: We performed Treponema pallidum molecular detection and genome sequencing from multiple specimens collected from early syphilis patients and isolates obtained by rabbit inoculation. Our results support the use of whole genome sequencing from rabbit isolates to inform syphilis vaccine development.

Clinical and genomic diversity of Treponema pallidum subsp. pallidum: A global, multi-center study of early syphilis to inform vaccine research.

Background: The continuing increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We conducted a multi-center, observational study to explore Treponema pallidum subsp. pallidum ( TPA ) molecular epidemiology essential for vaccine research by analyzing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. Methods: We enrolled patients with primary (PS), secondary (SS) or early latent (ELS) syphilis from clinics in China, Colombia, Malawi and the United States between November 2019 - May 2022. Inclusion criteria included age ≥18 years, and syphilis confirmation by direct detection methods and/or serological testing. TPA detection and WGS were conducted on lesion swabs, skin biopsies/scrapings, whole blood, and/or rabbit-passaged isolates. We compared our WGS data to publicly available genomes, and analysed TPA populations to identify mutations associated with lineage and geography. Findings: We screened 2,820 patients and enrolled 233 participants - 77 (33%) with PS, 154 (66%) with SS, and two (1%) with ELS. Median age of participants was 28; 66% were cis -gender male, of which 43% reported identifying as "gay", "bisexual", or "other sexuality". Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants demonstrated a predominance of SS14-lineage strains with geographic clustering. Phylogenomic analysis confirmed that Nichols-lineage strains are more genetically diverse than SS14-lineage strains and cluster into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models demonstrated population-specific substitutions, some in outer membrane proteins (OMPs) of interest. Interpretation: Our study involving participants from four countries substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains will be vital for vaccine development and improved understanding of syphilis pathogenesis on a population level. Funding: National Institutes of Health, Bill and Melinda Gates Foundation.

Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia.

Diagnosis and treatment of Plasmodium falciparum infections are required for effective malaria control and are pre-requisites for malaria elimination efforts; hence we need to monitor emergence, evolution and spread of drug- and diagnostics-resistant parasites. We deep sequenced key drug-resistance mutations and 1,832 SNPs in the parasite genomes of 609 malaria cases collected during a diagnostic-resistance surveillance study in Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused by P. falciparum carrying the candidate artemisinin partial-resistance kelch13 (K13) 622I mutation, which was less common in diagnostic-resistant parasites mediated by histidine-rich proteins 2 and 3 (pfhrp2/3) deletions than in wild-type parasites (P = 0.03). Identity-by-descent analyses showed that K13 622I parasites were significantly more related to each other than to wild type (P < 0.001), consistent with recent expansion and spread of this mutation. Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Of concern, 8.2% of K13 622I parasites also carried the pfhrp2/3 deletions. Close monitoring of the spread of combined drug- and diagnostic-resistant parasites is needed.

High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny.

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 ß-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.

Extracellular Loops of the Treponema pallidum FadL Orthologs TP0856 and TP0858 Elicit IgG Antibodies and IgG+-Specific B-Cells in the Rabbit Model of Experimental Syphilis.

The resurgence of syphilis in the new millennium has called attention to the importance of a vaccine for global containment strategies. Studies with immune rabbit serum (IRS) indicate that a syphilis vaccine should elicit antibodies (Abs) that promote opsonophagocytosis of treponemes by activated macrophages. The availability of three-dimensional models for Treponema pallidum's (Tp) repertoire of outer membrane proteins (OMPs) provides an architectural framework for identification of candidate vaccinogens with extracellular loops (ECLs) as the targets for protective Abs. Herein, we used Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to display Tp OMP ECLs to interrogate sera and peripheral blood mononuclear cells (PBMCs) from immune rabbits for ECL-specific Abs and B cells. We validated this approach using a PfTrx scaffold presenting ECL4 from BamA, a known opsonic target. Using scaffolds displaying ECLs of the FadL orthologs TP0856 and TP0858, we determined that ECL2 and ECL4 of both proteins are strongly antigenic. Comparison of ELISA and immunoblot results suggested that the PfTrx scaffolds present conformational and linear epitopes. We then used the FadL ECL2 and ECL4 PfTrx constructs as "hooks" to confirm the presence of ECL-specific B cells in PBMCs from immune rabbits. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for circumventing bottlenecks in vaccine development associated with large-scale production of folded OMPs. They also lay the groundwork for production of rabbit monoclonal Abs (MAbs) to characterize potentially protective ECL epitopes at the atomic level. IMPORTANCE 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 ß-barrel-forming OMPs and, more specifically, their surface exposed regions, the extracellular loops (ECLs). In this study, using Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to display Tp OMP ECLs, we interrogated immune rabbit sera and peripheral blood mononuclear cells for the presence of antibodies (Abs) and circulating rare antigen-specific B cells. Our results pinpoint immunogenic ECLs of two newly discovered OMPs, while advancing the utility of the rabbit model for surveying the entire Tp OMPeome for promising OMP vaccinogens. This work represents a major advancement toward characterizing potentially protective OMP ECLs and future vaccine studies. Additionally, this strategy could be applied to OMPs of nonspirochetal bacterial pathogens.

A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping.

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 2•5-18•8 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. FUNDING: National Institutes of Health (T32GM007092, R21AI148579, K24AI134990, R01AI121558, UL1TR002489, P30CA016086).