Genome sequence analyses identify novel risk loci for multiple system atrophy.

Multiple system atrophy (MSA) is an adult-onset, sporadic synucleinopathy characterized by parkinsonism, cerebellar ataxia, and dysautonomia. The genetic architecture of MSA is poorly understood, and treatments are limited to supportive measures. Here, we performed a comprehensive analysis of whole genome sequence data from 888 European-ancestry MSA cases and 7,128 controls to systematically investigate the genetic underpinnings of this understudied neurodegenerative disease. We identified four significantly associated risk loci using a genome-wide association study approach. Transcriptome-wide association analyses prioritized USP38-DT, KCTD7, and lnc-KCTD7-2 as novel susceptibility genes for MSA within these loci, and single-nucleus RNA sequence analysis found that the associated variants acted as cis-expression quantitative trait loci for multiple genes across neuronal and glial cell types. In conclusion, this study highlights the role of genetic determinants in the pathogenesis of MSA, and the publicly available data from this study represent a valuable resource for investigating synucleinopathies.

Keratoconus characteristics and associations: A cross-sectional keratoconus study in western India (CKSWI).

PURPOSE: To study the clinical and tomographic characteristics and associations of keratoconus (KC) patients visiting a tertiary eye care hospital. METHODS: This was a cross-sectional, observational study that included 242 newly diagnosed clinical KC patients. Detailed ocular and systemic history, visual complaints, habit of eye rubbing, best spectacle-corrected visual acuity (BSCVA), retinoscopy reflex, detailed slit-lamp examination and tomographic findings, and presence of any ocular and systemic associations were documented for the included patients. Severity of KC was further graded into stages 0, 1, 2, 3, and 4 according to the ABCD grading system. RESULTS: The most common visual complaint was blurred vision (82.64%), followed by itching (48.76%), tearing (43.80%), and photophobia (41.32%). A total of 44.62% of patients had the habit of eye rubbing and 4.54% had a family history of KC. Most common clinical signs were scissor reflex (94.21%), Rizutti's sign (77.27%), corneal protrusion (69.83%), and Fleischer ring (67.35%). A total of 14.05% of patients had stage 0, 42.15% had stage 1, 19.83% had stage 2, 18.60% had stage 3, and 5.37% had stage 4 KC. Of these patients, 70.25% had a BSCVA visual acuity of ≥6/12 or better; 26.45% had a BSCVA of ≥6/60 to <6/12; and 3.30% had a BSCVA of <6/60. Ocular allergies, atopy, and asthma were found in 32.32%, 4.96%, and 2.48% cases, respectively. CONCLUSION: This study gives an overview of the clinical findings of KC cases in western India. Our results suggest that use of retinoscopy should be promoted in early KC detection in primary eyecare screening programs.

Differential methylation analysis in neuropathologically confirmed dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is a common form of dementia in the elderly population. We performed genome-wide DNA methylation mapping of cerebellar tissue from pathologically confirmed DLB cases and controls to study the epigenetic profile of this understudied disease. After quality control filtering, 728,197 CpG-sites in 278 cases and 172 controls were available for the analysis. We undertook an epigenome-wide association study, which found a differential methylation signature in DLB cases. Our analysis identified seven differentially methylated probes and three regions associated with DLB. The most significant CpGs were located in ARSB (cg16086807), LINC00173 (cg18800161), and MGRN1 (cg16250093). Functional enrichment evaluations found widespread epigenetic dysregulation in genes associated with neuron-to-neuron synapse, postsynaptic specialization, postsynaptic density, and CTCF-mediated synaptic plasticity. In conclusion, our study highlights the potential importance of epigenetic alterations in the pathogenesis of DLB and provides insights into the modified genes, regions and pathways that may guide therapeutic developments.

A technological approach to "Reaching the Unreached" - Leveraging teleophthalmology services in Rural Gujarat.

Purpose: Early detection of sight-threatening disorders by technological applications like teleophthalmology and prompt treatment can help decrease visual impairment. This study evaluated the role of teleophthalmology in underserved rural areas along with cost-saving estimates for the end user. Methods: A prospective, observational, cross-sectional hospital-based study was conducted over 3 months. First 1000 teleconsultations were included. None of the patients denied providing informed consent. The patients were consulted at the eight vision centers and three satellite centers of the hospital in the nearby rural and tribal regions closer to their residential places. These vision and satellite centers were connected to the base hospital like a hub and spoke model with a teleophthalmology network. Results: Cataract (n = 301, 30.1%) and refractive error (n = 290, 29%) were the most common diagnosis. 42.1% of patients were referred to base hospital for further evaluation. Thus, a total of 57.9% of patients were not required to visit the base hospital for initial consultation, saving time and money. Furthermore, 15.1% of patients were provided medical treatment at the vision center and satellite center, which helped in making teleophthalmology cost-saving for the patients. An average of Rs. 621/- were saved per patient for the community in our study. Conclusion: Networked teleophthalmology model can be an affordable and feasible tool for providing eye care delivery services in rural and tribal regions of Gujarat and the whole country, especially for the end user. Thus, it may be a workable model in ophthalmology practice with substantial cost saving to the community.

HLA in isolated REM sleep behavior disorder and Lewy body dementia.

Synucleinopathies-related disorders such as Lewy body dementia (LBD) and isolated/idiopathic REM sleep behavior disorder (iRBD) have been associated with neuroinflammation. In this study, we examined whether the human leukocyte antigen (HLA) locus plays a role in iRBD and LBD. In iRBD, HLA-DRB1*11:01 was the only allele passing FDR correction (OR = 1.57, 95% CI = 1.27-1.93, p = 2.70e-05). We also discovered associations between iRBD and HLA-DRB1 70D (OR = 1.26, 95%CI = 1.12-1.41, p = 8.76e-05), 70Q (OR = 0.81, 95%CI = 0.72-0.91, p = 3.65e-04) and 71R (OR = 1.21, 95%CI = 1.08-1.35, p = 1.35e-03). Position 71 (pomnibus = 0.00102) and 70 (pomnibus = 0.00125) were associated with iRBD. Our results suggest that the HLA locus may have different roles across synucleinopathies.

HLA in isolated REM sleep behavior disorder and Lewy body dementia.

Background and Objectives: Isolated/idiopathic REM sleep behavior disorder (iRBD) and Lewy body dementia (LBD) are synucleinopathies that have partial genetic overlap with Parkinson's disease (PD). Previous studies have shown that neuroinflammation plays a substantial role in these disorders. In PD, specific residues of the human leukocyte antigen ( HLA ) were suggested to be associated with a protective effect. This study examined whether the HLA locus plays a similar role in iRBD, LBD and PD. Methods: We performed HLA imputation on iRBD genotyping data (1,072 patients and 9,505 controls) and LBD whole-genome sequencing (2,604 patients and 4,032 controls) using the multi-ethnic HLA reference panel v2 from the Michigan Imputation Server. Using logistic regression, we tested the association of HLA alleles, amino acids and haplotypes with disease susceptibility. We included age, sex and the top 10 principal components as covariates. We also performed an omnibus test to examine which HLA residue positions explain the most variance. Results: In iRBD, HLA-DRB1 *11:01 was the only allele passing FDR correction (OR=1.57, 95% CI=1.27-1.93, p =2.70e-05). We also discovered associations between iRBD and HLA-DRB1 70D (OR=1.26, 95%CI=1.12-1.41, p =8.76e-05), 70Q (OR=0.81, 95% CI=0.72-0.91, p =3.65e-04) and 71R (OR=1.21, 95% CI=1.08-1.35, p =1.35e-03). In HLA-DRB1 , position 71 ( p omnibus =0.00102) and 70 ( p omnibus =0.00125) were associated with iRBD. We found no association in LBD. Discussion: This study identified an association between HLA-DRB1 11:01 and iRBD, distinct from the previously reported association in PD. Therefore, the HLA locus may play different roles across synucleinopathies. Additional studies are required better to understand HLA's role in iRBD and LBD.

Screening Vaccinees for Non-Communicable Diseases and Risk Factors at a Covid-19 Vaccination Center in Vadodara, Gujarat.

Context: Adult population visiting COVID vaccination center is a potential teachable moment for screening and preventive advice on non-communicable diseases. Objectives: The objective of this study was to assess the proportion of vaccinees volunteering for screening and to know the proportion of newly detected hypertensives and diabetics among the screened vaccinees at COVID-19 vaccination center. Setting and Design: This descriptive, cross-sectional, operational research study was carried out at the COVID vaccination center at a medical college in central Gujarat from July to September 2021. Methods: After receiving the vaccine, the vaccinee was offered screening through a community-based assessment checklist for risk factors of non-communicable diseases, blood pressure, and blood sugar measurement. Those volunteering for this screening received a slip mentioning their risk score, blood pressure and blood sugar reading, and relevant health information and disease prevention advice. The study variables were acceptability (proportion of vaccinees volunteering for screening) and yield (newly detected hypertensives and diabetics among those screened). Results: Among vaccinees, 27.7% volunteered for risk scoring and blood pressure measurement, whereas 8.3% volunteered for blood sugar measurement. Around 15.5% of vaccinees had high-risk scores as per the community-based assessment checklist. The yield of freshly detected high blood pressure and high blood sugar was 19.3% and 10.5%, respectively. The yield was similar even among vaccinees under 30 years of age. Conclusions: Vaccinees demonstrated interest in undergoing screening for non-communicable diseases. Yield indicates that such screening is worth the effort.

GRN Mutations Are Associated with Lewy Body Dementia.

BACKGROUND: Loss-of-function mutations in GRN are a cause of familial frontotemporal dementia, and common variants within the gene have been associated with an increased risk of developing Alzheimer's disease and Parkinson's disease. Although TDP-43-positive inclusions are characteristic of GRN-related neurodegeneration, Lewy body copathology has also been observed in many GRN mutation carriers. OBJECTIVE: The objective of this study was to assess a Lewy body dementia (LBD) case-control cohort for pathogenic variants in GRN and to test whether there is an enrichment of damaging mutations among patients with LBD. METHODS: We analyzed whole-genome sequencing data generated for 2591 European-ancestry LBD cases and 4032 neurologically healthy control subjects to identify disease-causing mutations in GRN. RESULTS: We identified six heterozygous exonic GRN mutations in seven study participants (cases: n = 6; control subjects: n = 1). Each variant was predicted to be pathogenic or likely pathogenic. We found significant enrichment of GRN loss-of-function mutations in patients with LBD compared with control subjects (Optimized Sequence Kernel Association Test P = 0.0162). Immunohistochemistry in three definite LBD cases demonstrated Lewy body pathology and TDP-43-positive neuronal inclusions. CONCLUSIONS: Our findings suggest that deleterious GRN mutations are a rare cause of familial LBD. © 2022 International Parkinson Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Genetic evaluation of dementia with Lewy bodies implicates distinct disease subgroups.

The APOE locus is strongly associated with risk for developing Alzheimer's disease and dementia with Lewy bodies. In particular, the role of the APOE ε4 allele as a putative driver of α-synuclein pathology is a topic of intense debate. Here, we performed a comprehensive evaluation in 2466 dementia with Lewy bodies cases versus 2928 neurologically healthy, aged controls. Using an APOE-stratified genome-wide association study approach, we found that GBA is associated with risk for dementia with Lewy bodies in patients without APOE ε4 (P = 6.58 × 10-9, OR = 3.41, 95% CI = 2.25-5.17), but not with dementia with Lewy bodies with APOE ε4 (P = 0.034, OR = 1.87, 95%, 95% CI = 1.05-3.37). We then divided 495 neuropathologically examined dementia with Lewy bodies cases into three groups based on the extent of concomitant Alzheimer's disease co-pathology: pure dementia with Lewy bodies (n = 88), dementia with Lewy bodies with intermediate Alzheimer's disease co-pathology (n = 66) and dementia with Lewy bodies with high Alzheimer's disease co-pathology (n = 341). In each group, we tested the association of the APOE ε4 against the 2928 neurologically healthy controls. Our examination found that APOE ε4 was associated with dementia with Lewy bodies + Alzheimer's disease (P = 1.29 × 10-32, OR = 4.25, 95% CI = 3.35-5.39) and dementia with Lewy bodies + intermediate Alzheimer's disease (P = 0.0011, OR = 2.31, 95% CI = 1.40-3.83), but not with pure dementia with Lewy bodies (P = 0.31, OR = 0.75, 95% CI = 0.43-1.30). In conclusion, although deep clinical data were not available for these samples, our findings do not support the notion that APOE ε4 is an independent driver of α-synuclein pathology in pure dementia with Lewy bodies, but rather implicate GBA as the main risk gene for the pure dementia with Lewy bodies subgroup.

Prevalence of Corneal Topographic Patterns in Ectatic Corneal Diseases.

SIGNIFICANCE: The article highlights various topographic patterns and their prevalence in a large spectrum of ectatic corneal diseases (ECDs). Knowledge of these patterns can help clinicians for quicker diagnosis and selection of appropriate contact lens design. PURPOSE: This study aimed to determine various corneal topography patterns and their prevalence in patients with ECDs who visited a tertiary eye hospital in western India. METHODS: Keratoconus, pellucid marginal degeneration, keratoglobus, and post-refractive surgery progressive corneal ectasia are considered under ECDs. This cross-sectional retrospective study reviewed records of 632 consecutive patients with clinical ECDs at their first presentation. The right eye was considered for pattern analysis. In cases with suspected or forme fruste ectasia in the right eye, the fellow eye was considered. A sagittal map with standard scale of Atlas 9000 topographer (Carl Zeiss Meditec AG, Jena, Germany) was used for pattern analysis. They were classified into 18 categories and grouped under five groups. The prevalence of these patterns was calculated and assessed with 95% confidence interval (CI). RESULTS: The mean ± SD age of patients was 23.6 ± 8.2 years. The highest prevalence was of asymmetric patterns (39.6% [95% CI, 35.7 to 43.5%]; asymmetric bowtie [AB] with steepest radial axis index [SRAX], 18.8%; AB with inferior steep, 16.0%; AB with superior steep [SS], 3.2%; symmetric bowtie with SRAX, 1.6%) and of central or paracentral patterns (28.6% [95% CI, 25.1 to 32.3%]; inferior steep, 12.2%; heart, 7.4%; oval, 4.1%; symmetric bowtie, 2.4%; round, 1.6%; irregular, 0.9%) followed by advanced patterns (17.3% [95% CI, 14.4 to 20.4%; nonmeasurable, 5.4%; globus, 4.9%; indiscriminate, 7.0%). The peripheral patterns were 11.7% (95% CI, 9.3 to 14.4%) (claw, 6.3%; junctional, 3.2%; butterfly, 1.9%; SS, 0.3%). Rare patterns were 2.8% (95% CI, 1.7 to 4.5%) (superior [junctional, claw, and heart], AB with SS with SRAX, and AB with SRAX located temporally). CONCLUSIONS: Asymmetric and central or paracentral are the most common patterns in our study. The higher prevalence of advanced patterns indicates the need for earlier diagnosis of ECDs in our population. The peripheral patterns also have significant prevalence.

STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families.

BACKGROUND: RIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods. RESULTS: STRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis. CONCLUSIONS: STRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

Assessment and Correlation of Corneal Endothelial Cell Changes in Different Stages of Keratoconus in Non-contact Lens Wearers.

SIGNIFICANCE: The endothelial changes in keratoconus are of interest because these can affect the surgical plan in management. Previously, many studies have evaluated the endothelium in eyes with keratoconus, but there is no common consensus on change in endothelial cell density (ECD) with studies showing variable results. PURPOSE: This study aimed to compare and correlate endothelial cell parameters in different stages of keratoconus using specular microscope. METHODS: This cross-sectional, cohort, comparative study included 162 eyes of 96 patients with keratoconus in one or both eyes. Corneal endothelial cell parameters were assessed with SP-1P specular microscope (Topcon Co., Tokyo, Japan). Corneal topography and thickness data were obtained from Sirius tomographer (Costruzione Strumenti Oftalmici, Florence, Italy). Eyes were divided into keratoconus stage 0 to 4 according to ABCD classification. Comparison and correlation of endothelial cell parameters in different stages were done. RESULTS: There were 21.6% eyes (35) in stage 0, 29.6% eyes (48) in stage 1, 29.0% eyes (47) in stage 2, and 19.8% eyes (32) in stage 3 of keratoconus. Measurement was not possible in stage 4 keratoconus. Among the stages 0, 1, 2, and 3, the ECD was 3024, 3051, 3025, and 3043 cells/mm2; coefficient of variation was 27.2, 27.6, 26.8, and 27.4; and frequency of hexagon cells was 61.5, 63.7, 63.3, and 62.3, respectively (P > .05). The number of analyzed cells was 232, 209, 185, and 169 in stages 0, 1, 2, and 3, respectively (P < .001). No significant difference was found in minimum, maximum, and average cell area between the stages (P > .05). A weak Spearman rank correlation of ECD, coefficient of variation, and frequency of hexagon cells was found between eyes with keratoconus stages 0 and 1 and stages 2 and 3 (r = -0.05, P = .65; r = -0.11, P = .37; r = 0.05, P = .67, respectively). No significant correlation was found in the number of cells analyzed and minimum, maximum, and average cell area between the stages (P > .05). CONCLUSIONS: Endothelial cell parameters do not show any significant changes and correlation up to stage 3 of keratoconus in non-contact lens wearers.

Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies possible targets of allele-specific immunity to clinical malaria.

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. Immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode possible targets of naturally acquired immunity that should be validated immunologically and further characterized for their potential as vaccine candidates.

Genome sequencing analysis identifies new loci associated with Lewy body dementia and provides insights into its genetic architecture.

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia, and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer's disease and Parkinson's disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.

Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia.

BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Comparison of Percent Tissue altered in Topography Guided and Wavefront Optimized Laser-assisted in situ Keratomileusis using Zeiss MEL 80 Excimer Laser.

INTRODUCTION: Laser-assisted in situ Keratomileusis (LASIK) is the most commonly performed refractive surgical procedure. The amount of tissue ablated in LASIK affects the safety and long-term outcome. The objective of this study was to compare the percent tissue altered (PTA) in topography guided (TG) and wavefront optimized (WFO) LASIK using Zeiss MEL 80 excimer laser. MATERIALS AND METHODS: This retrospective observational study was conducted at a tertiary eye center. Patients with moderate myopia who underwent LASIK between June 2016 and January 2019 were divided into two groups (Group I: TG LASIK, 69 eyes; Group II: WFO LASIK, 70 eyes). The groups were compared for preoperative parameters [spherical equivalent (SE), keratometry and pachymetry], intraoperative parameters [ablation depth (AD), PTA and residual stromal bed thickness (RSBT)] and postoperative parameters (vision, SE). RESULTS: Among preoperative parameters, SE and keratometry were similar while thinnest pachymetry was significantly less in group I. Among the intraoperative parameters, PTA (P < 0.01) and AD (P < 0.01) were significantly less in group I while RSBT (P = 0.54) was not significantly different. Postoperatively at 6 months, 92.75% (64) eyes in group I and 90% (63) eyes in group II had visual acuity of 6/6 or better (P = 0.57). 98.55% (68) and 97.14% (68) eyes in group I and group II respectively had SE refraction within ± 0.5 dioptres. CONCLUSION: TG LASIK induces less tissue alteration for given refractive error with similar visual outcome as compared to WFO LASIK which makes TG apparently safer and is the preferred technique for borderline thin corneas.

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples.

BACKGROUND: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. METHODS: DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. RESULTS: MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI + sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. CONCLUSION: This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

BACKGROUND: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions. METHODS: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia. RESULTS: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8+ T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region. CONCLUSIONS: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.