Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

Helicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.

Evolution of Beak and Feather Disease Virus across Three Decades of Conservation Intervention for Population Recovery of the Mauritius Parakeet

Emerging infectious diseases (EIDs) are key contributors to the current global biodiversity crisis. Psittaciformes (parrots) are one of the most vulnerable avian taxa and psittacine beak and feather disease (PBFD) is the most common viral disease in wild parrots. PBFD is caused by the beak and feather disease virus (BFDV), which belongs to the Circoviridae family and comprises a circular, single-stranded DNA genome. BFDV is considered to have spread rapidly across the world and, in 2005, an outbreak of PBFD was documented in the recovering population of the Mauritius parakeet (Alexandrinus eques). The Mauritius parakeet was once the world’s rarest parrot and has been successfully recovered through 30 years of intensive conservation management. Molecular surveillance for the prevalence of BFDV was carried out across a 24-year sample archive spanning the period from 1993 to 2017, and DNA sequencing of positive individuals provided an opportunity to assess patterns of phylogenetic and haplotype diversity. Phylogenetic analyses show variation in the extent of viral diversification within the replicase protein (Rep). Timeseries of BFDV prevalence and number of haplotypes reveal that two subsequent waves of infection occurred in 2010/2011 and 2013/2014 following the initial outbreak in 2005. Continued disease surveillance to determine the frequency and intensity of subsequent waves of infection may benefit future translocation/reintroduction planning. The continued growth of the Mauritius parakeet population despite the presence of BFDV bodes well for its long-term persistence.

Malaria molecular surveillance in the Peruvian Amazon with novel highly multiplexed Plasmodium falciparum Ampliseq assay

Background: Malaria molecular surveillance has great potential to support local national malaria control programs (NMCPs) to inform policy for malaria control and elimination. Molecular markers associated with drug resistance are good predictors of treatment responses. In addition, molecular detection of deletions in hrp2 and hrp3 genes are indicative of potential failure of HRP2-based rapid diagnostic tests. However, there is an urgent need for feasible, cost-effective and fast molecular surveillance tools that NMCPs can implement. Methods: Here we present a new 3-day workflow for targeted resequencing of markers in 13 resistance-associated genes, hrp2&3, a country-specific 28 SNP-barcode for population genetic analysis, and ama1. The assay was applied to control isolates and retrospective samples collected between 2003-2018 in the Loreto region (n = 254) in Peru. Pf AmpliSeq libraries were prepared using a multiplex PCR simultaneously amplifying a high number of targets from dried blood spots and sequenced at high coverage (median 1336, range 20-43795). Results: There was no evidence suggesting the emergence of artemisinin resistance in Peru. However, alleles in ubp1 and coronin contributed to recent genetic differentiation of the parasite population. After 2008, predominant parasite lineages in Peru are resistant to sulfadoxine-pyrimethamine (sextuple dhfr/dhps mutant) and chloroquine (SVMNT in crt and NDFCDY in mdr1) and can escape HRP2 based RDTs. Conclusions: These findings indicate a parasite population under drug pressure, and demonstrates the added value of molecular surveillance systems and offers a highly multiplexed surveillance tool. The targets in the assay can be easily adjusted to suit the needs of other settings. Funding: This work was funded by the Belgium Development Cooperation (DGD) under the Framework Agreement Program between DGD and ITM (FA4 Peru, 2017-2021) and the sample collections in 2018 were supported by VLIR-UOS (project PE2018TEA470A102; University of Antwerp). Funding for the sample collections lead by the U.S. Naval Medical Research Unit 6 (NAMRU-6) in 2011 and 2012 was provided by the Armed Forces Health Surveillance Division (AFHSD) and its Global Emerging Infections Surveillance and Response (GEIS) Section (P0144_20_N6_01, 2020-2021).

The Predictive Value of Mutation Screening for Anticipating COVID-19 Waves

Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and associated deaths. Whole Genome Sequencing (WGS) continues to be the gold standard for molecular surveillance of the pandemics but other assays such as mutation genotyping can be used to reduce costs and allocated time. This study investigates the efficiency of mutation screening tests compared to WGS and their predictive value to anticipate future waves. A very high degree of fidelity for this type of assay was found, regardless of the method used. The positive predictive value (PPV) of 4/5 markers was over 95% for the detection of Alpha and Delta variants. By estimating the prevalence of the Alpha and Delta strains using genotyping assays and fitting the data to a mathematical model, a five week period between the point of exponential growth of variant prevalence and a drastic increase in case numbers was found. For that reason, raising awareness about the efficacy of mutation screening could help authorities adopt better measures in the future.

Molecular Surveillance and Ex Vivo Drug Susceptibilities of Plasmodium vivax Isolates From the China–Myanmar Border

Drug resistance in Plasmodium vivax may pose a challenge to malaria elimination. Previous studies have found that P. vivax has a decreased sensitivity to antimalarial drugs in some areas of the Greater Mekong Sub-region. This study aims to investigate the ex vivo drug susceptibilities of P. vivax isolates from the China–Myanmar border and genetic variations of resistance-related genes. A total of 46 P. vivax clinical isolates were assessed for ex vivo susceptibility to seven antimalarial drugs using the schizont maturation assay. The medians of IC50 (half-maximum inhibitory concentrations) for chloroquine, artesunate, and dihydroartemisinin from 46 parasite isolates were 96.48, 1.95, and 1.63 nM, respectively, while the medians of IC50 values for piperaquine, pyronaridine, mefloquine, and quinine from 39 parasite isolates were 19.60, 15.53, 16.38, and 26.04 nM, respectively. Sequence polymorphisms in pvmdr1 (P. vivax multidrug resistance-1), pvmrp1 (P. vivax multidrug resistance protein 1), pvdhfr (P. vivax dihydrofolate reductase), and pvdhps (P. vivax dihydropteroate synthase) were determined by PCR and sequencing. Pvmdr1 had 13 non-synonymous substitutions, of which, T908S and T958M were fixed, G698S (97.8%) and F1076L (93.5%) were highly prevalent, and other substitutions had relatively low prevalences. Pvmrp1 had three non-synonymous substitutions, with Y1393D being fixed, G1419A approaching fixation (97.8%), and V1478I being rare (2.2%). Several pvdhfr and pvdhps mutations were relatively frequent in the studied parasite population. The pvmdr1 G698S substitution was associated with a reduced sensitivity to chloroquine, artesunate, and dihydroartemisinin. This study suggests the possible emergence of P. vivax isolates resistant to certain antimalarial drugs at the China–Myanmar border, which demands continuous surveillance for drug resistance.

Investigation of MALDI-TOF Mass Spectrometry for Assessing the Molecular Diversity of Campylobacter jejuni and Comparison with MLST and cgMLST: A Luxembourg One-Health Study

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLSTCC, MLSTST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

ONTdeCIPHER: An amplicon-based nanopore sequencing pipeline for tracking pathogen variants

Motivation: Amplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis. Results: Here, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology (ONT) sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogenes. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses.