Polyclonal Infections Research Articles

Phylogenetic evaluation and genotypic identification of burn-related Pseudomonas aeruginosa strains isolated from post-burn human infections during hospitalization.

Cutaneous burn trauma, compromise of dermal layers and immune defense system is a physical and fiscal burden on healthcare systems. Burn-wound infections are a serious complication of thermal-injury and contribute significantly to care burden. After burn-induced trauma, sepsis by Pseudomonas aeruginosa impairs patient recovery and contributes to mortality and morbidity. Past studies show positive correlation between detection of Pseudomonas species and healing-impaired traumatic wounds. P. aeruginosa is a resilient opportunistic human pathogen and a nosocomial agent involved in pathology of healing-impaired wounds, especially in burn-patients. Expansive array of virulence determinants have resulted in gentamicin- and silver-resistant P. aeruginosa outbreaks. Knowledge of molecular dynamics and phylogeny of P. aeruginosa associated with burn-wounds is limited. Therefore, we conducted whole-genome sequencing for genotyping and phylogenetic analysis of P. aeruginosa burn-associated strains (n=19) isolated from 7 burn cases during hospitalization. Comparison of genetic features in P. aeruginosa strains in the core genome and mobilome detected genetic variations within some clonal infections overtime. Genetic variations were observed among different burn cases, with some features identified in severe lung infections. Polyclonal infections were also observed, with differing genotypes and virulence potentials, highlighting the importance of reasoned sampling of isolates for clinical testing.

Variability of macrolide-resistant profile in Mycobacterium avium complex pulmonary disease.

This single-center retrospective study aimed to analyze the variability of macrolide resistance (MR) in 68 patients with Mycobacterium avium complex pulmonary disease. Among 25 patients treated without macrolides, 13 (52%) reverted to macrolide-susceptible (MS) profiles. Only one (2%) of 43 patients who continued macrolide treatment showed this change. We compared 30 MR isolates with recent specimens. Among them, seven shifted to MS (five attributed to clonally related strains; two resulting from reinfection or polyclonal infection).

Genotyping methods to distinguish Plasmodium falciparum recrudescence from new infection for the assessment of antimalarial drug efficacy: an observational, single-centre, comparison study

Distinguishing Plasmodium falciparum recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against Pfalciparum. We aimed to compare the efficacy of different genotyping methods to assess their effect on drug efficacy estimates, particularly in patients from high-transmission settings with polyclonal infections. In this head-to-head comparison study, we compared five different genotyping methods currently used: fast capillary electrophoresis (F-CE) using msp1, msp2, and glurp; high-resolution capillary electrophoresis (H-CE) using msp1, msp2, and glurp; H-CE using microsatellites; targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers; and high-resolution melting (HRM) analysis using msp1 and msp2. We assessed their sensitivity in detecting minority clones in polyclonal infections, their reproducibility, and the genetic diversity of the markers used. Our study used four well characterised Pfalciparum laboratory strains mixed in varying ratios, and 20paired samples collected from an in-vivo clinical trial. The experiments were performed at the Swiss Tropical and Public Health Institute in Basel, Switzerland between May 5, 2020, and Aug 23,2021. H-CE using msp1 and msp2 and TADS revealed the highest sensitivity in detecting minority clones (up to ratios of 1:100 for H-CE and 50:1:1:1 for TADS in the FCB1:HB3 and 3D7:K1:HB3:FCB1 laboratory strain mixtures, respectively), highest reproducibility (intra-assay: 99% and 91% for H-CE and TADS, respectively; inter-assay: 98% and 92% for H-CE and TADS, respectively), and highest genetic diversity in the used markers (up to 36and32unique genotypes in 20paired samples for H-CE using msp2 and TADS using cpmp, respectively). Microsatellites assessed by H-CE had a lower genetic diversity compared with msp1, msp2, and glurp assessed by H-CE and the SNP-rich markers assessed by TADS, with a maximum of 13unique genotypes, and some genotypes having allelic frequencies larger than 30%.Markers used by TADS gave the most consistent results in distinguishing recrudescence from new infection across all methods (in 18of 20pairs of samples vs 15of 20pairs for H-CE). WHO currently recommends replacing glurp with microsatellites. However, in this study, the replacement of glurp with microsatellites did not change the genotyping outcome, probably due to the lower genetic diversity of microsatellites. More studies with large sample sizes are required to identify the most suitablemicrosatellites that could replace glurp. Our study indicates that TADS should be considered the gold standardfor genotyping to distinguish recrudescence from new infection, and that it should be used to validate other methods. Swiss Tropical and Public Health Institute.

Genomic epidemiology demonstrates spatially clustered, local transmission of Plasmodium falciparum in forest-going populations in southern Lao PDR.

While there has been significant progress in controlling falciparum malaria in the Lao People's Democratic Republic (PDR), sporadic cases persist in southern provinces where the extent and patterns of transmission remain largely unknown. To assess parasite transmission in this area, 53 Plasmodium falciparum (Pf) positive cases detected through active test and treat campaigns from December 2017 to November 2018 were sequenced, targeting 204 highly polymorphic amplicons. Two R packages, MOIRE and Dcifer, were applied to assess the multiplicity of infections (MOI), effective MOI (eMOI), within-host parasite relatedness, and between-host parasite relatedness ([Formula: see text]). Genomic data were integrated with survey data to characterize the temporal and spatial structures of identified clusters. The positive cases were mainly captured during the focal test and treat campaign conducted in 2018, and in the Pathoomphone area, which had the highest test positivity and forest activity. About 30% of the cases were polyclonal infections, with over half of theses (63%) showing within-host relatedness greater than 0.6, suggesting that cotransmission rather than superinfection was primarily responsible for maintaining polyclonality. A large majority of cases (81%) were infected by parasites genetically linked to one or more other cases. We identified five genetically distinct clusters in forest fringe villages within the Pathoomphone district, characterized by a high degree of genetic relatedness between parasites (mean [Formula: see text] = 0.8). Four smaller clusters of 2-3 cases linked Moonlapamok and Pathoomphone districts, with an average [Formula: see text] of 0.6, suggesting cross-district transmission. Most of the clustered cases occurred within 20 km and 2 months of each other, consistent with focal transmission. Transmission clusters identified in this study confirm the role of ongoing focal parasite transmission occurring within the forest or forest-fringe in the highly mobile population.

Single-cell analyses of polyclonal Plasmodium vivax infections and their consequences on parasite transmission

Most Plasmodium vivax infections contain genetically distinct parasites, but the consequences of this polyclonality on the development of asexual parasites, their sexual differentiation, and their transmission remain unknown. We describe infections of Saimiri monkeys with two strains of P. vivax and the analyses of 80,024 parasites characterized by single cell RNA sequencing and individually genotyped. In our model, consecutive inoculations fail to establish polyclonal infections. By contrast, simultaneous inoculations of two strains lead to sustained polyclonal infections, although without detectable differences in parasite regulation or sexual commitment. Analyses of sporozoites dissected from mosquitoes fed on coinfected monkeys show that all genotypes are successfully transmitted to mosquitoes. However, after sporozoite inoculation, not all genotypes contribute to the subsequent blood infections, highlighting an important bottleneck during pre-erythrocytic development. Overall, these studies provide new insights on the mechanisms regulating the establishment of polyclonal P. vivax infections and their consequences for disease transmission.

Assessing the therapeutic efficacy of artemether-lumefantrine for uncomplicated malaria in Lagos, Nigeria: a comprehensive study on treatment response and resistance markers

BackgroundThe burden of malaria persists in sub-Saharan Africa and the emergence of artemisinin resistance has introduced complexity to control efforts. Monitoring the efficacy of artemisinin-based treatment for malaria is crucial to address this challenge. This study assessed treatment efficacy of artemether-lumefantrine (AL) and genetic diversity of Plasmodium falciparum isolates in a Nigerian population.MethodsParticipants presenting with clinical symptoms of uncomplicated malaria at a health centre in Lagos, Nigeria, were screened for P. falciparum. Enrolled participants were treated with AL and monitored through scheduled check-up visits, clinical and laboratory examinations for 28 days. Parasite clearance and genetic diversity were assessed through polymerase chain reaction (PCR) analysis of merozoite surface proteins (msp1 and msp2). The prevalence of drug resistance mutations was assessed by P. falciparum multidrug resistance gene 1 (mdr1) genotyping followed by P. falciparum ubiquitin-specific protease 1 (ubp1) gene sequencing.ResultsThe PCR-uncorrected treatment outcome revealed 94.4% adequate clinical and parasitological response (ACPR) and 5.6% late parasitological failure (LPF) rates. After PCR correction, no suspected LPF case was detected and ACPR 67/67 (100%) was achieved in all the individuals. Moreover, a high prevalence of wild-type alleles for mdr1 N86Y (93.7%), and mdr1 D1246Y (87.5%) was observed. Genetic diversity analysis revealed predominant K1 allelic family for msp1 (90.2%) and FC27 for msp2 (64.4%). Estimated multiplicity of infection (MOI) was 1.7, with the highest MOI observed in the 5–15 years age group. ubp1 sequence analysis identified one nonsynonymous E1528D polymorphism at a low frequency (1.6%).ConclusionThe study demonstrated sustained efficacy of AL for treating uncomplicated P. falciparum malaria. Genetic diversity analysis revealed various allelic types, suggesting occurrences of polyclonal infections. Nonetheless, the detection of a significant ubp1 polymorphism could have future implications for the epidemiology of anti-malarial drug resistance in the population.

Lineage-informative microhaplotypes for recurrence classification and spatio-temporal surveillance of Plasmodium vivax malaria parasites

Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50–250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median HE = 0.70–0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources.

Additional blood meals increase sporozoite infection in Anopheles mosquitoes but not Plasmodium falciparum genetic diversity

The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.

Genetic surveillance reveals low, sustained malaria transmission with clonal replacement in Sao Tome and Principe.

Despite efforts to eliminate malaria in Sao Tome and Principe (STP), cases have recently increased. Understanding residual transmission structure is crucial for developing effective elimination strategies. This study collected surveillance data and generated amplicon sequencing data from 980 samples between 2010 and 2016 to examine the genetic structure of the parasite population. The mean multiplicity of infection (MOI) was 1.3, with 11% polyclonal infections, indicating low transmission intensity. Temporal trends of these genetic metrics did not align with incidence rates, suggesting that changes in genetic metrics may not straightforwardly reflect changes in transmission intensity, particularly in low transmission settings where genetic drift and importation have a substantial impact. While 88% of samples were genetically linked, continuous turnover in genetic clusters and changes in drug-resistance haplotypes were observed. Principal component analysis revealed some STP samples were genetically similar to those from Central and West Africa, indicating possible importation. These findings highlight the need to prioritize several interventions such as targeted interventions against transmission hotspots, reactive case detection, and strategies to reduce the introduction of new parasites into this island nation as it approaches elimination. This study also serves as a case study for implementing genetic surveillance in a low transmission setting.

Plasmodium vivax populations in the western Greater Mekong Subregion evaluated using a genetic barcode.

An improved understanding of the Plasmodium vivax populations in the Great Mekong Subregion (GMS) is needed to monitor the progress of malaria elimination. This study aimed to use a P. vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics and explore the gene flow among P. vivax parasite populations in the western GMS (China, Myanmar and Thailand). A total of 315 P. vivax patient samples collected in 2011 and 2018 from four regions of the western GMS were genotyped for 42 SNPs using the high-throughput MassARRAY SNP genotyping technology. Population genetic analysis was conducted to estimate the genetic diversity, effective population size, and population structure among the P. vivax populations. Overall, 291 samples were successfully genotyped at 39 SNPs. A significant difference was observed in the proportion of polyclonal infections among the five P. vivax populations (P = 0.0012, Pearson Chi-square test, χ2 = 18.1), with western Myanmar having the highest proportion (96.2%, 50/52) in 2018. Likewise, the average complexity of infection was also highest in western Myanmar (1.31) and lowest in northeast Myanmar (1.01) in 2018. The older samples from western China in 2011 had the highest pairwise nucleotide diversity (π, 0.388 ± 0.046), expected heterozygosity (He, 0.363 ± 0.02), and the largest effective population size. In comparison, in the neighboring northeast Myanmar, the more recent samples in 2018 showed the lowest values (π, 0.224 ± 0.036; He, 0.220 ± 0.026). Furthermore, the 2018 northeast Myanmar parasites showed high and moderate genetic differentiation from other populations with FST values of 0.162-0.252, whereas genetic differentiation among other populations was relatively low (FST ≤ 0.059). Principal component analysis, phylogeny, and STRUCTURE analysis showed that the P. vivax population in northeast Myanmar in 2018 substantially diverged from other populations. Although the 42 SNP barcode is a valuable tool for tracking parasite origins of worldwide parasite populations, a more extended barcode with additional SNPs is needed to distinguish the more related parasite populations in the western GMS.

Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring

ObjectivesThe accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. MethodsMultiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. ResultsqPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. ConclusionsMultiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Genomic epidemiology of Mycobacterium abscessus at an adult cystic fibrosis programme reveals low potential for healthcare-associated transmission.

Nontuberculous mycobacteria (NTM) has been reported to be transmitted between people with cystic fibrosis (CF) attending CF centres. A suspected Mycobacterium abscessus outbreak was investigated at the University of Texas Southwestern (UTSW) Adult CF Program using a combination of pathogen genomic sequencing and epidemiologic methods. The objectives of the present study were to apply the Healthcare-Associated Links in Transmission of NTM (HALT NTM) study to investigate the occurrence of potential healthcare-associated transmission and/or acquisition of NTM among people with CF infected with genetically similar NTM isolates. Whole-genome sequencing of respiratory M. abscessus isolates from 50 people with CF receiving care at UTSW was performed to identify genetically similar isolates. Epidemiologic investigation, comparison of respiratory and environmental isolates, and home residence watershed mapping were studied. Whole-genome sequencing analysis demonstrated seven clusters of genetically similar M. abscessus (four ssp. abscessus and three ssp. massiliense). Epidemiologic investigation revealed potential opportunities for healthcare-associated transmission within three of these clusters. Healthcare environmental sampling did not recover M. abscessus, but did recover four human disease-causing species of NTM. No subjects having clustered infections lived in the same home residence watershed. Some subjects were infected with more than one M. abscessus genotype, both within and outside of the dominant circulating clones. Healthcare-associated person-to-person transmission of M. abscessus appears to be rare at this centre. However, polyclonal infections of M. abscessus species and subspecies, not originating from the endemic hospital environment, suggest multiple shared modes of acquisition outside the healthcare setting.

Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing

BackgroundUnderstanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing.MethodsA data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach.ResultsA 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92–0.99] and 0.92 [95% CI: 0.83–0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28–0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35–3.68].ConclusionsA region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Using a mobile nanopore sequencing lab for end-to-end genomic surveillance of Plasmodium falciparum: A feasibility study.

Genomic epidemiology holds promise for malaria control and elimination efforts, for example by informing on Plasmodium falciparum genetic diversity and prevalence of mutations conferring anti-malarial drug resistance. Limited sequencing infrastructure in many malaria-endemic areas prevents the rapid generation of genomic data. To address these issues, we developed and validated assays for P. falciparum nanopore sequencing in endemic sites using a mobile laboratory, targeting key antimalarial drug resistance markers and microhaplotypes. Using two multiplexed PCR reactions, we amplified six highly polymorphic microhaplotypes and ten drug resistance markers. We developed a bioinformatics workflow that allows genotyping of polyclonal malaria infections, including minority clones. We validated the panels on mock dried blood spot (DBS) and rapid diagnostic test (RDT) samples and archived DBS, demonstrating even, high read coverage across amplicons (range: 580x to 3,212x median coverage), high haplotype calling accuracy, and the ability to explore within-sample diversity of polyclonal infections. We field-tested the feasibility of rapid genotyping in Zanzibar in close collaboration with the local malaria elimination program using DBS and routinely collected RDTs as sample inputs. Our assay identified haplotypes known to confer resistance to known antimalarials in the dhfr, dhps and mdr1 genes, but no evidence of artemisinin partial resistance. Most infections (60%) were polyclonal, with high microhaplotype diversity (median HE = 0.94). In conclusion, our assays generated actionable data within a few days, and we identified current challenges for implementing nanopore sequencing in endemic countries to accelerate malaria control and elimination.

Genomics of Plasmodium vivax in Colombia reveals evidence of local bottle-necking and inter-country connectivity in the Americas.

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation.

Genotype-Phenotype Characterization of Serial Mycobacterium tuberculosis Isolates in Bedaquiline-Resistant Tuberculosis.

Emerging resistance to bedaquiline (BDQ) threatens to undermine advances in the treatment of drug-resistant tuberculosis (DRTB). Characterizing serial Mycobacterium tuberculosis (Mtb) isolates collected during BDQ-based treatment can provide insights into the etiologies of BDQ resistance in this important group of DRTB patients. We measured mycobacteria growth indicator tube (MGIT)-based BDQ minimum inhibitory concentrations (MICs) of Mtb isolates collected from 195 individuals with no prior BDQ exposure who were receiving BDQ-based treatment for DRTB. We conducted whole-genome sequencing on serial Mtb isolates from all participants who had any isolate with a BDQ MIC >1 collected before or after starting treatment (95 total Mtb isolates from 24 participants). Sixteen of 24 participants had BDQ-resistant TB (MGIT MIC ≥4 µg/mL) and 8 had BDQ-intermediate infections (MGIT MIC = 2 µg/mL). Participants with pre-existing resistance outnumbered those with resistance acquired during treatment, and 8 of 24 participants had polyclonal infections. BDQ resistance was observed across multiple Mtb strain types and involved a diverse catalog of mmpR5 (Rv0678) mutations, but no mutations in atpE or pepQ. Nine pairs of participants shared genetically similar isolates separated by <5 single nucleotide polymorphisms, concerning for potential transmitted BDQ resistance. BDQ-resistant TB can arise via multiple, overlapping processes, including transmission of strains with pre-existing resistance. Capturing the within-host diversity of these infections could potentially improve clinical diagnosis, population-level surveillance, and molecular diagnostic test development.

In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.

Standardized clinical procedures for antibiotic administration rely on pathogen identification and antibiotic susceptibility testing, often performed on single-colony bacterial isolates. For respiratory pathogens, this could be questionable, as chronic patients may be persistently colonized by multiple clones or lineages from the same bacterial pathogen species. Indeed, multiple strains of nontypeable Haemophilus influenzae, with different antibiotic susceptibility profiles, can be co-isolated from cystic fibrosis and chronic obstructive pulmonary disease sputum specimens. Despite this clinical evidence, we lack information about the dynamics of H. influenzae polyclonal infections, which limits the optimization of therapeutics. Here, we present the engineering and validation of a plasmid toolkit (pTBH, toolbox for Haemophilus), with standardized modules consisting of six reporter genes for fluorescent or bioluminescent labeling of H. influenzae. This plasmid set was independently introduced in a panel of genomically and phenotypically different H. influenzae strains, and two of them were used as a proof of principle to analyze mixed biofilm growth architecture and antibiotic efficacy, and to visualize the dynamics of alveolar epithelial co-infection. The mixed biofilms showed a bilayer architecture, and antibiotic efficacy correlated with the antibiotic susceptibility of the respective single-species strains. Furthermore, differential kinetics of bacterial intracellular location within subcellular acidic compartments were quantified upon co-infection of cultured airway epithelial cells. Overall, we present a panel of novel plasmid tools and quantitative image analysis methods with the potential to be used in a whole range of bacterial host species, assay types, and/or conditions and generate meaningful information for clinically relevant settings. IMPORTANCE Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

Multiplicity of infection of Plasmodium knowlesi in Malaysia: an application of Pkmsp-1 block IV.

In Malaysia presently, the main cause of human malaria is by the zoonotic monkey parasite Plasmodium knowlesi. A previous study has suggested that the P. knowlesi merozoite surface protein 1 (Pkmsp-1) block IV to be a suitable multiplicity of infection (MOI) genotyping marker for knowlesimalaria. This study therefore aimed to investigate the usefulness of Pkmsp-1 block IV in assessing the MOI of P. knowlesi in clinical isolates from Malaysia. Two allele-specific PCR primer pairs targeting the two allelic families of block IV (T1 and T2) were designed, and used to genotype P. knowlesi in 200 blood samples (100 from Peninsular Malaysia and 100 from Malaysian Borneo). Results showed that the mean MOI in Malaysian Borneo was slightly higher as compared to Peninsular Malaysia (1.58 and 1.40, respectively). Almost half of the total blood samples from Malaysian Borneo (52%) had polyclonal infections (i.e., more than one allele of any family type) as compared to Peninsular Malaysia (33%) samples. The T1 allelic family was more prevalent in Peninsular Malaysia (n=75) than in Malaysian Borneo (n=60). The T2 allelic family, however, was more prevalent in the Malaysian Borneo (n=87 vs n=53 respectively). This study shows that the single locus Pkmsp-1 block IV can serve as a simple alternative genetic marker for estimating knowlesi malaria MOI in a population. Future MOI studies should focus on macaque populations as macaques are the natural host of P. knowlesi.

Plasmodium falciparum msp-1 and msp-2 genetic diversity and multiplicity of infection in isolates from Congolese patients in the Republic of Congo.

With limited up to date data from the Republic of Congo, the aim of this study was to investigate allelic polymorphism of merozoite surface protein-1 (msp-1) and merozoite surface protein-2 (msp-2). This will help assess the genetic diversity and multiplicity of Plasmodium falciparum infection (MOI), from uncomplicated malaria individuals living in Brazzaville. Between March and October 2021, a cross-sectional study was carried out at a health center in Madibou District located in the south of Brazzaville. Plasmodium infection was diagnosed in human blood by microscopy and the block 2 of P. falciparum msp-1 and block 3 of msp-2 genes were genotyped by nested PCR. Overall, 57 genotypes with fragment sizes ranging from 110 to 410 bp were recorded for msp-1, among which 25, 21, and 11 genotypes identified for K1, MAD20, and RO33 allelic families respectively. RO33 (34.3%) and MAD20 (34.3%) allelic families were more frequent compared to K1 (31.4%) although the difference was not statistically significant. Also, 47 msp-2 genotypes were identified, including 26 FC27 genotypes type, and 21 genotypes belonging to the 3D7 allelic family. FC27 was more frequent (52.3%) compared to 3D7 (47.7%). The prevalence of the polyclonal infection was 90.0% while the MOI was 2.90 ± 1.0. The MOI and polyclonal infection were not significantly associated with the parasitaemia and anaemia. This study reveals a high genetic diversity and the trend of increasing MOI of P. falciparum isolates from the south of Brazzaville, compared to the reports from the same setting before the COVID-19 pandemic.