Bias-corrected maximum-likelihood estimation of multiplicity of infection and lineage frequencies

Background The UN’s Sustainable Development Goals are devoted to eradicate a range of infectious diseases to achieve global well-being. These efforts require monitoring disease transmission at a level that differentiates between pathogen variants at the genetic/molecular level. In fact, the advantages of genetic (molecular) measures like multiplicity of infection (MOI) over traditional metrics, e.g., R0, are being increasingly recognized. MOI refers to the presence of multiple pathogen variants within an infection due to multiple infective contacts. Maximum-likelihood (ML) methods have been proposed to derive MOI and pathogen-lineage frequencies from molecular data. However, these methods are biased. Methods and findings Based on a single molecular marker, we derive a bias-corrected ML estimator for MOI and pathogen-lineage frequencies. We further improve these estimators by heuristical adjustments that compensate shortcomings in the derivation of the bias correction, which implicitly assumes that data lies in the interior of the observational space. The finite sample properties of the different variants of the bias-corrected estimators are investigated by a systematic simulation study. In particular, we investigate the performance of the estimator in terms of bias, variance, and robustness against model violations. The corrections successfully remove bias except for extreme parameters that likely yield uninformative data, which cannot sustain accurate parameter estimation. Heuristic adjustments further improve the bias correction, particularly for small sample sizes. The bias corrections also reduce the estimators’ variances, which coincide with the Cramér-Rao lower bound. The estimators are reasonably robust against model violations. Conclusions Applying bias corrections can substantially improve the quality of MOI estimates, particularly in areas of low as well as areas of high transmission—in both cases estimates tend to be biased. The bias-corrected estimators are (almost) unbiased and their variance coincides with the Cramér-Rao lower bound, suggesting that no further improvements are possible unless additional information is provided. Additional information can be obtained by combining data from several molecular markers, or by including information that allows stratifying the data into heterogeneous groups.

Phage Cocktail Development against Aeromonas salmonicida subsp. salmonicida Strains Is Compromised by a Prophage

Aquaculture is a rapidly growing food production sector. Fish farmers are experiencing increasing problems with antibiotic resistance when fighting against pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis. Phage therapy may provide an alternative, but effective use must be determined. Here, we studied the inhibition of A. salmonicida subsp. salmonicida strains by five phages (HER98 [44RR2.8t.2], HER110 [65.2], SW69-9, L9-6 and Riv-10) used individually or as combinations of two to five phages. A particular combination of four phages (HER98 [44RR2.8t.2], SW69-9, Riv-10, and HER110 [65.2]) was found to be the most effective when used at an initial multiplicity of infection (MOI) of 1 against the A. salmonicida subsp. salmonicida strain 01-B526. The same phage cocktail is effective against other strains except those bearing a prophage (named Prophage 3), which is present in 2/3 of the strains from the province of Quebec. To confirm the impact of this prophage, we tested the effectiveness of the same cocktail on strains that were either cured or lysogenized with Prophage 3. While the parental strains were sensitive to the phage cocktail, the lysogenized ones were much less sensitive. These data indicate that the prophage content of A. salmonicida subsp. salmonicida can affect the efficacy of a cocktail of virulent phages for phage therapy purposes.

Properties of Two Broad Host Range Phages of Yersinia enterocolitica Isolated from Wild Animals

Yersinia (Y.) enterocolitica and Y. pseudotuberculosis are important zoonotic agents which can infect both humans and animals. To combat these pathogens, the application of strictly lytic phages may be a promising tool. Since only few Yersinia phages have been described yet, some of which demonstrated a high specificity for certain serotypes, we isolated two phages from game animals and characterized them in terms of their morphology, host specificity, lytic activity on two bio-/serotypes and genome composition. The T7-related podovirus vB_YenP_Rambo and the myovirus vB_YenM_P281, which is very similar to a previously described phage PY100, showed a broad host range. Together, they lysed all the 62 tested pathogenic Y. enterocolitica strains belonging to the most important bio-/serotypes in Europe. A cocktail containing these two phages strongly reduced cultures of a bio-/serotype B4/O:3 and a B2/O:9 strain, even at very low MOIs (multiplicity of infection) and different temperatures, though, lysis of bio-/serotype B2/O:9 by vB_YenM_P281 and also by the related phage PY100 only occurred at 37 °C. Both phages were additionally able to lyse various Y. pseudotuberculosis strains at 28 °C and 37 °C, but only when the growth medium was supplemented with calcium and magnesium cations.

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10−4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

Application of bacteriophage as food preservative to control enteropathogenic Escherichia coli (EPEC)

Objectives This study was conducted to characterize lytic bacteriophages infecting enteropathogenic Escherichia coli (EPEC) on several types of food and analyze their ability as phage biocontrol to be used as a food preservative. Characterization was done for bacteriophage morphology and stability, along with the determination of minimum multiplicity of infection (miMOI), and application of bacteriophage in the food matrix. Results Out of the five samples, BL EPEC bacteriophage exhibited the highest titer of 2.05 × 109 PFU/mL, with a wide range of pH tolerance, and high thermal tolerance. BL EPEC also showed the least reduction after 168 h of incubation, with a rate of 0.90 × 10–3 log10 per hour. Bacteriophages from BL EPEC and CS EPEC showed an ideal value of miMOI of 0.01. As a food preservative, BL EPEC bacteriophage was able to reduce bacteria in food samples with a reduction above 0.24 log10 in lettuce and approximately 1.84 log10 in milk. From this study we found that BL EPEC bacteriophage showed the greatest potential to be used as phage biocontrol to improve food safety

Markov chain Monte Carlo Gibbs sampler approach for estimating haplotype frequencies among multiple malaria infected human blood samples

Background Malaria patients can have two or more haplotypes in their blood sample making it challenging to identify which haplotypes they carry. In addition, there are challenges in measuring the type and frequency of resistant haplotypes in populations. This study presents a novel statistical method Gibbs sampler algorithm to investigate this issue. Results The performance of the algorithm is evaluated on simulated datasets consisting of patient blood samples characterized by their multiplicity of infection (MOI) and malaria genotype. The simulation used different resistance allele frequencies (RAF) at each Single Nucleotide Polymorphisms (SNPs) and different limit of detection (LoD) of the SNPs and the MOI. The Gibbs sampler algorithm presents higher accuracy among high LoD of the SNPs or the MOI, validated, and deals with missing MOI compared to previous related statistical approaches. Conclusions The Gibbs sampler algorithm provided robust results when faced with genotyping errors caused by LoDs and functioned well even in the absence of MOI data on individual patients.

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

Plasmodium falciparum, the main causative agent of malaria is an important public health vector in Khyber Pakhtunkhwa, Pakistan. Identification of the genetic diversity of malaria parasites can inform the intensity of transmission and identify potential deficiencies in malaria control programs. The aim of this study was to investigate the genetic diversity, allele frequencies and multiplicity of infection (MOI) of P. falciparum in Khyber Pakhtunkhwa, Pakistan. Methods: A total of 85 isolates from patients presenting to the local health centers with P. falciparum species were collected from 2017 to 2019. Parasite DNA was extracted from a total of 200 micro litter whole blood per patient using the Qiagen DNA extraction kit according to manufactures instructions. The polymorphic region of msp-1, msp-2 and glurp loci were genotyped by using nested polymerase chain reactions followed by gel electrophoresis for fragment analysis. Results: Genetic diversity and allelic frequencies of msp-1, msp-2 and glurp were identified in 85 blood samples. A total of 62 msp alleles were detected in which 30 for msp-1 and 32 for msp-2. For msp-1 the successful amplification occurred in (75/85) 88.23% isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp. For msp-1, the K1 allelic family was predominant at 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75) and 10.67% (8/75) respectively. In msp-2, the FC27 allelic family was the most abundant with 70.14% (47/67) compared to 3D7 with 67.16% (45/67). Nine glurp RII region genotypes were identified. The overall mean multiplicity of infection was 2.6 with1.8 and 1.4 for msp-1 and msp-2 respectively while for glurp RII genes (MOI=1.03). There was no significant association between multiplicity of infection and age group (Spearman rank coefficient = 0.050; P = 0.6). There was significant correlation between MOI and parasite density for msp-2 allelic family. Conclusion: Our study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates from malaria patients in Khyber Pakhtunkhwa Pakistan. In the present study the genotype data provided the valuable information which is essential for monitoring the impact of malaria eradication efforts in this region.

Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019–2020

Background Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. Methods A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2–10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher’s exact test as appropriate at P ≤ 0.05. Results Overall, 75.5% (95% CI 61.1–85.8) and 24.5% (95% CI14.2–38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8–89.1), P < 0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI 1.7–2.1), P = 0.01 that was similar between age groups (1.8 vs 1.9), P = 0.60 and regions (1.9 vs 1.8), P = 0.43 for the < 5 and ≥ 5 years and Eastern and Western regions, respectively. Genomic sequencing of the pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT−/PCR + samples (1.9% vs 1.5%) and (13% vs 8%), P < 0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. Conclusion High level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies.

An optimized protocol for dual RNA-Seq of human macrophages infected with Mycobacterium avium

Recently, dual RNA-sequencing (RNA-Seq) has been identified as a powerful tool to study host- pathogen interactions, which is particularly interesting for intracellular pathogens such as mycobacteria. However, due to the complexity of obtaining human host cells, many models rely on the usage of host cells derived from animals or cell lines, which does not accurately mimic the situation in the patient. Furthermore, due to the severe disbalance in host and pathogen RNA content, it is difficult to obtain sufficient sequencing depth for the infecting pathogen. Here, we present an optimized method to perform dual RNA-sequencing on human monocyte-derived macrophages (hMDMs) infected with Mycobacterium avium (M. avium). It is likely that, with slight modifications in multiplicity of infection (MOI) to account for differences in virulence, this methodology will be applicable for other difficult-to-lyse intracellular mycobacteria.