Mixed-strain Infections Research Articles

Coagulase-negative staphylococci are the main causes of bacterial meningitis in duck

Since September 2018, serious meningitis has been found on some breeding-duck farms in Shandong Province, China. A large number of ducks exhibit severe neurological symptoms. The ducks were randomly selected for laboratory testing. Duck brain samples were collected using standard sterile techniques, and the staphylococci isolates were detected in 404 (70.14%) out of 576 brain samples. A total of 525 coagulase-negative staphylococci (CoNS) strains were isolated, including 6 species: Staphylococcus sciuri (S. sciuri) (67.24%, 353/525), Staphylococcus epidermidis (S. epidermidis) (9.71%, 51/525), Staphylococcus saprophyticus (S. saprophyticus) (8.38%, 44/525), Staphylococcus lentus (S. lentus) (7.62%, 40/525), Staphylococcus haemolyticus (S. haemolyticus) (2.48%, 13/525), and Staphylococcus xylosus (S. xylosus) (4.57%, 24/525). Mixed strain infections were detected in 121 (29.95%) infected presentations. The antimicrobial susceptibility testing indicated that 40.38% of the isolates exhibited multi-drug resistance, and 53.90% of the strains were methicillin-resistant strains by amplification of the methicillin resistance gene (mecA) gene. Through experimental reproduction of the disease, we determined that the CoNS strains were the leading pathogens causing bacterial meningitis in ducks. Although these CoNS strains does not directly cause the death of sick ducks, they still cause large economic losses due to the retarded growth and development of the sick ducks, lower feed returns, and lower grades of processed duck products. The results of this study will contribute to our understanding of the epidemiology and pathogenesis of CoNS and be helpful in the prevention and treatment of the infection.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda

BackgroundMixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays.MethodsThis was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit–Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed.ResultsA total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male’s 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88–64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection.ConclusionsThe presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

Mixed infections in genotypic drug-resistant Mycobacterium tuberculosis

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major global public health problem, resulting in more than 1 million deaths each year. Drug resistance (DR), including multi-drug (MDR-TB), is making TB control difficult and accounts for 16% of new and 48% of previously treated cases. To further complicate treatment decision-making, many clinical studies have reported patients harbouring multiple distinct strains of M. tuberculosis across the main lineages (L1 to L4). The extent to which drug-resistant strains can be deconvoluted within mixed strain infection samples is understudied. Here, we analysed M. tuberculosis isolates with whole genome sequencing data (n = 50,723), which covered the main lineages (L1 9.1%, L2 27.6%, L3 11.8%, L4 48.3%), with genotypic resistance to isoniazid (HR-TB; n = 9546 (29.2%)), rifampicin (RR-TB; n = 7974 (24.4%)), and at least MDR-TB (n = 5385 (16.5%)). TB-Profiler software revealed 531 (1.0%) isolates with potential mixed sub-lineage infections, including some with DR mutations (RR-TB 21/531; HR-TB 59/531; at least MDR-TB 173/531). To assist with the deconvolution of such mixtures, we adopted and evaluated a statistical Gaussian Mixture model (GMM) approach. By simulating 240 artificial mixtures of different ratios from empirical data across L1 to L4, a GMM approach was able to accurately estimate the DR profile of each lineage, with a low error rate for the estimated mixing proportions (mean squared error 0.012) and high accuracy for the DR predictions (93.5%). Application of the GMM model to the clinical mixtures (n = 531), found that 33.3% (188/531) of samples consisted of DR and sensitive lineages, 20.2% (114/531) consisted of lineages with only DR mutations, and 40.6% (229/531) consisted of lineages with genotypic pan-susceptibility. Overall, our work demonstrates the utility of combined whole genome sequencing data and GMM statistical analysis approaches for providing insights into mono and mixed M. tuberculosis infections, thereby potentially assisting diagnosis, treatment decision-making, drug resistance and transmission mapping for infection control.

Investigating in vivo Mycobacterium avium subsp. paratuberculosis microevolution and mixed strain infections.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's Disease (JD) in ruminants, which is responsible for significant economic loss to the global dairy industry. Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with genetically distinct strains of a pathogen, whereas within-host changes in an infecting strain leading to genetically distinguishable progeny is called microevolution. The two processes can influence host-pathogen dynamics, disease progression and outcomes, but not much is known about their prevalence and impact on JD. Therefore, we obtained up to 10 MAP isolates each from 14 high-shedding animals and subjected them to whole-genome sequencing. Twelve of the 14 animals examined showed evidence for the presence of MSIs and microevolution, while the genotypes of MAP isolates from the remaining two animals could be attributed solely to microevolution. All MAP isolates that were otherwise isogenic had differences in short sequence repeats (SSRs), of which SSR1 and SSR2 were the most diverse and homoplastic. Variations in SSR1 and SSR2, which are located in ORF1 and ORF2, respectively, affect the genetic reading frame, leading to protein products with altered sequences and computed structures. The ORF1 gene product is predicted to be a MAP surface protein with possible roles in host immune modulation, but nothing could be inferred regarding the function of ORF2. Both genes are conserved in Mycobacterium avium complex members, but SSR1-based modulation of ORF1 reading frames seems to only occur in MAP, which could have potential implications on the infectivity of this pathogen. IMPORTANCE Johne's disease (JD) is a major problem in dairy animals, and concerns have been raised regarding the association of Mycobacterium avium subsp. paratuberculosis (MAP) with Crohn's disease in humans. MAP is an extremely slow-growing bacterium with low genome evolutionary rates. Certain short sequence repeats (SSR1 and SSR2) in the MAP chromosome are highly variable and evolve at a faster rate than the rest of the chromosome. In the current study, multiple MAP isolates with genetic variations such as single-nucleotide polymorphisms, and more noticeably, diverse SSRs, could simultaneously infect animals. Variations in SSR1 and SSR2 affect the products of the respective genes containing them. Since multiple MAP isolates can infect the same animal and the possibility that the pathogen undergoes further changes within the host due to unstable SSRs, this could provide a compensative mechanism for an otherwise slow-evolving pathogen to increase phenotypic diversity for overcoming host responses.

Prevalence of Borrelia burgdorferi and diversity of its outer surface protein C (ospC) alleles in blacklegged ticks (Ixodes scapularis) in Delaware

Characterizing the diversity of genes associated with virulence and transmission of a pathogen across the pathogen's distribution can inform our understanding of host infection risk. Borrelia burgdorferi is a vector-borne bacterium that causes Lyme disease in humans and is common in the United States. The outer surface protein C (ospC) gene of B. burgdorferi exhibits substantial genetic variation across the pathogen's distribution and plays a critical role in virulence and transmission in vertebrate hosts. In fact, B. burgdorferi infections that disseminate across host tissues in humans are associated with only a subset of ospC alleles. Delaware has a high incidence of Lyme disease, but the diversity of ospC in B. burgdorferi in the state has not been evaluated. We used PCR to amplify ospC in B. burgdorferi-infected blacklegged ticks (Ixodes scapularis) in sites statewide and used short-read sequencing to identify ospC alleles. B. burgdorferi prevalence in blacklegged ticks varied across sites, but not significantly so. We identified 15 previously characterized ospC alleles accounting for nearly all of the expected diversity of alleles across the sites as estimated using the Chao1 index. Nearly 40% of sequenced infections (23/58) had more than one ospC allele present suggesting mixed strain infections and the relative frequencies of alleles in single infections were positively correlated with their relative frequencies in mixed infections. Turnover of ospC alleles was positively related to distance between sites with closer sites having more similar allele compositions than more distant sites. This suggests a degree of B. burgdorferi dispersal limitation or habitat specialization. OspC alleles known to cause disseminated infections in humans were found at the highest frequencies across sites, corresponding to Delaware's high incidence of Lyme disease.

Culture-Independent Genotyping Improves Surveillance of Neisseria gonorrhoeae, Especially in Oropharyngeal Samples, the Netherlands, 2017 to 2018.

It is important i to monitor the transmission and antimicrobial resistance of Neisseria gonorrhoeae (NG). Current surveillance relies on culturing, which frequently fails. Previously, a culture-independent genotyping method was developed based on NG multi-antigen sequence typing (NG-MAST). To determine whether crucial sequence types (STs) are missed during culture-dependent surveillance, NG-positive NAAT samples were genotyped, and the results of the culture-positive and culture-negative samples were compared. In total, 196 NG-positive NAAT samples, from January 2017 until August 2018, which were also routinely cultured, were retrospectively included. Genotyping was successful in 152 NAAT samples (77.0%), 33 NAAT samples failed, and 11 NAAT samples showed possible mixed strain infections. Oropharyngeal samples (n = 16) showed the largest increase in typing rate from 6.3% (1/16) success in culture-dependent genotyping to 81.3% (13/16) in culture-independent genotyping. Nine genogroups (n ≥ 5 samples) were found; all included both culture-positive and culture-negative NG. However, culture-independent surveillance revealed 14 additional STs in the culture-negative samples. Overall, culture-dependent surveillance could detect all genogroups, indicating that major trends could be identified with culture-dependent surveillance. However, culture-independent surveillance provides more STs, mixed infections and more oropharyngeal samples, giving a more detailed view and could result in an earlier detection of outbreaks and transmission.

Transcriptomic Analysis of Liver Indicates Novel Vaccine to Porcine Reproductive and Respiratory Virus Promotes Homeostasis in T-Cell and Inflammatory Immune Responses Compared to a Commercial Vaccine in Pigs.

One of the largest impediments for commercial swine production is the presence of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a devastating RNA viral infection that is responsible for over $1 billion in loss in the U.S. annually. The challenge with combating PRRSV is a combination of the effect of an extraordinary rate of mutation, the ability to infect macrophages, and subversion of host immune response through a series of actions leading to both immunomodulation and immune evasion. Currently there are a handful of commercial vaccines on the market that have been shown to be effective against homologous infections, but struggle against heterologous or mixed strain infections. However, vaccination is the current best strategy for combating PRRSV, making research into new vaccine technology key. To address these issues with PRRSV and host antiviral functions a novel modified-live vaccine (MLV) able to stimulate known antiviral interferons was created and examined for its ability to potentiate effective immunity and better protection. Here, we examine gene expression in the liver of pigs vaccinated with our novel vaccine, given the liver's large role in antiviral responses and vaccine metabolism. Our study indicated that pigs administered the novel vaccine experience homeostatic gene expression consistent with less inflammation and T-cell depletion risk than pigs administered the commercial vaccine.

Analysis of Citrus Tristeza Virus Incidences within Asian Citrus Psyllid (Diaphorina citri) Populations in Florida via High-Throughput Sequencing.

Simple SummaryThe iconic citrus crop of Florida is suffering from an incurable disease called Huanglongbing (HLB), or citrus greening. HLB is caused by a bacterium Candidatus Liberibacter asiaticus (CLas), which is efficiently vectored by a small insect, the Asian citrus psyllid (ACP). To control the spread of CLas and ultimately HLB, citrus growers primarily depend on environmentally harmful insecticides against the ACP, which can induce insecticide resistance. An alternative, biological approach involves insect-specific viruses (ISVs) of the ACP for future exploitation to exclusively target this insect pest. To identify these ISVs, we have subjected ACPs to high-throughput sequencing, a powerful sequencing technology. This current study follows an initial detection of citrus tristeza virus (CTV) amongst the viruses detected in the ACP during HTS and continues to show the persistent and diverse presence of CTV in Florida ACPs across subsequent years. We suggest that the ACP may be utilized as a tool or lens of CTV presence throughout Florida citrus groves.The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida’s citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.

Mycobacterium tuberculosis mixed infections and drug resistance in sub-Saharan Africa: a systematic review.

Sub-Saharan Africa, is a region that records high rates of TB infection. Mycobacterium tuberculosis mixed strain infection, especially when the strains involved are of different susceptibilities, is an area of great interest because it is linked with an increased risk of treatment failure and transmission of resistant strains within the population. This paper reviewed original studies that reported MTB mixed infection and heteroresistance in the region between 2010 and 2020 to understand the extent of mixed strain infection and heteroresistance in the region. This information is very critical in the control of TB and ending the TB epidemic by 2035 as per the World Health Organization's vision. pubmed, Scopus, JSTOR, AJOL, and Google Scholar databases were searched through both key terms and subject headings. The literature was screened, assessed for the quality and evidence synthesized. Eighteen original articles were included in this review after having met the inclusion criteria. The frequency of mixed strain infection reported in these studies varied between 2.8% and 21.1% while drug resistance range between 0.06% to 19% depending on the study design and the drug susceptibility screening technique utilized. The majority of the studies (50%) utilized Spoligotyping in conjunction with MIRU-VNTR typing in the detection of mixed infections. Despite the scarcity of data on mixed infections and heteroresistance in sub-Saharan Africa, various studies have revealed that these conditions are frequent in the region than previously thought. Given the evidence of the effect of mixed infections on drug resistance and treatment outcome, we conclude that mixed infection is an unavoidable topic for future studies.

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area, a historical sleeping sickness focus of Côte d'Ivoire.

BackgroundThe existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s.MethodsFor the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations.ResultsNo HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively.DiscussionFree-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.

Common Polymorphisms in the Glycoproteins of Human Cytomegalovirus and Associated Strain-Specific Immunity.

Human cytomegalovirus (HCMV), one of the most prevalent viruses across the globe, is a common cause of morbidity and mortality for immunocompromised individuals. Recent clinical observations have demonstrated that mixed strain infections are common and may lead to more severe disease progression. This clinical observation illustrates the complexity of the HCMV genome and emphasizes the importance of taking a population-level view of genotypic evolution. Here we review frequently sampled polymorphisms in the glycoproteins of HCMV, comparing the variable regions, and summarizing their corresponding geographic distributions observed to date. The related strain-specific immunity, including neutralization activity and antigen-specific cellular immunity, is also discussed. Given that these glycoproteins are common targets for vaccine design and anti-viral therapies, this observed genetic variation represents an important resource for future efforts to combat HCMV infections.

Nonparametric Binary Classification to Distinguish Closely Related versus Unrelated Plasmodium falciparum Parasites.

ABSTRACTAssessing genetic relatedness of Plasmodium falciparum genotypes is a key component of antimalarial efficacy trials. Previous methods have focused on determining a priori definitions of the level of genetic similarity sufficient to classify two infections as sharing the same strain. However, factors such as mixed-strain infections, allelic suppression, imprecise typing methods, and heterozygosity complicate comparisons of apicomplexan genotypes. Here, we introduce a novel method for nonparametric statistical testing of relatedness for P. falciparum parasites. First, the background distribution of genetic distance between unrelated strains is computed. Second, a threshold genetic distance is computed from this empiric distribution of distances to demarcate genetic distances that are unlikely to have arisen by chance. Third, the genetic distance between paired samples is computed, and paired samples with genetic distances below the threshold are classified as related. The method is designed to work with any arbitrary genetic distance definition. We validated this procedure using two independent approaches to calculating genetic distance. We assessed the concordance of the novel nonparametric classification with a gold-standard Bayesian approach for 175 pairs of recurrent P. falciparum episodes from previously published malaria efficacy trials with microsatellite data from five studies in Guinea and Angola. The novel nonparametric approach was 98% sensitive and 84–89% specific in correctly identifying related genotypes compared with a gold-standard Bayesian algorithm. The approach provides a unified and systematic method to statistically assess relatedness of P. falciparum parasites using arbitrary genetic distance methodologies.

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into the Ogawa and Inaba serotypes. The O-antigen is V. cholerae's immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogeneous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germfree mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single- and mixed-strain infection assays, we found that all three HaitiV variants-InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa)-were immunogenic and protective. None of the vaccine serotypes were superior across both of these vaccine metrics, suggesting that the impact of O1-serotype variation in OCV design, although detectable, is subtle. However, all three live vaccines significantly outperformed formalin-killed HikoV, supporting the idea that live OCV usage will bolster current cholera control practices. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Effect of mixed strain infections on clinical and epidemiological features of tuberculosis in Florida.

Mixed infections with genetically distinct Mycobacterium tuberculosis (MTB) strains within a single host have been documented in different settings; however, this phenomenon is rarely considered in the management and care of new and relapse tuberculosis (T.B.) cases. This study aims to establish the epidemiological and clinical features of mixed infections among culture-confirmed T.B. patients enrolled in tuberculosis care at the Florida Department of Health (FDOH) and measure its association with T.B. mortality. We analyzed de-identified surveillance data of T.B. cases enrolled in T.B. care from April 2008 to January 2018. Mixed MTB infection was determined by the presence of more than one Copy Number Variant (CNV) in at least one locus, based on the genotype profile pattern of at least one isolate using 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR). The prevalence of mixed MTB infections among the 4944 culture-confirmed TB cases included in this analysis was 2.6% (129). Increased odds of mixed infections were observed among middle-aged patients, 45-64years (AOR=2.38; 95% CI: 0.99, 5.69; p=0.0513), older adults 65years and above (AOR=3.95; 95% CI: 1.63, 9.58; p=0.0023) and patients with diabetes (OR=1.77; 95% CI: 1.12, 2.80; p=0.0150). There was no significant association between mixed infections and death. Our study provides insight into the epidemiological and clinical characteristics of patients with mixed MTB infections, which is essential in the management of T.B. patients.

Pseudomonas aeruginosa Strain-sharing in Early Infection Among Children With Cystic Fibrosis.

We previously identified Pseudomonas aeruginosa isolates with characteristics typical of chronic infection in some early infections in children with cystic fibrosis (CF), suggesting that these isolates may have been acquired from other patients. Our objective was to define the extent of P. aeruginosa strain-sharing in early CF infections and its impact on antibiotic eradication treatment failure rates. We performed whole genome sequencing on isolates from early pediatric CF pulmonary infections and from the following comparator groups in the same hospital: chronic CF infection, sink drains, sterile site infections, and asymptomatic carriage. Univariate logistic regression was used to assess factors associated with treatment failure. In this retrospective, observational study, 1029 isolates were sequenced. The CF clones strain B and clone C were present. In 70 CF patients with early infections, 14 shared strains infected 29 (41%) patients over 5 years; 16% (n = 14) of infections had mixed strains. In the 70 children, approximately one-third of shared-strain infections were likely due to patient-to-patient transmission. Mixed-strain infections were associated with strain-sharing (odds ratio, 8.50; 95% confidence interval, 2.2-33.4; P = .002). Strain-sharing was not associated with antibiotic eradication treatment failure; however, nosocomial strain transmission was associated with establishment of chronic infection in a CF sibling pair. Although early P. aeruginosa CF infection is thought to reflect acquisition of diverse strains from community reservoirs, we identified frequent early CF strain-sharing that was associated with the presence of mixed strains and instances of possible patient-to-patient transmission.

Genetic diversity and genotype multiplicity of Plasmodium falciparum infection in patients with uncomplicated malaria in\xa0Chewaka district, Ethiopia

BackgroundGenetic diversity in Plasmodium falciparum poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity of P. falciparum strains can be used to assess intensity of parasite transmission and identify potential deficiencies in malaria control programmes, which provides vital information to evaluating malaria elimination efforts. This study investigated the P. falciparum genetic diversity and genotype multiplicity of infection in parasite isolates from cases with uncomplicated P. falciparum malaria in Southwest Ethiopia.MethodsA total of 80 P. falciparum microscopy and qPCR positive blood samples were collected from study participants aged 6 months to 60 years, who visited the health facilities during study evaluating the efficacy of artemether-lumefantrine from September–December, 2017. Polymorphic regions of the msp-1 and msp-2 were genotyped by nested polymerase chain reactions (nPCR) followed by gel electrophoresis for fragment analysis.ResultsOf 80 qPCR-positive samples analysed for polymorphisms on msp-1 and msp-2 genes, the efficiency of msp-1 and msp-2 gene amplification reactions with family-specific primers were 95% and 98.8%, respectively. Allelic variation of 90% (72/80) for msp-1 and 86.2% (69/80) for msp-2 were observed. K1 was the predominant msp-1 allelic family detected in 20.8% (15/72) of the samples followed by MAD20 and RO33. Within msp-2, allelic family FC27 showed a higher frequency (26.1%) compared to IC/3D7 (15.9%). Ten different alleles were observed in msp-1 with 6 alleles for K1, 3 alleles for MAD20 and 1 allele for RO33. In msp-2, 19 individual alleles were detected with 10 alleles for FC27 and 9 alleles for 3D7. Eighty percent (80%) of isolates had multiple genotypes and the overall mean multiplicity of infection was 3.2 (95% CI 2.87–3.46). The heterozygosity indices were 0.43 and 0.85 for msp-1 and msp-2, respectively. There was no significant association between multiplicity of infection and age or parasite density.ConclusionsThe study revealed high levels of genetic diversity and mixed-strain infections of P. falciparum populations in Chewaka district, Ethiopia, suggesting that both endemicity level and malaria transmission remain high and that strengthened control efforts are needed in Ethiopia.

Prediction of the hidden genotype of mixed infection strains in Iranian tuberculosis patients

BackgroundPatients with mixed-strain Mycobacterium tuberculosis infections may be at a high risk of poor treatment outcomes. However, the mechanisms through which mixed infections affect the clinical manifestations are not well recognized. Evidence suggests that failure to detect the pathogen diversity within the host can influence the clinical results. We aimed to investigate the effects of different genotypes in mixed infections and determine their relationship with heteroresistance in the treatment of Iranian tuberculosis patients. MethodsOne of the genotypes was identified in the culture and another genotype pattern in the mixed infection was predicted by comparing the pattern of MIRU-VNTR between the clinical specimens and their respective cultures in each patient. For all patients, the drug susceptibility testing was carried out on three single colonies from each clinical sample. The follow-up of patients was carried out during six months of treatment. ResultsBased on MIRU-VNTR profiles of clinical samples, we showed that 55.6% (25/45) of the Iranian patients included in the study had mixed infections. Patients with mixed infections had a higher rate of treatment failure, compared to others (P=0.03). By comparing clinical sample profiles to profiles obtained after culture, we were able to distinguish between major and hidden genotypes. Among hidden genotypes, Haarlem (L4.1.2) and Beijing (L2) were associated to treatment failure (6/8 patients). ConclusionsTo conclude, we propose a procedure using the MIRU-VNTR method to identify the different genotypes in mixed infections. The present findings suggest that genotypes with potentially higher pathogenicity may not be detected when performing experimental culture in patients with mixed infections.

Differential Responses of Tobacco to the Citrus Variegated Chlorosis and Coffee Stem Atrophy Strains of Xylella fastidiosa.

Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. No mixed infections were detected. In two experiments, the citrus and coffee strains infected only their original hosts as well as tobacco. The usefulness of this tobacco bioassay as a tool to study X. fastidiosa spread was demonstrated. It provided evidence that, over the years, the CVC and CSA pathogens have remained limited to their original hosts, despite crop proximity and the presence of sharpshooter vectors that favor transmission of the bacteria to and between both host species.

Genome sequencing of Mycobacterium pinnipedii strains: genetic characterization and evidence of superinfection in a South American sea lion (Otaria flavescens)

BackgroundMycobacterium pinnipedii, a member of the Mycobacterium tuberculosis Complex (MTBC), is capable of infecting several host species, including humans. Recently, ancient DNA from this organism was recovered from pre-Columbian mummies of Peru, sparking debate over the origin and frequency of tuberculosis in the Americas prior to European colonization.ResultsWe present the first comparative genomic study of this bacterial species, starting from the genome sequencing of two M. pinnipedii isolates (MP1 and MP2) obtained from different organs of a stranded South American sea lion. Our results indicate that MP1 and MP2 differ by 113 SNPs (single nucleotide polymorphisms) and 46 indels, constituting the first report of a mixed-strain infection in a sea lion. SNP annotation analyses indicate that genes of the VapBC family, a toxin-antitoxin system, and genes related to cell wall remodeling are under evolutionary pressure for protein sequence change in these strains. OrthoMCL analysis with seven modern isolates of M. pinnipedii shows that these strains have highly similar proteomes. Gene variations were only marginally associated with hypothetical proteins and PE/PPE (proline-glutamate and proline-proline-glutamate, respectively) gene families. We also detected large deletions in ancient and modern M. pinnipedii strains, including a few occurring only in modern strains, indicating a process of genome reduction occurring over the past one thousand years. Our phylogenomic analyses suggest the existence of two modern clusters of M. pinnipedii associated with geographic location, and possibly host species, and one basal node associated with the ancient M. pinnipedii strains. Previously described MiD3 and MiD4 deletions may have occurred independently, twice, over the evolutionary course of the MTBC.ConclusionThe presence of superinfection (i.e. mixed-strain infection) in this sea lion suggests that M. pinnipedii is highly endemic in this population. Mycobacterium pinnipedii proteomes of the studied isolates showed a high degree of conservation, despite being under genomic decay when compared to M. tuberculosis. This finding indicates that further genomes need to be sequenced and analyzed to increase the chances of finding variably present genes among strains or that M. pinnipedii genome remodeling occurred prior to bacterial speciation.

Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.

Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.