Novel detection of Leishmania RNA virus-1 (LRV-1) in clinical isolates of Leishmania Viannia panamensis.

American tegumentary leishmaniasis comprises a discrete set of clinical presentations endemic to Latin America. Leishmania RNA virus-1 (LRV-1) is a double-stranded RNA virus identified in 20­25% of the Leishmania Viannia braziliensis and L. V. guyanensis, however not in L. V. panamensis. This is the first report of LRV-1 in L. V. panamensis and its associations with clinical phenotypes of ATL. Unique surplus discard clinical isolates of L. V. panamensis were identified from the Public Health Ontario Laboratory (PHOL) and the Leishmania Clinic of the Instituto de Medicina Tropical 'Alexander von Humboldt' between 2012 and 2019 and screened for LRV-1 by real-time polymerase chain reaction. Patient isolates were stratified according to clinical phenotype. Of 30 patients with L. V. panamensis, 14 (47%) and 16 (53%) patients had severe and non-severe ATL, respectively. Five (36%) of 14 severe cases and 2 (12%) of 16 non-severe cases were positive for LRV-1, respectively. No differences in sex were observed for clinical phenotype and LRV-1 status. Although an association between LRV-1 status and clinical phenotype was not demonstrated, this is the first description of the novel detection of LRV-1 in L. V. panamensis, a species that has been documented predominantly in Central America.

Genomic Surveillance of Recent Dengue Outbreaks in Colombo, Sri Lanka.

All four serotypes of the dengue virus (DENV1-4) cause a phenotypically similar illness, but serial infections from different serotypes increase the risk of severe disease. Thus, genomic surveillance of circulating viruses is important to detect serotype switches that precede community outbreaks of disproportionate magnitude. A phylogenetic analysis was conducted on near full length DENV genomes sequenced from serum collected from a prospective cohort study from the Colombo district, Sri Lanka during a 28-month period using Oxford nanopore technology, and the consensus sequences were analyzed using maximum likelihood and Bayesian evolutionary analysis. From 523 patients, 328 DENV sequences were successfully generated (DENV1: 43, DENV2: 219, DENV3:66). Most circulating sequences originated from a common ancestor that was estimated to have existed from around 2010 for DENV2 and around 2015/2016 for DENV1 and DENV3. Four distinct outbreaks coinciding with monsoon rain seasons were identified during the observation period mostly driven by DENV2 cosmopolitan genotype, except for a large outbreak in 2019 contributed by DENV3 genotype I. This serotype switch did not result in a more clinically severe illness. Phylogeographic analyses showed that all outbreaks started within Colombo city and then spread to the rest of the district. In 2019, DENV3 genotype I, previously, rarely reported in Sri Lanka, is likely to have contributed to a disease outbreak. However, this did not result in more severe disease in those infected, probably due to pre-existing DENV3 immunity in the community. Targeted vector control within Colombo city before anticipated seasonal outbreaks may help to limit the geographic spread of outbreaks.

Comparación de la eficacia y toxicidad del estibogluconato de sodio y antimoniato de meglumina en el tratamiento de leishmaniasis cutánea en Perú / Efficacy and safety of sodium stibogluconate compared to meglumine antimoniate in the treatment of cutaneous leishmaniasis in Peru

Objetivos : Comparar la eficacia y toxicidad del antimoniato de meglumina (AM) y estibogluconato sódico (EGS) en el tratamiento de leishmaniasis cutánea (LC) en un hospital general. Material y métodos : Serie de casos comparativa de 193 pacientes con LC tratados en tres ensayos clínicos con AM (n=69) y EGS (n=124) durante 2001-2010. La administración de ambas drogas fue vía endovenosa lenta de 20 mg Sb5+/kg/día por 20 días consecutivos siguiendo las normativas de la OPS y OMS. La información clínica, toxicidad y eficacia fue obtenida de las historias clínicas almacenadas en el centro de investigación según la normativa local e internacional. Resultados : Las características demográficas fueron similares entre grupos, pero el tamaño y número de lesiones fueron mayores en el grupo AM. La eficacia del tratamiento con AM fue 76,0% versus 68,4% con EGS (p=0,340) y 55,1% versus 50,8% (p=0,570) en el análisis por protocolo y de intención de tratar, respectivamente. No se observaron efectos adversos inmediatos. Los síntomas más frecuentemente reportados fueron disgeusia (37,0%), mareos (32,0%), cefalea (36,0%), artralgias (31,0%) y linfangitis (21,0%). Los tres primeros síntomas, así como elevación de transaminasas, leucopenia, trombocitopenia y QTc prolongado fueron frecuentes en el grupo EGS, pero clínica y estadísticamente no significativos. El tratamiento fue suspendido definitivamente por toxicidad severa únicamente con EGS por emesis refractaria (2 participantes) y QTc prolongado con extrasístoles (1 participante). Conclusiones : La eficacia del tratamiento con AM y EGS fue comparable. La administración endovenosa de ambos no produjo efectos adversos inmediatos, aunque sí alteraciones clínicas y laboratoriales usuales.

SUMMARY Objectives : To compare the efficacy and safety of sodium stibogluconate (SS) and meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL) in a general hospital. Methods: Case-series of 193 patients with CL treated in three clinical trials with MA (n=69) and SS (n=124) during 2001-2010. Both study drugs were administered intravenously at a slow speed at 20 mg Sb5+/kg/day for 20 consecutive days following WHO-PAHO recommendations. Clinical and safety data were gathered from clinical files. Results: Demographic characteristics were similar between the study groups, but the size and number of lesions were higher in the MA group. Efficacy was 76.0% in the MA vs. 68.4% in the SS group (p=0.340) and 55.1% vs. 50.8% (p=0.570) in the per protocol and intention to treat analysis. respectively. Side effects more frequently reported were dysgeusia (37.0%). dizziness (32.0%). headache (36.0%). arthralgia (31.0%) and lymphangitis (21.0%). These first three symptoms as well as elevation of transaminases, leukopenia, thrombocytopenia and prolonged QTc were numerically more frequent in the SS group but without reaching statistical significance. Treatment was stopped definitively for severe toxicity in the SS group due to refractory emesis (two patients) and prolonged QTc (one patient). Conclusions: The efficacy of MA and SS is comparable. The intravenous administration of these compounds did not produce immediate reactions, but it was associated with unusual clinical and laboratory abnormalities.

Interventions to enhance testing and linkage to treatment for hepatitis C infection for people who inject drugs: A systematic review and meta-analysis.

BACKGROUND: With the advent of direct acting antiviral (DAA) therapies for the treatment of hepatitis C virus (HCV), the World Health Organization recommended a goal to eliminate HCV as a public health threat globally by 2030. With the majority of new and existing infections in high income countries occurring among people who inject drugs, achieving this goal will require the design and implementation of interventions which address the unique barriers to HCV care faced by this population. METHODS: In this systematic review and meta-analysis, we searched bibliographic databases and conference abstracts to July 21, 2020 for studies assessing interventions to improve the following study outcomes: HCV antibody testing, HCV RNA testing, linkage to care, and treatment initiation. We included both randomised and non-randomised studies which included a comparator arm. We excluded studies which enrolled only paediatric populations (<18 years old) and studies where the intervention was conducted in a different healthcare setting than the control or comparator. This analysis was restricted to studies conducted among people who inject drugs. Data were extracted from the identified records and meta-analysis was used to pool the effect of interventions on study outcomes. This study was registered in PROSPERO (CRD42020178035). FINDINGS: Of 15,342 unique records, 45 studies described the implementation of an intervention to improve HCV testing, linkage to care and treatment initiation among people who inject drugs. These included 27 randomised trials and 18 non-randomised studies with the risk of bias rated as "critical" for most non-randomised studies. Patient education and patient navigation to address patient-level barriers to HCV care were shown to improve antibody testing uptake and linkage to HCV care respectively although patient education did not improve antibody testing when restricted to randomised studies. Provider care coordination to address provider level barriers to HCV care was effective at improving antibody testing uptake. Three different interventions to address systems-level barriers to HCV care were effective across different stages of HCV care: point-of-care antibody testing (linkage to care); dried blood-spot testing (antibody testing uptake); and integrated care (linkage to care and treatment initiation). INTERPRETATION: Multiple interventions are available that can address the barriers to HCV care for people who inject drugs at the patient-, provider-, and systems-level. The design of models of care to improve HCV testing and treatment among people who inject drugs must consider the unique barriers to care that this population faces. Further research, including high-quality randomised controlled trials, are needed to robustly assess the impact these interventions can have in varied populations and settings.

Effect of prior Zika and dengue virus exposure on the severity of a subsequent dengue infection in adults.

Given the structural similarity between Zika and dengue viruses, prior infection from one virus is hypothesized to modulate the severity of a subsequent infection from the other virus. A previous paediatric cohort study observed that a prior Zika infection may increase the risk of a subsequent symptomatic or severe dengue infection. The Colombo Dengue study is a prospective hospital-based cohort study in Sri Lanka that recruits symptomatic adult dengue patients within the first three days of fever. Anti-Dengue Envelope and anti-Zika NS1 IgG antibodies were tested by ELISA (Euroimmun, Lubeck, Germany) in all recruited patients. Associations between pre-morbid seroprevalence for either or both infections and adverse clinical outcomes of the current dengue infection were explored. A total of 507 dengue infected patients were assessed of whom 342 (68%) and 132 (26%) patients had anti-dengue IgG and anti-Zika IgG respectively. People with combined prior dengue and zika exposure as well as prior dengue exposure alone, were at increased risk of plasma leakage, compensated and uncompensated shock, and severe dengue (p < 0·05), compared to people without prior exposure to either infection. The effect of prior Zika exposure alone could not be established due to the small the number of primary dengue infections with prior Zika exposure.

Predictors of Chronic Fatigue Syndrome and Mood Disturbance After Acute Infection.

Prospective cohort studies following individuals from acute infections have documented a prevalent post-infective fatigue state meeting diagnostic criteria for chronic fatigue syndrome (CFS) - that is, a post-infective fatigue syndrome (PIFS). The Dubbo Infection Outcomes Study (DIOS) was a prospective cohort following individuals from acute infection with Epstein-Barr virus (EBV), Ross River virus (RRV), or Q fever through to assessment of caseness for CFS designated by physician and psychiatrist assessments at 6 months. Previous studies in DIOS have revealed that functional genetic polymorphisms in both immunological (pro- and anti-inflammatory cytokines) and neurological (the purinergic receptor, P2X7) genes are associated with both the severity of the acute infection and subsequent prolonged illness. Principal components analysis was applied to self-report data from DIOS to describe the severity and course of both the overall illness and concurrent mood disturbance. Associations between demographics and acute infection characteristics, with prolonged illness course as well as the PIFS outcome were examined using multivariable statistics. Genetic haplotype-driven functional variations in the neuropeptide Y (NPY) gene previously shown to be associated with brain responses to stress, and to trait anxiety were also examined as predictors. The sample included 484 subjects (51% female, median age 32, IQR 19-44), of whom 90 (19%) met diagnostic criteria for CFS at 6 months. Participants with greater overall illness severity and concurrent mood disturbance in the acute illness had a more prolonged illness severity (HR = 0.39, 95% CI: 0.34-0.46, p < 0.001) and mood disturbance (HR = 0.36, 95% CI: 0.30-0.42, p < 0.001), respectively. Baseline illness severity and RRV infection were associated with delayed recovery. Female gender and mood disturbance in the acute illness were associated with prolonged mood disturbance. Logistic regression showed that the odds of an individual being diagnosed with PIFS increased with greater baseline illness severity (OR = 2.24, 95% CI: 1.71-2.94, p < 0.001). There was no association between the NPY haplotypes with overall illness severity or mood disturbance either during the acute illness phase or with prolonged illness (p > 0.05). Severe acute infective illnesses predicted prolonged illness, prolonged mood disturbance and PIFS. These factors may facilitate early intervention to manage both PIFS and mood disturbances.

Elevated baseline expression of seven virulence factor RNA transcripts in visceralizing species of Leishmania: a preliminary quantitative PCR study.

Introduction: Leishmaniasis is a neglected tropical disease that manifests as three major disease phenotypes: cutaneous, mucocutaneous, and visceral. In this preliminary study, we quantified virulence factor (VF) RNA transcript expression in Leishmania species, stratified by geographic origin and propensity for specific disease phenotypes. Methods: Cultured promastigotes of 19 Leishmania clinical and ATCC isolates were extracted for total cellular RNA, cDNA was reverse transcribed, and qPCR assays were performed to quantify VF RNA transcript expression for hsp23, hsp70, hsp83, hsp100, mpi, cpb, and gp63. Results: Comparison of visceralizing species (Leishmania donovani, Leishmania chagasi, and Leishmania infantum) versus non-visceralizing species [Leishmania (Viannia) spp., Leishmania tropica, Leishmania major, Leishmania mexicana, and Leishmania amazonensis] revealed a significantly greater pooled transcript expression for visceralizing species (p = 0.0032). Similarly, Old World species demonstrated significantly higher VF RNA transcript expression than New World species (p = 0.0015). On a per-gene basis, species with a propensity to visceralize ubiquitously expressed higher levels of gp63 (p = 0.005), cpb (p = 0.0032), mpi (p = 0.0032), hsp23 (p = 0.0039), hsp70 (p = 0.0032), hsp83 (p = 0.0032), and hsp100 (p = 0.0032). Conclusion: Here, we provide quantitative, preliminary evidence of elevated VF RNA transcript expression driven largely by the visceralizing causative species of Leishmania. This work highlights the extensive heterogeneity in pathogenicity mechanisms between Leishmania species, which may partly underpin the fatal progression of visceral leishmaniasis.

Interventions to enhance testing, linkage to care, and treatment initiation for hepatitis C virus infection: a systematic review and meta-analysis.

BACKGROUND: Despite the goal set by WHO to eliminate hepatitis C virus (HCV) as a public health threat, uptake of HCV testing and treatment remains low. To achieve this target, evidence-based interventions are needed to address the barriers to care for people with, or at risk of, HCV infection. We aimed to assess the efficacy of interventions to improve HCV antibody testing, HCV RNA testing, linkage to HCV care, and treatment initiation. METHODS: In this systematic review and meta-analysis, we searched MEDLINE (PubMed), Scopus, Web of Science, the Cochrane Central Register of Controlled Trials, and PsycINFO without language restrictions for reports published between database inception and July 21, 2020, assessing the following primary outcomes: HCV antibody testing; HCV RNA testing; linkage to HCV care; and direct-acting antiviral treatment initiation. We also searched key conference abstracts. We included randomised and non-randomised studies assessing non-pharmaceutical interventions that included a comparator or control group. Studies were excluded if they enrolled only paediatric populations (aged <18 years) or if they conducted the intervention in a different health-care setting to that of the control or comparator. Authors were contacted to clarify study details and to obtain additional population-level data. Data were extracted from the records identified into a pre-piloted and standardised data extraction form and a random-effects meta-analysis was used to pool the effects of the interventions on study outcomes. This study is registered in PROSPERO, CRD42020178035. FINDINGS: Of 15 342 unique records identified, 142 were included, which reported on 148 unique studies (47 randomised controlled trials and 101 non-randomised studies). Medical chart reminders, provider education, and point-of-care antibody testing significantly improved at least three study outcomes compared with a comparator or control. Interventions that simplified HCV testing, including dried blood spot testing, point-of-care antibody testing, reflex RNA testing, and opt-out screening, significantly improved testing outcomes compared with a comparator or control. Enhanced patient and provider support through patient education, provider care coordination, and provider education also significantly improved testing outcomes compared with a comparator or control. Integrated care and patient navigation or care coordination significantly improved linkage to care and the uptake of direct-acting antiviral treatment compared with a comparator or control. INTERPRETATION: Several interventions to improve HCV care that address several key barriers to HCV care were identified. New models of HCV care must be designed and implemented to address the barriers faced by the population of interest. Further high-quality research, including rigorously designed randomised studies, is still needed in key populations. FUNDING: None.

A phase II multicenter randomized study to evaluate the safety and efficacy of combining thermotherapy and a short course of miltefosine for the treatment of uncomplicated cutaneous leishmaniasis in the New World.

BACKGROUND: Systemic pentavalent antimonials, mainly meglumine antimoniate, continue to be the first-choice drugs for treatment of cutaneous leishmaniasis (CL) despite their toxicity, difficulty of administration and high cost. In the search for therapeutic alternatives, combining two treatment interventions has emerged as a potential alternative to either reduce the use of antimonials with the associated toxicities, or to increase efficacy. Here, we report the results of a recently completed trial assessing the efficacy and safety of a combination of thermotherapy (TT) plus a short course of miltefosine (MLT) for the treatment of uncomplicated CL in Colombia and Peru. METHODS: A multicenter, randomized, evaluator-blinded, phase II, controled clinical trial was conducted. Adult volunteers with a parasitologically confirmed diagnosis of uncomplicated CL were randomly allocated to receive either a single session of TT or a combination of TT plus a short course of MLT (3 weeks). Therapeutic response outcomes and safety were assessed. RESULTS: 130 subjects were included in the study, of whom 64 were randomly assigned to the TT arm and 66 to the TT + MLT arm. Cure at 3 months' follow-up was achieved in 57.8% (n = 37) and 80.3% (n = 53) in the TT and TT + MLT groups, respectively, in the intention to treat analysis. The TT + MLT regimen was better that TT alone (p = 0.0055). The presence of vesicles at the site of heat application was the most common adverse event reported associated with the use of TT; while vomiting (31.8%) and elevation of liver enzymes (28.8%) were the most frequent adverse events reported associated with the use of MLT. CONCLUSION: The combination of TT plus a short course of MLT was shown to be significantly better than TT alone for the treatment of uncomplicated CL in the New World. TRIAL REGISTRATION: Registered in clinicaltrials.gov NCT02687971.

Clinical and Public Health Implications of Human T-Lymphotropic Virus Type 1 Infection.

Human T-lymphotropic virus type 1 (HTLV-1) is estimated to affect 5 to 10 million people globally and can cause severe and potentially fatal disease, including adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The burden of HTLV-1 infection appears to be geographically concentrated, with high prevalence in discrete regions and populations. While most high-income countries have introduced HTLV-1 screening of blood donations, few other public health measures have been implemented to prevent infection or its consequences. Recent advocacy from concerned researchers, clinicians, and community members has emphasized the potential for improved prevention and management of HTLV-1 infection. Despite all that has been learned in the 4 decades following the discovery of HTLV-1, gaps in knowledge across clinical and public health aspects persist, impeding optimal control and prevention, as well as the development of policies and guidelines. Awareness of HTLV-1 among health care providers, communities, and affected individuals remains limited, even in countries of endemicity. This review provides a comprehensive overview on HTLV-1 epidemiology and on clinical and public health and highlights key areas for further research and collaboration to advance the health of people with and at risk of HTLV-1 infection.

Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.

Evaluation of a point-of-care molecular detection device for Leishmania spp. and intercurrent fungal and mycobacterial organisms in Peruvian patients with cutaneous ulcers.

PURPOSE: Overlapping clinical features of cutaneous leishmaniasis (CL) with ulcers caused by fungi and mycobacteria necessitate confirmatory diagnostic testing. We evaluated a handheld battery-operated device for detection of CL and common fungal and mycobacterial causes of ulcers. METHODS: We validated Palm PCR™ for detection of common ulcerative skin pathogens using ATCC® reference and clinical strains of Leishmania, mycobacteria, and fungi in the lab and field. Amplified products were Sanger sequenced. Performance characteristics were calculated using conventional PCR as a reference standard. RESULTS: Palm PCR™ detected 100% of ATCC® strains of Leishmania, fungi, and mycobacteria, with sensitivity and specificity of 90% and 91.7%, respectively. In the field, the sensitivity for detection of Leishmania in patients with suspected CL was 100%. In 61% of CL patients, co-colonization with genera such as Malassezia, Aspergillus, Candida, and Cladosporium was detected. In 50% of CL patients with an inflammatory (secondarily infected) phenotype, detected fungal species had known associations with human cutaneous disease. CONCLUSIONS: Palm PCR™ performs comparably to conventional PCR for detection of Leishmania, fungi, and mycobacteria. This work has implications for the diagnostic approach to tropical ulcers, and has the potential to improve field detection of ulcerative pathogens in resource constrained areas.

Comparison of Whole Genome Sequencing versus Standard Molecular Diagnostics for Species Identification in the Leishmania Viannia Subgenus.

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.

The severity of the pathogen-induced acute sickness response is affected by polymorphisms in genes of the NLRP3 inflammasome pathway.

The acute sickness response (ASR) is a stereotyped set of symptoms including fatigue, pain, and disturbed mood, which are present in most acute infections. The immunological mechanisms of the ASR are conserved, with variations in severity determined partly by the pathogen, but also by polymorphisms in host genes. The ASR was characterised in three different serologically-confirmed acute infections in Caucasians (n = 484) across four symptom domains or endophenotypes (termed 'Fatigue', 'Musculoskeletal pain', 'Mood disturbance', and 'Acute sickness'). Correlations were sought with functional single nucleotide polymorphisms in the NLRP3 inflammasone pathway and severity of the endophenotypes. Individuals with severe Fatigue, Musculoskeletal pain, or Mood endophenotypes were more likely to have prior episodes of significant fatigue (11.4 vs. 3.8%, p = 0.07), pain (14.3 vs. 1.2%, p = 0.001), or Mood disturbance (13 vs 1%, p=0.001), suggesting trait characteristics. The high functioning allele of the rs35829419 SNP in NLRP3 was more common in those with severe Fatigue (OR = 13.3, 95% CI: 1.7-104), particularly in a dominant inheritance pattern (OR = 13.4, 95% CI: 1.8-586.3). In a multivariable analysis assuming dominant inheritance, both rs35829419 and the rs4848306 SNP in Interleukin(IL)-1ß, were independently associated with severe Fatigue (OR = 29.6, 95% CI: 2.6-330.9 and OR = 13, 95% CI: 2.7-61.8, respectively). The severity of fatigue in acute infection is influenced by genetic polymorphisms in NLRP3 and IL-1ß.

Single molecule, near full-length genome sequencing of dengue virus.

Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.

Leishmania RNA Virus 1 (LRV-1) in Leishmania (Viannia) braziliensis Isolates from Peru: A Description of Demographic and Clinical Correlates.

RNA virus 1-1 (LRV-1-1) is a dsRNA virus identified in isolates of Leishmania (Viannia) braziliensis and thought to advance localized cutaneous leishmaniasis (LCL) to mucocutaneous or mucosal leishmaniasis (MCL/ML). We examined the prevalence of LRV-1 and its correlation to phenotypes of American tegumentary leishmaniasis caused by L. (V.) braziliensis from Peru to better understand its epidemiology. Clinical isolates of L. (V.) braziliensis were screened for LRV-1 by real-time polymerase chain reaction (PCR) and stratified according to the phenotype: LCL (< 4 ulcers in number) MCL/ML; inflammatory ulcers (erythematous, purulent, painful ulcers with or without lymphatic involvement) or multifocal ulcers (≥ 4 in ≥ 2 anatomic sites). Proportionate LRV-1 positivity was compared across phenotypes. Of 78 L. (V.) braziliensis isolates, 26 (54.2%) had an inflammatory phenotype, 22 (28%) had the MCL/ML phenotype, whereas 30 (38.5%) had LCL. Mucocutaneous or mucosal leishmaniasis was found exclusively in adult male enrollees. Leishmania RNA virus 1 positivity by phenotype was as follows: 9/22 (41%) with MCL/ML; 5/26 (19%) with an inflammatory/multifocal cutaneous leishmaniasis phenotype; and 7/30 (23%) with LCL (P = 0.19). Leishmania RNA virus 1 positivity was not associated with age (P = 0.55) or gender (P = 0.49). Relative LRV-1 copy number was greater in those with MCL/ML than those with inflammatory/multifocal CL (P = 0.02). A direct association between LRV-1 status and clinical phenotype was not demonstrated; however, relative LRV-1 copy number was highest in those with MCL/ML. Future analyses to understand the relationship between viral burden and pathogenesis are required to determine if LRV-1 is truly a contributor to the MCL/ML phenotype.

Virulence factor RNA transcript expression in the Leishmania Viannia subgenus: influence of species, isolate source, and Leishmania RNA virus-1.

BACKGROUND: Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus identified in 20-25% of Viannia-species endemic to Latin America, and is believed to accelerate cutaneous to mucosal leishmaniasis over time. Our objective was to quantify known virulence factor (VF) RNA transcript expression according to LRV1 status, causative species, and isolate source. METHODS: Eight cultured isolates of Leishmania were used, four of which were LRV1-positive (Leishmania Viannia braziliensis [n = 1], L. (V.) guyanensis [n = 1], L. (V.) panamensis [n = 2]), and four were LRV1-negative (L. (V.) panamensis [n = 3], L. (V.) braziliensis [n = 1]). Promastigotes were inoculated into macrophage cultures, and harvested at 24 and 48 h. RNA transcript expression of hsp23, hsp70, hsp90, hsp100, mpi, cpb, and gp63 were quantified by qPCR. RESULTS: RNA transcript expression of hsp100 (p = 0.012), cpb (p = 0.016), and mpi (p = 0.022) showed significant increases from baseline pure culture expression to 24- and 48-h post-macrophage infection, whereas hsp70 (p = 0.004) was significantly decreased. A trend toward increased transcript expression of hsp100 at baseline in isolates of L. (V.) panamensis was noted. Pooled VF RNA transcript expression by L. (V.) panamensis isolates was lower than that of L. (V.) braziliensis and L. (V.) guyananesis at 24 h (p = 0.03). VF RNA transcript expression did not differ by LRV1 status, or source of cultured isolate at baseline, 24, or 48 h; however, a trend toward increased VF RNA transcript expression of 2.71- and 1.93-fold change of mpi (p = 0.11) and hsp90 (p = 0.11), respectively, in LRV1 negative isolates was noted. Similarly, a trend toward lower levels of overall VF RNA transcript expression in clinical isolates (1.15-fold change) compared to ATCC® strains at 24 h was noted (p = 0.07). CONCLUSIONS: Our findings suggest that known VF RNA transcript expression may be affected by the process of macrophage infection. We were unable to demonstrate definitively that LRV-1 presence affected VF RNA transcript expression in the species and isolates studied. L. (V.) guyanensis and L. (V.) braziliensis demonstrated higher pooled VF RNA transcript expression than L. (V.) panamensis; however, further analyses of protein expression to corroborate this finding are warranted.

Regulatory T-Cell Dynamics in Cutaneous and Mucocutaneous Leishmaniasis due to Leishmania braziliensis.

To evaluate the dynamics of regulatory T cells (Tregs) during tegumentary leishmaniasis, we assessed peripheral blood and biopsies from 54 patients. Patients with cutaneous leishmaniasis (CL) had a decreased proportion of Tregs in the peripheral blood, but the proportion was higher in the biopsies of lesions. During treatment of CL, circulating Tregs increased reaching normal proportions, whereas antigen-specific interferon-γ responses diminished. By contrast, circulating Tregs from mucosal leishmaniasis patients failed to normalize during treatment. C-C chemokine receptor type 5 was expressed on a large proportion of Tregs at the site of infection. These results demonstrate increased Tregs at the site of infection, possibly homing from the peripheral circulation.

Influence of Leishmania RNA Virus 1 on Proinflammatory Biomarker Expression in a Human Macrophage Model of American Tegumentary Leishmaniasis.

Backgound: Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Methods: Expression of proinflammatory biomarkers, including interleukin (IL) 1ß, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. Results: LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1ß (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Conclusions: Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.