ABSTRACT Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance ...mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.
Abstract
IL13 KO BALB/c mice developed severe myosin-induced experimental autoimmune myocarditis (EAM). This severe disease is characterized by increased inflammation, increased total intracardiac ...CD45+ leukocytes, and progression to dilated cardiomyopathy. Hearts of IL13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL1β, IL18, IFNγ, TGFβ1, IL4 and histamine. However, another proinflammatory cytokine, IL17 is significantly decreased in both hearts (p= 0.0005) and spleens of IL13 KO mice on day 21 of EAM. The decrease of IL17 is significantly correlated with decrease of another Th17 cytokine – TNF α in the hearts and spleens of IL13 KO mice. The substantial decrease of the Th17 population in the absence of IL13 is not due to the absence of cytokines required to induce Th17, since IL13 KO mice have increased levels of IL1β and TGFβ. This Th17 defect is also not caused by increased IL27, which is known to impair Th17 development, since IL27 is actually significantly decreased in IL13 KO mice. IL23, which is necessary for sustaining the Th17 population, is significantly decreased in IL13 KO mice. It has been shown that IL13 induces production of IL23 by dendritic cells. We have observed severely impaired DC maturation both in vivo as well as in vitro in IL13 KO mice. Thus, we hypothesize that IL13 is required for maturation and IL23 production by DCs; therefore, IL13 is required for Th17 differentiation.
(Supported by 2R01 HL67290).
IL‐13 knockout (KO) BALB/c mice developed severe myosin‐induced experimental autoimmune myocarditis (EAM) and coxsackievirus B3 ‐induced myocarditis with impaired cardiac function in some of the ...IL‐13 KO mice. Total CD45+ leukocyte, B cell and macrophage were increased in the hearts of IL‐13 KO mice. Spleen T cells from IL‐13 KO mice were highly activated and had increased proliferation in vitro to myosin. Hearts of IL‐13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL‐1b, IL‐18, IFN‐g, TGF‐a, IL‐4 and histamine. Severe EAM in IL‐13 KO was not due to increased IL‐4, as myocarditis was mild in IL‐4 KO mice. CD4+CD25+Foxp3+ regulatory T cell numbers were not altered in the heart, but decreased in the spleen of IL‐13 KO mice. Thus, IL‐13 protects against severe myocarditis in BALB/c mice by inhibiting macrophage, T cells and B cell/antibody‐mediated damage to the heart.
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Interleukin‐17 (IL‐17) drives the pathogenesis of a number of autoimmune disease models, including experimental autoimmune myocarditis (EAM). We have previously found that IL‐13 ...deficient (IL‐13 KO) mice developed severe EAM. To assess whether IL‐17 modulates the pathogenic response in heartss of IL‐13 KO mice, we determined the levels of IL‐17. Interestingly, IL‐17 was significantly decreased in spleen, heart (R&D Systems ELISA), and draining lymph nodes (by RT‐PCR) of IL‐13 KO mice at day 21 of EAM. Naïve IL‐13 deficient CD4+ T cells differentiated into Th‐17 cells and CD4+ FOXP 3+ T regulatory cells similar to naïve WT BALB/c T cells. However, bone marrow derived dendritic cells (BMDCs) from IL‐13KO mice were deficient in driving OVA323–339‐specific Th‐17 differentiation from naïve DO11.10 CD4+ T cells. This defect was partially restored when the BMDCs were cultured in the presence of recombinant IL‐13. IL‐13 deficient BMDCs expressed less MHCII, CD80, CD86 and CD40 upon maturation with LPS, b‐glucan curdlan, or antiCD40 mAb. Furthermore, IL‐13 deficient BMDCs produced less proinflammatory cytokines IL‐6 and TNFalpha after maturation with LPS. Thus, IL‐13 is required for DC maturation and cytokine production, which limits its ability to induce Th17. In summary, a decrease of IL‐17 in hearts of IL‐13 KO mice during EAM represents a general defect of IL‐13 KO DCs in inducing Th‐17.
Supported by 2R01 HL67290
Article
Military personnel deployed to the Amazon Basin are at high risk for cutaneous leishmaniasis (CL). We responded
to an outbreak among Peruvian Army personnel returning from short-term training ...in the Amazon, conducting
active case detection, lesion sample collection, and risk factor assessment. The attack rate was 25% (76/303); the
incubation period was 2–36 weeks (median = 8). Most cases had one lesion (66%), primarily ulcerative (49%), and
in the legs (57%). Real-time polymerase chain reaction (PCR) identified Leishmania (Viannia) braziliensis (59/61 =
97%) and L. (V.) guyanensis (2/61 = 3%). Being male (risk ratio RR = 4.01; P = 0.034), not wearing long-sleeve
clothes (RR = 1.71; P = 0.005), and sleeping in open rooms (RR = 1.80; P = 0.009) were associated with CL.
Sodium stibogluconate therapy had a 41% cure rate, less than previously reported in Peru (70%; P < 0.001). After
emphasizing pre-deployment education and other basic prevention measures, trainees in the following year had
lower incidence (1/278 = 0.4%; P < 0.001). Basic prevention can reduce CL risk in deployed militaries.
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