The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst.

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30-50 nm in diameter. We identified 548 P. kellicotti-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of P. kellicotti and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between Paragonimus and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.

Comparative proteomics of adult Paragonimus kellicotti excretion/secretion products released in vitro or present in the lung cyst nodule.

Paragonimus kellicotti is a zoonotic lung fluke infection, the agent of North American paragonimiasis, and an excellent model for other Paragonimus infections. The excretory/secretory proteins (ESP) released by parasites and presented at the parasite-host interface are frequently proposed to be useful targets for drugs and/or vaccines In vitro culture conditions may alter ESP compared to those produced in vivo. In order to investigate ESPs produced in vivo we took advantage of the fact that adult P. kellicotti reproduce in the lungs of experimentally infected gerbils in tissue cysts. We performed a mass-spectrometric analysis of adult P. kellicotti soluble somatic protein (SSPs) extracts, excreted/secreted proteins (ESPs) produced by adult worms during in vitro culture, and lung cyst fluid proteins (CFPs) from experimentally infected gerbils. We identified 2,137 P. kellicotti proteins that were present in at least two of three biological replicates and supported by at least two peptides. Among those were 1,914 proteins found in SSP, 947 in ESP and 37 in CFP. In silico analysis predicted that only 141 of the total 2,137 proteins were secreted via classical or non-classical pathways. The most abundant functional categories in SSP were storage and oxidative metabolism. The most abundant categories in ESP were proteins related to metabolism and signal transduction. The 37 parasite-related proteins in CFP belonged to 11 functional categories. The largest groups were proteins with unknown function, cytoskeletal proteins and proteasome machinery. 29 of these 37 proteins were shared among all three sample types. To our knowledge, this is the first study that compares in vitro and in vivo ESP for any Paragonimus species. This study has provided new insights into ESPs of food-borne trematodes that are produced and released in vivo. Proteins released at the host-parasite interface may help the parasite evade host immunity and may represent new targets for novel treatments or diagnostic tests for paragonimiasis.

Community-based trial assessing the impact of annual versus semiannual mass drug administration with ivermectin plus albendazole and praziquantel on helminth infections in northwestern Liberia.

We assessed the impact of three annual vs five semiannual rounds of mass drug administration (MDA) with ivermectin plus albendazole followed by praziquantel for the control or elimination of lymphatic filariasis (LF), onchocerciasis, soil-transmitted helminth (STH) infections and schistosomiasis in Lofa County, Liberia. The study started in 2012 and was interrupted in 2014 during the Ebola virus outbreak. Repeated cross-sectional surveys were conducted in individuals 5 years and older to measure infection markers. Wuchereria bancrofti antigenemia prevalences decreased from 12.5 to 1.2% (90% reduction) and from 13.6 to 4.2% (69% reduction) one year after three rounds of annual or five rounds of semiannual MDA, respectively. Mixed effects logistic regression models showed decreases in odds of antigenemia positivity were 91 and 74% at that time in the annual and semiannual treatment zones, respectively (p < 0.001). Semiannual MDA was slightly more effective for reducing Onchocerca volvulus microfiladermia prevalence and at follow-up 3 were 74% (from 14.4 to 3.7%) and 83% (from 23.6 to 4.5%) in the annual and semiannual treatment zones, respectively. Both treatment schedules had similar beneficial effects on hookworm prevalence. Thus, annual and semiannual MDA with ivermectin and albendazole had similar beneficial impacts on LF, onchocerciasis, and STH in this setting. In contrast, MDA with praziquantel had little impact on hyperendemic Schistosoma mansoni in the study area. Results from a long-term follow-up survey showed that improvements in infection parameters were sustained by routine annual MDA provided by the Liberian Ministry of Health after our study endpoint.

Characterization and localization of antigens for serodiagnosis of human paragonimiasis.

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76-99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Changes in Cytokine, Filarial Antigen, and DNA Levels Associated With Adverse Events Following Treatment of Lymphatic Filariasis.

Background: Mild to moderate adverse events (AEs) are common after treatment of lymphatic filariasis (LF) and pose a major challenge for the global LF elimination program. We studied changes in cytokine levels and filarial worm components in plasma of subjects with and without AEs following treatment of LF. Methods: Participants (n = 24) were hospitalized and monitored for AEs following treatment. Cytokines (27), filarial DNA, circulating filarial antigen (CFA), and immune complexes were measured in plasma samples collected before and after treatment. Results: Levels for 16 cytokines increased after treatment in individuals with moderate AEs compared to individuals with no and/or mild AEs. These included 3 major proinflammatory cytokines (interleukin 6, tumor necrosis factor α, and interleukin 1ß). Eotaxin-1 levels were elevated at baseline in individuals who developed moderate AEs after treatment; thus, eotaxin-1 is a potential biomarker for AE risk. CFA and filarial DNA levels increased more in individuals with moderate AEs after treatment than in people with no/mild AEs. Conclusions: Increases in cytokine, filarial DNA, and CFA levels were associated with development of AEs following treatment of LF. Improved understanding of the pathogenesis of AEs may lead to improved methods for their prevention or management that could increase compliance in elimination programs.

Systems biology studies of adult paragonimus lung flukes facilitate the identification of immunodominant parasite antigens.

BACKGROUND: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. CONCLUSIONS: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future.

Serological diagnosis of North American Paragonimiasis by Western blot using Paragonimus kellicotti adult worm antigen.

Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.