Diagnosis of Parasitic Diseases: Old and New Approaches

Methods for the diagnosis of infectious diseases have stagnated in the last 20–30 years. Few major advances in clinical diagnostic testing have been made since the introduction of PCR, although new technologies are being investigated. Many tests that form the backbone of the “modern” microbiology laboratory are based on very old and labour-intensive technologies such as microscopy for malaria. Pressing needs include more rapid tests without sacrificing sensitivity, value-added tests, and point-of-care tests for both high- and low-resource settings. In recent years, research has been focused on alternative methods to improve the diagnosis of parasitic diseases. These include immunoassays, molecular-based approaches, and proteomics using mass spectrometry platforms technology. This review summarizes the progress in new approaches in parasite diagnosis and discusses some of the merits and disadvantages of these tests

Usefulness of polymerase chain reaction tests in Chagas disease studies

The Polymerase Chain Reaction (PCR) test is a highly sensitive, specific, and rapid diagnostic tool for Chagas disease. Chagas disease is caused by the protozoan flagellate Trypanosoma cruzi and is endemic to the Americas. While conventional serological methods are still used in the diagnosis of Chagas disease, they are being gradually replaced by molecular methods like PCR. PCR can detect the parasite’s DNA in blood or tissue samples from humans and animals, including asymptomatic infections and animal reservoirs. In a study conducted on a colony of New World monkeys, PCR analysis was found to be superior to conventional screening tools for trypanosome infection, although false negatives can still occur. In clinical studies, PCR has been used to assess the effectiveness of Nifurtimox and Benznidazole in treating acute and chronic Chagas patients. However, the presence of low-grade and intermittent parasitemia in peripheral blood, even in the absence of treatment, renders PCR an unreliable test for evaluating successful treatment. Based on this limiting factor, among others, we do not believe that PCR is an appropriate gold standard test for Chagas in clinical and preclinical studies. Other diagnostic methods, such as serological and biomarker tests, should be used in conjunction with PCR techniques for more accurate diagnosis of Chagas

Validation of apolipoprotein A-1 and fibronectin fragments as markers of parasitological cure for congenital chagas disease in children treated with benznidazole

Background. No reliable tests or validated biomarkers exist to ensure parasitological cure following treatment of Chagas disease (CD) patients chronically infected with Trypanosoma cruzi. As seroreversion, the only marker of cure, happens more quickly in children, we investigated the correlation between previously identified biomarkers and seroreversion in children. Methods. Thirty CD children (age 1 month to 10 years) diagnosed as T. cruzi positive (time point S0) were treated with benznidazole (BZ) 5-8 mg/kg/d for 60 days. At least 2 serological tests were used to evaluate treatment efficacy from the end of treatment (S1) until seroreversion (S2). Thirty children (age 1 month to 10 years) and 15 adults were used as healthy controls (HCs). Immunoblot and a proteomic-based assay were used to validate previously identified fragments of apolipoprotein A-1 (ApoA1) and fibronectin (FBN) as CD biomarkers. Results. Correlation between seroreversion and absence of ApoA1 and FBN fragments by immunoblot was observed in 30/30 (100%) and 29/30 (96.6%) CD children, respectively. ApoA1 and FBN fragments were absent at the end of BZ treatment in 20/30 (66.6%) and 16/30 (53.3%) children, respectively. Absence of fragments in serum profiles was confirmed by mass spectrometry. Using intact protein analysis, a 28 109-Da protein identified as full-length ApoA1 by tandem mass spectrometry was detected in HC serum samples. Conclusions. These data confirm that ApoA1 and FBN fragments can discriminate between healthy and T. cruzi-infected samples. Correlation with seroreversion was shown for the first time; results suggest predictive capacity potentially superior to serology, making them potentially useful as surrogate biomarkers.Fil: Ruiz Lancheros, Elizabeth. National Reference Centre For Parasitology; CanadáFil: Rasoolizadeh, Asieh. National Reference Centre For Parasitology; CanadáFil: Chatelain, Eric. No especifíca;Fil: Garcia Bournissen, Facundo. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Moroni, Samanta. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Moscatelli, Guillermo. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Servicio de Parasitología y Chagas; ArgentinaFil: Ndao, Momar. National Reference Centre For Parasitology; Canad

Trypanosoma cruzi Infection in an Indigenous Kariña Community in Eastern Venezuela

We investigated the seroprevalence of Trypanosoma cruzi infection in an indigenous Kariña population in eastern Venezuela. A total of 175 serum samples were collected in the community of Piñantal during February 2009. Interviews targeting socioeconomic and environmental factors associated with the T. cruzi transmission were also conducted. Samples were evaluated using trypomastigote excreted/secreted antigens (TESAs) in an ELISA format. TESA-ELISA positive samples were confirmed by indirect haemagglutination (HAI) (Wiener). A nonsystematic collection of vectors was also undertaken. T. cruzi seroprevalence was 7.43% according to both assays, and the mean age of infected patients was 48.61±10.40 years (range 34 to 73 years). The vector infection rate was 20.00% (2/10). T. cruzi seropositivity was associated with a history of triatomine bites, the ability to recognize the vector and poor knowledge about Chagas disease, but no associations were found with gender, house type, knowledge of how the disease is transmitted, or the presence of vectors or animals inside dwellings. To our knowledge, this is the first study of the seroprevalence of T. cruzi in an indigenous population in eastern Venezuela. All of the epidemiological variables required for the establishment of active vectorial transmission of T. cruzi were present in this community

Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology.

BACKGROUND: Strongyloidiasis is a persistent human parasitic infection caused by the intestinal nematode, Strongyloides stercoralis. The parasite has a world-wide distribution, particularly in tropical and subtropical regions with poor sanitary conditions. Since individuals with strongyloidiasis are typically asymptomatic, the infection can persist for decades without detection. Problems arise when individuals with unrecognized S. stercoralis infection are immunosuppressed, which can lead to hyper-infection syndrome and disseminated disease with an associated high mortality if untreated. Therefore a rapid, sensitive and easy to use method of diagnosing Strongyloides infection may improve the clinical management of this disease. METHODOLOGY/PRINCIPAL FINDINGS: An immunological assay for diagnosing strongyloidiasis was developed on a novel diffraction-based optical bionsensor technology. The test employs a 31-kDa recombinant antigen called NIE derived from Strongyloides stercoralis L3-stage larvae. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis and control sera from healthy individuals or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis who were seronegative using the NIE ELISA assay. If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group These results were in excellent agreement (κ = 0.98) with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA). A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA. CONCLUSIONS/SIGNIFICANCE: In summary, this test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes, making this platform amenable to rapid near-patient screening with minimal technical expertise

Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

Parasitic diseases cause similar to 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.National Institutes of HealthStanford Univ, Sch Med, Dept Chem & Syst Biol, Stanford, CA 94305 USAUniv Sao Paulo, Inst Quim, Dept Bioquim, BR-05508 Sao Paulo, SP, BrazilMcGill Univ, Res Inst, Natl Reference Ctr Parasitol, Montreal, PQ, CanadaUniv Autonoma Yucatan, Ctr Invest Reg Dr Hideyo Noguchi, Parasitol Lab, Merida, Yucatan, MexicoStanford Univ, Biomat & Adv Drug Delivery Lab, Stanford, CA 94305 USAUniv Estadual Campinas, Inst Chem, Campinas, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Biol, Campus Diadema, Sao Paulo, BrazilMcGill Univ, Inst Parasitol, Quebec City, PQ, CanadaMcGill Univ, Ctr Host Parasite Interact, Quebec City, PQ, CanadaUniv Fed Sao Paulo, Dept Ciencias Biol, Campus Diadema, Sao Paulo, BrazilNIH: TW008781-01C-IDEANIH: AI078505Web of Scienc

Genetic Polymorphisms and Drug Susceptibility in Four Isolates of Leishmania tropica Obtained from Canadian Soldiers Returning from Afghanistan

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease transmitted by the bite of sandflies, resulting in sores on the skin. No vaccines are available, and treatment relies on chemotherapy. CL has been frequently diagnosed in military personnel deployed to Afghanistan and returning from duty. The parasites isolated from Canadian soldiers were characterized by pulsed field gels and by sequencing conserved genes and were identified as Leishmania tropica. In contrast to other Leishmania species, high allelic polymorphisms were observed at several genetic loci for the L. tropica isolates that were characterized. In vitro susceptibility testing in macrophages showed that all isolates, despite their genetic heterogeneity, were sensitive to most antileishmanial drugs (antimonials, miltefosine, amphotericin B, paromomycin) but were insensitive to fluconazole. This study suggests a number of therapeutic regimens for treating cutaneous leishmaniasis caused by L. tropica among patients and soldiers returning from Afghanistan. Canadian soldiers from this study were successfully treated with miltefosine