Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

Specificity of SARS-CoV-2 Antibody Detection Assays against S and N Proteins among Pre-COVID-19 Sera from Patients with Protozoan and Helminth Parasitic Infections.

We aimed to assess the specificity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody detection assays among people with tissue-borne parasitic infections. We tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 neutralization antibody detection kit [cPass], Abbott SARS-CoV-2 IgG assay [Abbott Architect], and Standard Q COVID-19 IgM/IgG combo rapid diagnostic test [SD RDT IgM/SD RDT IgG]) among 559 pre-COVID-19 seropositive sera for several parasitic infections. The specificity of assays was 95 to 98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95% CI, 75 to 95]) and visceral leishmaniasis (SD RDT IgG; 80% [95% CI, 30 to 99]), and from patients with recent malaria in areas of Senegal where malaria is holoendemic (ranging from 91% for Abbott Architect and SD RDT IgM to 98 to 99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all ≥96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98 to 100%. The majority (>85%) of false-positive results were positive by only one assay. The specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

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.

Reversible cysteine protease inhibitors show promise for a Chagas disease cure.

The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile "warhead" were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 µM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 µM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg.

The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease.

BACKGROUND: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. CONCLUSIONS/SIGNIFICANCE: A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.