Polymorphisms of the TLR4 gene: Risk factor for chronicity and severity in oral vectorial Chagas disease.

Chagas disease is one of the parasitic infections with the greatest socio-economic impact in Latin America. In Venezuela, epidemiological data has shown different sources of infection, such as the vectorial route by oral transmission. Given the importance of the TLR4 gene in the innate immune response triggered by infection with Trypanosoma cruzi, this work analyses the role of TLR4 polymorphisms and its possible effect on cytokine expression. Genomic DNA was extracted from the peripheral blood of patients from the main outbreak of oral Chagas disease in Venezuela (n = 90), as well as from a group of healthy individuals (n = 183). Subsequently, peripheral blood was also extracted from individuals with different TLR4 haplotypes and then stimulated with LPS to determine the cytokine concentration by ELISA. The internalization of TLR4 was evaluated by flow cytometry. In comparison to healthy individuals, the analysis showed a significantly increased frequency of the Asp/Gly genotype in symptomatic patients. Also, observed a correlation of the 299/399 haplotype with a significant decrease in cytokine concentration and disease severity. Finally, the parasites' trypomastigotes cause the internalization or negative regulation of TLR4. The variants of TLR4 associated with low production of cytokines may be a risk factor for chronicity and severity (cardiac involvement) in oral vectorial Chagas disease.

Specific Recognition of ß-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal ß-galactofuranosyl (ß-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic ß-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfß1,3Manpα-(CH2)3SH (glycan G29SH) and Galfß1,3Manpα1,2-[Galfß1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfß1,3Manpα1,2-[Galfß1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galß. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

Nifurtimox response of Trypanosoma cruzi isolates from an outbreak of Chagas disease in Caracas, Venezuela.

BACKGROUND & OBJECTIVES: In Venezuela, Chagas disease (ChD) is considered a serious health problem, with about 6 million people at risk; and acute outbreaks due to oral transmission of Chagas Disease (OChD) are becoming increasingly important. In 2007 there was a major outbreak of OChD and although patients from this episode were treated with nifurtimox (Lampit®-Bayer), about 70% therapeutic failure was registered. These results led us to examine whether parasite's drug susceptibility was related to this therapeutic failure. METHODS: The Trypanosoma cruzi parasites were isolated by haemoculture of the peripheral blood drawn from the pre- and post-nifurtimox treated patients infected in the 2007 OChD outbreak at Caracas, Venezuela. The in vitro assays for drug testing were performed by the MTT methodology followed by calculation of inhibitory concentration-50 (IC50) values. RESULTS: Parasite isolates obtained from the infected patients prior and after nifurtimox treatment when subjected to variable concentrations of the drug showed great heterogeneity in susceptibility with IC50 values ranging from 4.07 ± 1.82 to 94.92 ± 7.24 µM. INTERPRETATION & CONCLUSION: The high heterogeneity in nifurtimox IC50 values in the isolates and clones from the OChD patients, suggests that the therapeutic failure to nifurtimox could be due in part to a phenotypic variability that existed in the wild parasite population at the original source of contamination. Though, further pharmacological studies are needed to confirm the existence of natural nifurtimox resistance in the parasite.

Description of an oral Chagas disease outbreak in Venezuela, including a vertically transmitted case.

We describe the eleventh major outbreak of foodborne Trypanosoma cruzi transmission in urban Venezuela, including evidence for vertical transmission from the index case to her fetus. After confirming fetal death at 24 weeks of gestation, pregnancy interruption was performed. On direct examination of the amniotic fluid, trypomastigotes were detected. T. cruzi specific-polymerase chain reaction (PCR) also proved positive when examining autopsied fetal organs. Finally, microscopic fetal heart examination revealed amastigote nests. Acute orally transmitted Chagas disease can be life threatening or even fatal for pregnant women and unborn fetuses owing to vertical transmission. There is therefore an urgent need to improve national epidemiologic control measures.

Update on oral Chagas disease outbreaks in Venezuela: epidemiological, clinical and diagnostic approaches.

Orally transmitted Chagas disease has become a matter of concern due to outbreaks reported in four Latin American countries. Although several mechanisms for orally transmitted Chagas disease transmission have been proposed, food and beverages contaminated with whole infected triatomines or their faeces, which contain metacyclic trypomastigotes of Trypanosoma cruzi, seems to be the primary vehicle. In 2007, the first recognised outbreak of orally transmitted Chagas disease occurred in Venezuela and largest recorded outbreak at that time. Since then, 10 outbreaks (four in Caracas) with 249 cases (73.5% children) and 4% mortality have occurred. The absence of contact with the vector and of traditional cutaneous and Romana's signs, together with a florid spectrum of clinical manifestations during the acute phase, confuse the diagnosis of orally transmitted Chagas disease with other infectious diseases. The simultaneous detection of IgG and IgM by ELISA and the search for parasites in all individuals at risk have been valuable diagnostic tools for detecting acute cases. Follow-up studies regarding the microepidemics primarily affecting children has resulted in 70% infection persistence six years after anti-parasitic treatment. Panstrongylus geniculatus has been the incriminating vector in most cases. As a food-borne disease, this entity requires epidemiological, clinical, diagnostic and therapeutic approaches that differ from those approaches used for traditional direct or cutaneous vector transmission.

Molecular epidemiologic source tracking of orally transmitted Chagas disease, Venezuela.

Oral outbreaks of Chagas disease are increasingly reported in Latin America. The transitory presence of Trypanosoma cruzi parasites within contaminated foods, and the rapid consumption of those foods, precludes precise identification of outbreak origin. We report source attribution for 2 peri-urban oral outbreaks of Chagas disease in Venezuela via high resolution microsatellite typing.

Genetic Characterization of Trypanosoma cruzi I Populations from an Oral Chagas Disease Outbreak in Venezuela: Natural Resistance to Nitroheterocyclic Drugs.

The oral transmission of Chagas disease (oCD) in Venezuela announced its appearance in 2007. Different from other populations affected by oCD and despite close supervision during treatment with nitroheterocyclic drugs, the result was treatment failure. We studied genetic features of natural bloodstream parasite populations and populations after treatment of nine patients of this outbreak. In total, we studied six hemoculture isolates, eight Pre-Tx blood samples, and 17 samples collected at two or three Post-Tx time-points between 2007 and 2015. Parasitic loads were determined by quantitative polymerase chain reaction (qPCR), and discrete typing units (DTU), minicircle signatures, and Tcntr-1 gene sequences were searched from blood samples and hemocultures. Half-maximal inhibitory concentration (IC50) values were measured from the hemocultures. All patients were infected by TcI. Significant decrease in parasitic loads was observed between Pre-Tx and Post-Tx samples, suggesting the evolution from acute to chronic phase of Chagas disease. 60% of intra-DTU-I variability was observed between Pre-Tx and Post-Tx minicircle signatures in the general population, and 43 single-nucleotide polymorphisms (SNPs) were detected in a total of 12 Tcntr-1 gene sequences, indicative of a polyclonal source of infection. SNPs in three post-Tx samples produced stop codons giving rise to putative truncated proteins or displaced open reading frames, which would render resistance genes. IC50 values varied from 5.301 ± 1.973 to 104.731 ± 4.556 µM, demonstrating a wide range of susceptibility. The poor drug response in the Pre-Tx parasite populations may be associated with the presence of naturally resistant parasite clones. Therefore, any information that can be obtained on drug susceptibility from in vitro assays, in vivo assays, or molecular characterization of natural populations of Trypanosoma cruzi becomes essential when therapeutic guidelines are designed in a given geographical area.

Defects in immune response to Toxoplasma gondii are associated with enhanced HIV-1-related neurocognitive impairment in co-infected patients.

Human immunodeficiency virus-1 (HIV-1) and Toxoplasma gondii can invade the central nervous system and affect its functionality. Advanced HIV-1 infection has been associated with defects in immune response to T. gondii, leading to reactivation of latent infections and development of toxoplasmic encephalitis. This study evaluates relationship between changes in immune response to T. gondii and neurocognitive impairment in HIV-1/T. gondii co-infected patients, across different stages of HIV-1 infection. The study assessed the immune response to T. gondii by measuring cytokine production in response to parasite antigens, and also neurocognitive functions by performing auditory and visual P300 cognitive evoked potentials, short term memory (Sternberg) and executive function tasks (Wisconsin Card Sorting Test-WCST) in 4 groups of individuals: HIV-1/T. gondii co-infected (P2), HIV-1-infected/T. gondii-non-infected (P1), HIV-1-non-infected/T. gondii-infected (C2) and HIV-1-non-infected/T. gondii-non-infected (C1). Patients (P1 and P2) were grouped in early/asymptomatic (P1A and P2A) or late/symptomatic (P1B/C and P2B/C) according to peripheral blood CD4+ T lymphocyte counts (>350 or <350/µL, respectively). Groups were compared using T-student or U-Mann-Whitney tests as appropriate, p<0.05 was considered as significantly. For P300 waves, HIV-1-infected patients (P1) had significantly longer latencies and significantly smaller amplitudes than uninfected controls, but HIV-1/T. gondii co-infected patients (P2) had significantly longer latencies and smaller amplitude than P1. P1 patients had significantly poorer results than uninfected controls in Sternberg and WCST, but P2 had significantly worse results than P1. HIV-1 infection was associated with significantly lower production of IL-2, TNF-α and IFN-γ in response to T. gondii from early/asymptomatic stages, when comparing P2 patients to C2 controls. These findings may indicate impairment in anti-parasitic response in co-infected patients, facilitating early limited reactivation of the parasitic latent infection, therefore creating cumulative damage in the brain and affecting neurocognitive functions from asymptomatic stages of HIV-1 infection, as suggested by defects in co-infected patients in this study.

Ten years follow-up of the largest oral Chagas disease outbreak: Cardiological prospective cohort study.

BACKGROUND: Chagas disease (ChD) is the most important endemy in Latin America. Some patients, develop chronic Chagasic cardiopathy (CCC) years after the acute phase. It is unknown if patients infected by the oral route have higher risk of developing early CCC. METHODS AND FINDINGS: A prospective cohort study was conducted to assess morbidity and mortality during 10 years observation in 106 people simultaneously infected and treated in the largest known orally transmitted ChD outbreak in 2007. A preschooler died during the acute phase, but thereafter was no mortality associated to ChD. All acute phase findings improved in the first-year post-treatment. Each person was evaluated 8.7 times clinically, 6.4 by electrocardiogram (ECG)/Holter, and 1.7 by echocardiogram. Based on prevalence, the number of people who had any abnormalities (excluding repolarization abnormalities and atrial tachycardia which decreased) was higher than 2007, since they were found at least once between 2008-2017. However, when we evaluated incidence, except for clinical bradycardia and dizziness, it was observed that the number of new cases of all clinical and ECG findings decreased at the end of the follow-up. Between 2008-2017 there was not incidence of low voltage complex, 2nd degree AV block, long QT interval, left bundle branch block or left ventricular dysfunction that allowed the diagnosis of CCC. Total improvement prevailed over the persistence of all clinical and ECG/Holter findings, except for sinus bradycardia. Incomplete right bundle branch block, sinus bradycardia and/or T-wave inversion were diagnosed persistently in 9 children. The second treatment did not have significant influence on the incidence of clinical or ECG/Holter findings. CONCLUSIONS: At the end of the 10-year follow-up, there were not clinical or ECG/Holter criteria for classifying patients with CCC. The incidence of arrhythmias and repolarization abnormalities decreased. However, special attention should be paid on findings that not revert as sinus bradycardia, or those diagnosed persistently in all ECG as sinus bradycardia, incomplete right bundle branch block or T-wave inversion. Early diagnosis and treatment may have contributed to the rapid improvement of these patients. In ChD follow-up studies prevalence overestimates the real dimension of abnormalities, the incidence looks as a better indicator.

The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease.

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

Large urban outbreak of orally acquired acute Chagas disease at a school in Caracas, Venezuela.

BACKGROUND: Trypanosoma cruzi oral transmission is possible through food contamination by vector's feces. Little is known about the epidemiology and clinical features of microepidemics of orally acquired acute Chagas disease (CD). METHODS: A case-control, cohort-nested, epidemiological study was conducted during an outbreak of acute CD that affected a school community. Structured interviews were designed to identify symptoms and sources of infection. Electrocardiograms were obtained for all patients. Specific serum antibodies were assessed by immunoenzimatic and indirect hemagglutination tests. In some cases, parasitemia was tested directly or by culture, animal inoculation, and/or a polymerase chain reaction technique. RESULTS: Infection was confirmed in 103 of 1000 exposed individuals. Of those infected, 75% were symptomatic, 20.3% required hospitalization, 59% showed ECG abnormalities, parasitemia was documented in 44, and 1 child died. Clinical features differed from those seen in vectorial transmission. The infection rate was significantly higher among younger children. An epidemiological investigation incriminated contaminated fresh guava juice as the sole source of infection. CONCLUSIONS: This outbreak was unique, because it affected a large, urban, predominantly young, middle-class, otherwise healthy population and resulted in an unprecedented public health emergency. Rapid diagnosis and treatment avoided higher lethality. Food-borne transmission of T. cruzi may occur more often than is currently recognized.

Characterization and Follow-Up of Trypanosoma cruzi Natural Populations Refractory to Etiological Chemotherapy in Oral Chagas Disease Patients.

We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman's r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients' blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection.

Ten-year follow-up of the largest oral Chagas disease outbreak. Laboratory biomarkers of infection as indicators of therapeutic failure.

Trypanosoma cruzi uses various mechanisms of infection to access humans. Since 1967, food contaminated with metacyclic trypomastigotes has triggered several outbreaks of acute infection of Chagas disease by oral transmission. Follow-up studies to assess the effectiveness of anti-parasitic treatment of oral outbreaks are rather scarce. Here, we report a 10-year laboratory follow-up using parasitological, serological, and molecular tests of 106 individuals infected in 2007 of the largest known outbreak of orally transmitted Chagas disease, which occurred in Caracas city, Venezuela. Before treatment (2007), specific IgA, IgM and IgG, were found in 71% (75/106), 90% (95/106) and 100% (106/106), respectively, in addition to 21% (9/43) parasitemia, Complement Mediated Lysis (CML) in 98% (104/106) and 79% (34/43) parasitic DNA for PCR. Blood culture detected parasitemia up to 18 months post-treatment in 6% (6/106) of the patients. In 2017, the original number of cases in the follow-up decreased by 46% and due to the country's economic situation, not all the trials could be carried out in the entire population. During follow-up, IgA and IgM disappeared promptly, with IgM persisting in 19% (20/104) of the patients three years after treatment. The anti-T. cruzi IgG remained positive 10 years later in 41% (20/49) of the individuals evaluated. CML remained positive seven years later in 79% (65/82) of the cases. PCR positive cases decreased after treatment but progressively recovered, being positive in 69% (32/46) of the individuals evaluated in 2017. The group of children (under 18 years of age) showed the highest PCR positivity with 76% (26/34) of the cases, but their parasitic load tended to diminish, while in adults the parasitic load regained their initial values. The simultaneous evaluation of serological tests and PCR of the patients allowed us to separate patients among responders and non-responders to the anti-parasitic treatment, and this information prompted us to apply a second anti-parasitic treatment in the group of non-responders. In this population not subjected to the like lihood of re-infection, adult patients were more likely to be non-responders when compared to children. These results suggest that rigorous laboratory follow-up with T. cruzi infectious biomarkers is essential to detect cases of parasite persistence.

Genome of the fatal tapeworm Sparganum proliferum uncovers mechanisms for cryptic life cycle and aberrant larval proliferation.

The cryptic parasite Sparganum proliferum proliferates in humans and invades tissues and organs. Only scattered cases have been reported, but S. proliferum infection is always fatal. However, S. proliferum's phylogeny and life cycle remain enigmatic. To investigate the phylogenetic relationships between S. proliferum and other cestode species, and to examine the mechanisms underlying pathogenicity, we sequenced the entire genomes of S. proliferum and a closely related non-life-threatening tapeworm Spirometra erinaceieuropaei. Additionally, we performed larvae transcriptome analyses of S. proliferum plerocercoid to identify genes involved in asexual reproduction in the host. The genome sequences confirmed that the S. proliferum has experienced a clearly distinct evolutionary history from S. erinaceieuropaei. Moreover, we found that nonordinal extracellular matrix coordination allows asexual reproduction in the host, and loss of sexual maturity in S. proliferum are responsible for its fatal pathogenicity to humans. Our high-quality reference genome sequences should be valuable for future studies of pseudophyllidean tapeworm biology and parasitism.

Immunoinformatics and Pepscan strategies on the path of a peptide-based serological diagnosis of COVID19.

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Toward the Establishment of a Single Standard Curve for Quantification of Trypanosoma cruzi Natural Populations Using a Synthetic Satellite Unit DNA Sequence.

Accurate diagnostic tools and surrogate markers of parasitologic response to treatment are needed for managing Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (TcI) and CL-Brener (TcVI) stocks similar 95% limit of detection values [0.903 (0.745 to 1.497) and 0.667 (CI, 0.113 to 3.927) copy numbers/µL, respectively] when synthetic DNA was the standard for quantification, allowing direct comparison of loads in samples infected with different discrete typing units. This standard curve was evaluated in 205 samples (38 acute oral and 19 chronic Chagas disease patients) from different geographical areas infected with various genotypes, including samples obtained during treatment follow-up; high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase 3 clinical trials, to follow up patients under treatment or at risk of reactivation, and in experimental models using different parasite strains.

Rapid detection of Trypanosoma cruzi in human serum by use of an immunochromatographic dipstick test.

We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).

Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi Loopamp) kit for detection of congenital, acute and Chagas disease reactivation.

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally-infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen's kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77-99) and 100% (95% CI: 80-100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62-1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite populations. The T. cruzi Loopamp kit appears potentially useful for rapid detection of T. cruzi infection in congenital, acute and CD reactivation due to HIV infection.