The Immunoglobulin M-Shed Acute Phase Antigen (SAPA)-test for the Early Diagnosis of Congenital Chagas Disease in the Time of the Elimination Goal of Mother-to-Child Transmission.

BACKGROUND: Diagnosis of congenital Chagas disease (CChD) in most endemic areas is based on low-sensitive microscopy at birth and 9-month immunoglobulin G (IgG), which has poor adherence. We aim to evaluate the accuracy of the Immunoglobulin M (IgM)-Shed Acute Phase Antigen (SAPA) test in the diagnosis of CChD at birth. METHODS: Two cohort studies (training and validation cohorts) were conducted in 3 hospitals in the department of Santa Cruz, Bolivia. Pregnant women were screened for Chagas disease, and all infants born to seropositive mothers were followed for up to 9 months to diagnose CChD. A composite reference standard was used to determine congenital infection and was based on the parallel use of microscopy, quantitative polymerase chain reaction (qPCR), and IgM-trypomastigote excreted-secreted antigen (TESA) blot at birth and/or 1 month, and/or the detection of anti-Trypanosoma cruzi IgG at 6 or 9 months. The diagnostic accuracy of the IgM-SAPA test was calculated at birth against the composite reference standard. RESULTS: Adherence to the 6- or 9-month follow-up ranged from 25.3% to 59.7%. Most cases of CChD (training and validation cohort: 76.5% and 83.7%, respectively) were detected during the first month of life using the combination of microscopy, qPCR, and/or IgM-TESA blot. Results from the validation cohort showed that when only 1 infant sample obtained at birth was evaluated, the qPCR and the IgM-SAPA test have similar accuracy (sensitivity: range, 79.1%-97.1% and 76.7%-94.3%, respectively, and specificity: 99.5% and 92.6%, respectively). CONCLUSIONS: The IgM-SAPA test has the potential to be implemented as an early diagnostic tool in areas that currently rely only on microscopy.

Trypomastigote Excretory Secretory Antigen Blot Is Associated With Trypanosoma cruzi Load and Detects Congenital T. cruzi Infection in Neonates, Using Anti-Shed Acute Phase Antigen Immunoglobulin M.

Background: Congenital Trypanosoma cruzi infection accounts for an estimated 22% of new cases of Chagas disease in Latin America. However, neonatal diagnosis is challenging, as 9-month follow-up for immunoglobulin G testing is poor, quantitative polymerase chain reaction (qPCR) analysis is not routinely performed, and the micromethod misses ≥40% of congenital infections. Methods: Biorepository samples from new mothers and their infants from Piura, Peru, (an area of nonendemicity), and Santa Cruz, Bolivia (an area of endemicity) were accessed. Infant specimens were assessed using the micromethod, qPCR analysis, and a trypomastigote excretory secretory antigen (TESA) blot for detection of immunoglobulin M (IgM)-specific shed acute phase antigen (SAPA) bands, using qPCR as the gold standard. Results: When compared to qPCR, IgM TESA blot was both sensitive and specific for congenital Chagas disease diagnosis. Cumulative sensitivity (whether only 4 bands or all 6 bands were present) was 80% (95% confidence interval [CI], 59%-92%). Specificity was 94% (95% CI, 92%-96%) in the area of endemicity and 100% in the area of nonendemicity. SAPA bands occurred sequentially and in pairs, and parasite loads correlated highly with the number of SAPA bands present. The micromethod detected infection in fewer than half of infected infants. Conclusions: The IgM TESA blot for detection of SAPA bands is rapid, relatively inexpensive, and more sensitive than the micromethod and may be a useful point-of-care test for detection of congenital T. cruzi infection.

Metabolizer phenotype prediction in different Peruvian ethnic groups through CYP2C9 polymorphisms.

OBJECTIVES: The CYP2C9 gene have three common alleles, CYP2C9*1, CYP2C9*2 and CYP2C9*3, associated with different homozygous (*1/*1, *2/*2 and *3/*3) and heterozygous (*1/*2 and *1/*3) genotypes, which in turn are related to extensive (gEM), intermediate (gIM) and poor (gPM) metabolizers. Likewise, the inter-ethnic variability was intimately associated with different drug metabolism. Therefore, the aim of the present study was predict the metabolizer phenotypes in different Peruvian ethnic groups from lowland (<2,500 m) and highland (>2,500 m). METHODS: TaqMan genotyping assays were performed in a group of 174 healthy unrelated Peruvian individuals. RESULTS: In this study, the allelic comparison between the three eco-regions showed that the CYP2C9*1 was the most common in Andean (96.32%); the *2 was the most frequent in Coast (7.45%, p<0.05). Regarding the *3 was the most common in Amazonian (6.25%, p<0.05). In a corroborative manner, the gEM was the most common in Andean (94.74%), the gIM in Coast (17.02%) and gPM in Amazonian (6.25%) populations. CONCLUSIONS: Our study provides a valuable source of information about to metabolizer phenotype drugs in different Peruvian ethnic groups. In this way, it could be established suitable genetic-dosage medicaments for various common diseases in these heterogenetic populations.

IgG Subclasses and Congenital Transmission of Chagas Disease.

The mechanism of vertical transmission of Trypanosoma cruzi is poorly understood. In this study, we evaluated the role of IgG subclasses in the congenital transmission of Chagas disease. We conducted a case-control study in a public maternity hospital in Santa Cruz, Bolivia, enrolling women at delivery. Thirty women who transmitted T. cruzi to their newborns (cases), and 51 women who did not (controls) were randomly selected from 676 total seropositive women. Trypanosoma cruzi-specific IgG1, IgG2, and IgG3 levels were measured by in-house ELISA. The IgG4 levels were unmeasurable as a result of low levels in all participants. Quantitative polymerase chain reaction results and demographic factors were also analyzed. One-unit increases in normalized absorbance ratio of IgG1 or IgG2 levels increased the odds of congenital T. cruzi transmission in Chagas-seropositive women by 2.0 (95% CI: 1.1-3.6) and 2.27 (95% CI: 0.9-5.7), adjusted for age and previous blood transfusion. Odds of congenital transmission were 7.0 times higher in parasitemic mothers (95% CI: 2.3-21.3, P < 0.01) compared with nonparasitemic mothers. We observed that all mothers with IgG1 ≥ 4 were transmitters (sensitivity = 20%, specificity = 100%). Additionally, no mothers with IgG2 < 1.13 were transmitters (sensitivity = 100%, specificity = 21.6%). We demonstrated that IgG subclasses and parasite presence in blood are associated with vertical transmission of T. cruzi and could identify women at increased risk for congenital transmission by measuring IgG subclasses. These measures have potential as objective screening tests to predict the congenital transmission of Chagas.

α-Gal immunization positively impacts Trypanosoma cruzi colonization of heart tissue in a mouse model.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qß-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.

Geographic distribution of the 3435C>T polymorphism of the MDR1 gene in Peruvian populations.

Background The MDR1 gene presents several genetic polymorphisms with pharmacological implications. Therefore, the aim of the present study is to establish the genotype and allele frequencies of 3435C>T polymorphism of MDR1 gene into Peruvian populations (Coastal, Andean and Amazonian ecoregions), even considering the altitude (lowland <2500 m and highland >2500 m). Methods The polymorphism was analyzed by TaqMan genotyping assays in a group of 181 healthy unrelated Peruvian individuals. The comparison of genotype and allele frequencies of 3435C>T polymorphism was made with the Pearson test (X2), and, to calculate the genotype distributions, the Hardy-Weinberg equilibrium (HWE) was used. Results In all populations evaluated in this study, the genotype frequency distributions met HWE assumptions. The comparison between genotype and allele frequencies showed significant differences (p < 0.05), when the Andean, Coastal and Amazonian populations were compared. Also, significant differences (p < 0.05) were obtained when these populations were compared considering their altitudes. Likewise, in comparison with countries like USA, Finland, Nigeria and Kenya, the results showed significant differences (p < 0.05). Conclusions This investigation allowed us to establish the genotype and allele frequencies of 3435C>T polymorphism in different Peruvian populations, considering the geographic localization and even the altitude.

Use of a Chagas Urine Nanoparticle Test (Chunap) to Correlate with Parasitemia Levels in T. cruzi/HIV Co-infected Patients.

BACKGROUND: Early diagnosis of reactivated Chagas disease in HIV patients could be lifesaving. In Latin America, the diagnosis is made by microscopical detection of the T. cruzi parasite in the blood; a diagnostic test that lacks sensitivity. This study evaluates if levels of T. cruzi antigens in urine, determined by Chunap (Chagas urine nanoparticle test), are correlated with parasitemia levels in T. cruzi/HIV co-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi antigens in urine of HIV patients (N = 55: 31 T. cruzi infected and 24 T. cruzi serology negative) were concentrated using hydrogel particles and quantified by Western Blot and a calibration curve. Reactivation of Chagas disease was defined by the observation of parasites in blood by microscopy. Parasitemia levels in patients with serology positive for Chagas disease were classified as follows: High parasitemia or reactivation of Chagas disease (detectable parasitemia by microscopy), moderate parasitemia (undetectable by microscopy but detectable by qPCR), and negative parasitemia (undetectable by microscopy and qPCR). The percentage of positive results detected by Chunap was: 100% (7/7) in cases of reactivation, 91.7% (11/12) in cases of moderate parasitemia, and 41.7% (5/12) in cases of negative parasitemia. Chunap specificity was found to be 91.7%. Linear regression analysis demonstrated a direct relationship between parasitemia levels and urine T. cruzi antigen concentrations (p<0.001). A cut-off of > 105 pg was chosen to determine patients with reactivation of Chagas disease (7/7). Antigenuria levels were 36.08 times (95% CI: 7.28 to 64.88) higher in patients with CD4+ lymphocyte counts below 200/mL (p = 0.016). No significant differences were found in HIV loads and CD8+ lymphocyte counts. CONCLUSION: Chunap shows potential for early detection of Chagas reactivation. With appropriate adaptation, this diagnostic test can be used to monitor Chagas disease status in T. cruzi/HIV co-infected patients.

Un nuevo coronavirus, una nueva enfermedad: COVID-19 / A novel coronavirus, a novel disease: COVID-19

RESUMEN La nueva pandemia de COVID-19 causada por el virus SARS-CoV-2 desafía actualmente a la humanidad. Este virus originario de murciélagos ha sido transmitido a humanos, probablemente, a través del pangolín en el mercado marino de Wuhan (China) a fines del 2019. Se disemina por gotitas de saliva o a través de materiales contaminados, por lo que es sumamente importante aislar a las personas infectadas que pueden ser sintomáticas o asintomáticas. El cuadro clínico típico se caracteriza por fiebre, tos seca, dificultad respiratoria y malestar general. Este integrante de la familia de los coronavirus tiene mayor capacidad infectiva que sus predecesores, pero una menor mortalidad (2-3 %). Las pruebas de laboratorio para detectar la presencia del virus se basan en la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR, por su sigla en inglés) o en inmunoensayos.

ABSTRACT The novel COVID-19 pandemic caused by the SARS-CoV-2 virus is currently challenging humankind. This virus originated in bats has probably been transmitted to humans through pangolins in the Wuhan marine market (China) by the end of 2019. It is spread by droplets of saliva or through contaminated materials, making it extremely important to isolate infected people who may be symptomatic or asymptomatic. The typical clinical features of this disease are fever, dry cough, shortness of breath and general malaise. This member of the coronavirus family shows higher infectivity but lower mortality rates (2-3 %) than its predecessors. Laboratory tests to detect the virus include reverse transcriptase-polymerase chain reaction (RT-PCR) tests or immunoassays.

SARS-CoV-2 y sistema inmune: una batalla de titanes / SARS-CoV-2 and the immune system: a battle of titans

RESUMEN La humanidad se enfrenta, en la actualidad, a un reto que no diferencia fronteras, ideologías, sistemas socioeconómicos, etnias, religiones o culturas. Es una guerra que nos une como especie biológica, en la que las armas fundamentales provienen de la investigación puesta al servicio de los sistemas de salud. En esta guerra los gobiernos y las organizaciones internacionales de salud definen la estrategia; mientras que los sistemas de salud y las fuerzas del orden, de la mano de la ciudadanía, concretan las acciones y libran cada una de las batallas. Sin embargo, el más importante de los enfrentamientos se realiza en el interior de cada uno de nosotros. Como Perseo enfrentó a Hades en una batalla de titanes para evitar que convirtiera la Tierra en un infierno, el sistema inmune pone en marcha una poderosa maquinaria en la que moléculas y células del sistema innato y adquirido actúan de manera coordinada para combatir al SARS-CoV-2. Una maquinaria que sigue un guion escrito por la evolución, y que deja en nuestro sistema de defensa una memoria que nos fortalecerá como especie para enfrentar futuros Hades. Este trabajo resume la valiosa información que se ha publicado en los últimos meses respecto al coronavirus SARS-CoV-2 en su interacción con el sistema inmune. Se incluyen aspectos relacionados con la detección de la respuesta inmune como herramienta para el diagnóstico de esta infección, y la manipulación del sistema inmune en la prevención o el tratamiento de la misma.

ABSTRACT Humankind is currently facing a challenge that distinguishes no borders, ideologies, socioeconomic systems, ethnic groups, religions or cultures. It is a war that unites us as a biological species, in which the main weapons come from research aimed at improving health systems. In this war, governments and international health organizations define the strategy; and health systems and law enforcement agencies, together with citizens' support, carry out actions and fight the battles. However, the most important battle is fought inside each of us. Just like Perseus fought against Hades in a battle of titans to prevent the Earth from turning into hell, the immune system activates a powerful machinery in which molecules and cells of the innate and acquired immune system jointly act to defeat SARS-Cov-2: a machinery that follows a script written by evolution, and that will leave in our immune system a memory that will strengthen us as a species to face future Hades. This work summarizes valuable information published in recent months regarding the interaction between SARS-Cov-2 and the immune system. It also includes aspects related to the detection of the immune response as a tool for the diagnosis of this infection, as well as the manipulation of the immune system to prevent or treat the disease.

Pruebas diagnósticas para la COVID-19: la importancia del antes y el después / Diagnostic tests for COVID-19: the importance of the before and the after

RESUMEN En 2019, en la cuidad de Wuhan, China, se detectaron pacientes infectados con un nuevo coronavirus denominado 2019-nCoV, hoy SARS-CoV-2. El alarmante crecimiento de esta pandemia hace necesario conocer e implementar métodos de diagnóstico confiables para detectar y tratar adecuadamente a los pacientes, lo que contribuirá a frenar la propagación de la enfermedad. La necesidad de tener profesionales entrenados en la toma de muestras, en las buenas prácticas de laboratorio clínico y en el manejo de técnicas moleculares y hematológicas es de suma importancia para identificar adecuadamente la infección con este virus. La estandarización de los protocolos de obtención, traslado y almacenamiento de la muestra es igualmente relevante para detener la expansión de la pandemia de COVID-19 causada por el SARS-CoV-2

ABSTRACT In 2019, in the city of Wuhan, China, patients were diagnosed with a novel coronavirus originally named 2019-nCoV, currently known as SARS-CoV-2. The alarming expansion of this pandemic makes it necessary to find out and implement reliable diagnostic methods in order to properly detect and treat patients, thus contributing to slowing down the spread of the disease. It is extremely important to have trained professionals in sample collection, good clinical laboratory practices, and molecular and hematological techniques to adequately detect any cases of infection with this virus. Moreover, standardized protocols are essential for obtaining, transferring and storing samples to stop the spread of the COVID-19 pandemic caused by SARS-CoV-2

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

BACKGROUND: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). METHODS/PRINCIPAL FINDINGS: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. CONCLUSIONS/SIGNIFICANCE: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Use of a novel chagas urine nanoparticle test (chunap) for diagnosis of congenital chagas disease.

BACKGROUND: Detection of congenital T. cruzi transmission is considered one of the pillars of control programs of Chagas disease. Congenital transmission accounts for 25% of new infections with an estimated 15,000 infected infants per year. Current programs to detect congenital Chagas disease in Latin America utilize microscopy early in life and serology after 6 months. These programs suffer from low sensitivity by microscopy and high loss to follow-up later in infancy. We developed a Chagas urine nanoparticle test (Chunap) to concentrate, preserve and detect T. cruzi antigens in urine for early, non-invasive diagnosis of congenital Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: This is a proof-of-concept study of Chunap for the early diagnosis of congenital Chagas disease. Poly N-isopropylacrylamide nano-particles functionalized with trypan blue were synthesized by precipitation polymerization and characterized with photon correlation spectroscopy. We evaluated the ability of the nanoparticles to capture, concentrate and preserve T. cruzi antigens. Urine samples from congenitally infected and uninfected infants were then concentrated using these nanoparticles. The antigens were eluted and detected by Western Blot using a monoclonal antibody against T. cruzi lipophosphoglycan. The nanoparticles concentrate T. cruzi antigens by 100 fold (western blot detection limit decreased from 50 ng/ml to 0.5 ng/ml). The sensitivity of Chunap in a single specimen at one month of age was 91.3% (21/23, 95% CI: 71.92%-98.68%), comparable to PCR in two specimens at 0 and 1 month (91.3%) and significantly higher than microscopy in two specimens (34.8%, 95% CI: 16.42%-57.26%). Chunap specificity was 96.5% (71/74 endemic, 12/12 non-endemic specimens). Particle-sequestered T. cruzi antigens were protected from trypsin digestion. CONCLUSION/SIGNIFICANCE: Chunap has the potential to be developed into a simple and sensitive test for the early diagnosis of congenital Chagas disease.

Variabilidad genética y recurrencia de Plasmodium vivax durante la malaria asintomática en Mazán, Iquitos, Perú / Genetic variability of Plasmodium vivax and patterns of recurrence in asymptomatic malaria at Mazan, Iquitos, Peru

El Plasmodium vivax muestra una alta variabilidad genética durante episodios recurrentes de la enfermedad. Objetivos: Determinar la variabilidad genética de P. vivax y los patrones de recurrencia durante la malaria asintomática. Diseño: Estudio descriptivo analítico. Institución: Laboratorio de Investigación en Enfermedades Infecciosas-Universidad Peruana Cayetano Heredia. Población: Individuos provenientes de Mazán-Iquitos, región endémica en malaria. Intervención: Se analizó 222 individuos con dos muestras de sangre secuenciales, entre junio de 2006 y noviembre de 2008. Principales medidas de resultados: Identificación de P. vivax, genotipificación en base al gen pvmsp3-α, variabilidad genética de P. vivax y patrones de recurrencia. Resultados: Primera evaluación: Positivos a P. vivax 191/222 (86%), a P. falciparum 2/222 (0,9%), con infección mixta 21/222 (9,5%) y negativos 8/222 (3,6%). Segunda evaluación: Permanecieron positivos a P. vivax 180/191 (94,2%). La genotipificación por nested PCR y digestión enzimática mostró haplotipos policlonales en 17/180 (9,4%) y monoclonales en 163/180 (90,6%). Se observó haplotipos diferentes (reinfección) en 88/180 (48,9%) y haplotipos idénticos (relapso) en 75/180 (41,7%). Conclusiones: Existió una alta variabilidad genética de P. vivax, y los patrones de recurrencia, basados en la genotipificación, indicaron diferencias entre reinfecciones y relapsos en individuos asintomáticos para malaria en Mazán, Iquitos.

Plasmodium vivax displays a high genetic variability for recurrent episodes of illness. Objectives: To determine the genetic variability of P. vivax and patterns of recurrence in asymptomatic malaria. Design: Descriptive analytical. Setting: Laboratory of Infectious Disease Research, Universidad Peruana Cayetano Heredia. Population: Individuals from Mazan, Iquitos, Peru, a malaria endemic region. Intervention: Between June 2006 and November 2008, 222 individuals were analyzed with two sequential blood samples. Main outcome measures: Identification of P. vivax, genotyping based on gene pvmsp3-α, genetic variability of P. vivax and patterns of recurrence. Results: First evaluation: Positive for P. vivax 191/222 (86%), P. falciparum 2/222 (0.9%), mixed infection 21/222 (9.5%) and negative 8/222 (3.6%). Second evaluation: 180/191 (94.2%) remained positive for P. vivax. Genotyping by nested PCR and enzymatic digestion showed polyclonal haplotypes in 17/180 (9.4%) and monoclonal in 163/180 (90.6%). Different haplotypes (reinfection) were observed in 88/180 (48.9%) and identical haplotypes (relapse) in 75/180 (41.7%). Conclusions: There was P. vivax high genetic variability and patterns of malaria recurrence based on genotyping showed differences between reinfection and relapse in asymptomatic individuals in Mazan, Iquitos.