Comparative Analysis of Antimicrobial Antibodies between Mild and Severe COVID-19.

Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person's antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

Risk factors for mortality and clinical presentation of monkeypox.

OBJECTIVES: To describe risk factors for mortality and clinical characteristics in patients with mpox infection at a reference hospital in Mexico. DESIGN: A prospective cohort study was conducted from September to December 2022 at Hospital de Infectología La Raza National Medical Center. METHODS: Study participants were patients that met operational definition of confirmed case of mpox according to WHO criteria. Information was obtained through a case report form that included epidemiological, clinical, and biochemical information. The follow-up period was from initial evaluation for hospitalization until discharge due to clinical improvement or death. Written informed consent was obtained from all participants. RESULTS: Seventy-two patients were included in the analysis, 64 of 72 (88.9%) were people with HIV (PWH). Of the total of patients 71 of 72 (98.6%) were male, with a median age of 32 years old [95% confidence interval (CI), interquartile range (IQR) 27-37]. Coinfection with sexually transmitted infections was reported in 30 of 72 (41.7%). The overall mortality was five of 72 (6.9%). The incidence of mortality rate in PWH was 6.3%. Median days from onset of symptoms to death from any cause during hospitalization was 50 days (95% CI, IQR 38-62). Risk factors for mpox mortality in the bivariate analysis were CD4 + cells count ≤100 cells/µl at the time of assessment RR 20 (95% CI, IQR 6.6-60.2) ( P  < 0.001), absence of antiretroviral therapy RR 6.6 (95% CI, IQR 3.6-12.1) ( P  = 0.001) and ≥50 skin lesions at presentation RR 6.4 (95% CI, IQR 2.6-15.7) ( P  = 0.011). CONCLUSIONS: The clinical presentation between PWH and non-HIV patients was similar in this study, however, reported mortality was associated with advanced-HIV disease.

Herpesvirus Screening in Childhood Hematopoietic Transplant Reveals High Systemic Inflammation in Episodes of Multiple Viral Detection and an EBV Association with Elevated IL-1ß, IL-8 and Graft-Versus-Host Disease.

Infections remain a major cause of morbidity and mortality among hematopoietic stem cell transplant (HSCT) recipients. Unlike Epstein-Barr Virus (EBV) and Human Cytomegalovirus (HCMV), Human Herpesvirus (HHV) 6, HHV7 and HHV8 are not routinely monitored in many centers, especially in the pediatric population of low-medium income countries. We screened EBV, HCMV, HHV6, HHV7 and HHV8 in 412 leukocytes-plasma paired samples from 40 pediatric patients assisted in a tertiary hospital in Mexico. Thirty-two underwent allo-HSCT, whereas eight received auto-HSCT. Overall viral detection frequencies in allo- and auto-HSCT were: EBV = 43.7% and 30.0%, HCMV = 5.0% and 6.7%, HHV6 = 7.9% and 20.0% and HHV7 = 9.7% and 23.3%. HHV8 was not detected in any sample. Interestingly, HHV6 and HHV7 were more frequent in auto-HSCT, and HHV6 was observed in all episodes of multiple detection in auto-HSCT patients. We found EBV DNA in plasma samples, whereas HCMV, HHV6 and HHV7 DNA were predominantly observed in leukocytes, indicative of their expansion in cellular compartments. We also found that IL-1ß, IL-2, IL-6 and IL-8 were significantly increased in episodes in which multiple viruses were simultaneously detected, and samples positive for EBV DNA and graft-versus-host disease had a further increase of IL-1ß and IL-8. In conclusion, the EBV, HCMV, HHV6 and HHV7 burdens were frequently detected in allo- and auto-HSCT, and their presence associated with systemic inflammation.

Acute lymphoblastic leukemia-secreted miRNAs induce a proinflammatory microenvironment and promote the activation of hematopoietic progenitors.

Leukemogenesis is proposed to result from the continuous interplay between inducive bone marrow (BM) microenvironments and malignant precursor cells. Recent findings point toward an abnormal production of proinflammatory mediators within the BM from acute lymphoblastic leukemia (ALL) patients, although the mechanism underlying this phenomenon is uncertain. Here, we have identified 3 miRNAs, miR-146a-5p, miR-181b-5p, and miR-199b-3p, as potential candidates for TLR8 ligation, which are overexpressed in ALL and show agonist functional binding. When purified from ALL exosomes, they demonstrated their capacity of inducing cytokine production by both, hematopoietic and stromal BM cells. Of note, the exposure of BM cells from ALL patients to the proinflammatory milieu resulting from these miRNAs agonist activity revealed the proliferation of normal progenitors, while poor effects were recorded in the leukemic counterpart. The unconventional roles of the tumor-secreted miRNAs as TLR8 agonist ligands may provide a novel mechanism contributing a tumor-microenvironment feedback loop by switching on proinflammatory pathways that further activate normal hematopoietic precursors and support ALL progression. Secreted B-ALL TLR8-agonist miRNAs are involved in the promotion of proinflammatory microenvironments that target normal hematopoietic cells. B-lineage ALL cells secrete exosomes containing miRNAs endowed with the ability of functionally binding TLR8 in hematopoietic and BM mesenchymal stromal cells. Upon TLR8 signaling, the activation of the NF-kB pathway induces secretion of proinflammatory cytokines that, in turn, promotes cell proliferation in early hematopoietic cell populations, driving a tumor-microenvironment-hematopoietic activation feedback loop that may reduce the normal hematopoietic stem and progenitor cell compartment and facilitate cancer progression.

Contribution of APOA5, APOC3, CETP, ABCA1 and SIK3 genetic variants to hypertriglyceridemia development in Mexican HIV-patients receiving antiretroviral therapy.

OBJECTIVE: To investigate the impact of single nucleotide polymorphisms (SNPs) from APOA5, APOC3, CETP, ATP binding cassette transporter A1 and SIK3 genes in the development of hypertriglyceridemia in HIV patients under antiretroviral therapy. MATERIAL AND METHODS: A case-control study was developed. Leukocytic genomic DNA was extracted and genotyping for SNPs rs662799, rs964184, rs5128, rs2854116, rs2854117, rs3764261, rs4149310, rs4149267 and rs139961185 was performed by real time-PCR using TaqMan allelic discrimination assays, in Mexican mestizo patients with HIV infection, with hypertriglyceridemia (>1.7 mmol/L) under antiretroviral therapy. Genetic variants were also investigated in a control group of normolipidemic HIV patients (≤ 1.7 mmol/L). Haplotypes and gene interactions were analyzed. RESULTS: A total of 602 HIV patients were genotyped (316 cases and 286 controls). Age and antiretroviral regimen based on protease inhibitors were associated with hypertriglyceridemia (P = 0.0001 and P = 0.0002. respectively). SNP rs964184 GG genotype in APOA5 gene exhibited the highest association with hypertriglyceridemia risk (OR, 3.2, 95% CI, 1.7-5.8, P = 0.0001); followed by SNP rs139961185 in SIK3 gene (OR = 2.3; (95% CI, 1.1-4.8; P = 0.03 for AA vs. AG genotype; and APOC3 rs5128 GG genotype, (OR, 2.2; 95% CI, 1.1-4.9; P = 0.04) under codominant models. These associations were maintained in the adjusted analysis by age and protease inhibitors based antiretroviral regimens. CONCLUSIONS: This study reveals an association between rs964184 in APOA5; rs5128 in APOC3 and rs139961185 in SIK3 and high triglyceride concentrations in Mexican HIV-patients receiving protease inhibitors. These genetic factors may influence the adverse effects related to antiretroviral therapy.

Detection of cytomegalovirus in plasma of patient's HIV by duplex real time PCR / Detección de citomegalovirus en plasma de pacientes con VIH mediante PCR dúplex en tiempo real

BACKGROUND: Cytomegalovirus (CMV) is able to cause serious and even deadly diseases in immunocompromised patients. It is important to have a sensitive, specific and molecular viral tests for its detection, using as targets, key genes for viral replication. The following genes have been used in the molecular detection of CMV: UL122 (replication) and UL83 (most abundant protein of the tegument). OBJECTIVE: Detect and quantify CMV, by real-time duplex PCR, from a minimum amount of plasma. MATERIAL AND METHODS: The UL122 and UL83 genes were amplified with different fluorophores, by real-time duplex PCR. To quantify CMV, curves were generated, starting with DNA-CMV (1.0-0.0000001 ng). RESULTS: The dynamic range of "master" duplex straight had a pendent (m) −3.0, the amplification efficiency was 115.44% plasmas from patients with HIV viral load ≥ 100,000 copies/mL, 11.36% were true positive for CMV and 88.64% had no amplifications or they were outside of the linear range of molecular detection. CONCLUSIONS: This test identified two important CMV genes (UL122 and UL83) in a single reaction (FAM:VIC), viral detection was confirmed from a minimum amount of plasma. This mean a smaller amount of biological sample required and would add a tool to the clinical area, as well as a lower consumption of reagents and materials.

INTRODUCCIÓN: El citomegalovirus (CMV) es capaz de provocar enfermedades graves e incluso mortales en pacientes inmunocomprometidos. Es importante contar con pruebas moleculares de detección viral, sensibles y específicas, utilizando como blanco los genes clave para la replicación viral. En la detección molecular de CMV se han utilizado los genes UL122 (replicación) y UL83 (proteína más abundante del tegumento). OBJETIVO: Detectar y cuantificar el CMV mediante reacción en cadena de la polimerasa (PCR) dúplex en tiempo real, a partir de una mínima cantidad de plasma. MATERIAL Y MÉTODOS: Los genes UL122 y UL83 se amplificaron con diferentes fluoróforos mediante PCR dúplex en tiempo real. Para cuantificar el CMV se generó una recta estándar, a partir de DNA del CMV (1.0-0.0000001 ng). RESULTADOS: El rango dinámico de la «recta maestra¼ tuvo una pendiente (m) de -3.0; la eficiencia de amplificación fue del 115.44%; de los plasmas de pacientes con infección por el virus de la inmunodeficiencia humana (VIH) con una carga viral ≥ 100,000 copias/ml, el 11.36% fueron verdaderos positivos para CMV y el 88.64% no tuvieron amplificaciones o estuvieron fuera del rango lineal de detección molecular. CONCLUSIONES: Esta prueba identificó dos genes importantes del CMV (UL122 y UL83) en una sola reacción (FAM:VIC), y se ratificó la detección viral a partir de una mínima cantidad de plasma. Esto se traduce en una menor cantidad de muestra biológica requerida y sumaría una herramienta al área clínica, así como un menor consumo de reactivos y materiales.

Comparison of direct sequencing of the NS5B region with the Versant HCV genotype 2.0 assay for genotyping of viral isolates in Mexico.

Hepatitis C virus (HCV) infection affects an estimated 71 million people worldwide. HCV is classified into eight genotypes and >70 subtypes. Determination of HCV genotype is important for selection of type and duration of antiviral therapy, and genotype is also a predictor of treatment response. The most commonly used HCV genotyping method in clinical laboratories is a hybridization-based line probe assay (LiPA; Versant HCV Genotype 2.0). However, these methods have a lack of specificity in genotype identification and subtype assignment. Here, we compared the performance of Versant HCV Genotype 2.0 with the gold standard direct sequencing of the NS5B region, in 97 samples from Mexican patients. We found a genotypic concordance of 63.9% between these methods. While 68 samples (70%) were classified into HCV genotype 1 (GT1) by NS5B sequencing, it was not true for 17 samples (17.5%), which were not match HCV subtype by LiPA. Furthermore, nine of the 33 samples classified by NS5B sequencing as GT1a were not identified by LiPA. Use of direct sequencing could improve selection of the optimal therapy, avoid possible failures of therapy and avoid high costs resulting from incorrect genotyping tests in settings without broad access to pangenotypic regimens.

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection.

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.

Protective immunity against enteral stages of Trichinella spiralis elicited in mice by live attenuated Salmonella vaccine that secretes a 30-mer parasite epitope fused to the molecular adjuvant C3d-P28.

The development of a veterinary vaccine against T. spiralis infection is an alternative strategy to control trichinellosis. In an effort to develop an efficient vaccine, BALB/c mice were immunized with attenuated Salmonella enterica serovar Typhimurium SL3261 that expresses a 30-mer peptide (Ag30) derived from the gp43 of T. spiralis muscle larvae fused to three copies of the molecular adjuvant P28 (Ag30-P283) and it was either displayed on the surface or secreted by recombinant Salmonella strains. Salmonella strain secreting Ag30-P283, reduced the adult worm burden 92.8% following challenge with T. spiralis muscle larvae compared to 42% achieved by recombinant Salmonella displaying Ag30-P283 on the surface. The protection induced by secreted Ag30-P283 was associated with a mixed Th1/Th2 with predominance of Th2 phenotype, which was characterized by the production of IgG1, intestinal IgA antibodies and IL-5 secretion. This finding could provide an efficient platform technology for the design of novel vaccination strategies.

Prevalence of gene rearrangements in Mexican children with acute lymphoblastic leukemia: a population study-report from the Mexican Interinstitutional Group for the identification of the causes of childhood leukemia.

Mexico has one of the highest incidences of childhood leukemia worldwide and significantly higher mortality rates for this disease compared with other countries. One possible cause is the high prevalence of gene rearrangements associated with the etiology or with a poor prognosis of childhood acute lymphoblastic leukemia (ALL). The aims of this multicenter study were to determine the prevalence of the four most common gene rearrangements [ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL rearrangements] and to explore their relationship with mortality rates during the first year of treatment in ALL children from Mexico City. Patients were recruited from eight public hospitals during 2010-2012. A total of 282 bone marrow samples were obtained at each child's diagnosis for screening by conventional and multiplex reverse transcription polymerase chain reaction to determine the gene rearrangements. Gene rearrangements were detected in 50 (17.7%) patients. ETV6-RUNX1 was detected in 21 (7.4%) patients, TCF3-PBX1 in 20 (7.1%) patients, BCR-ABL1 in 5 (1.8%) patients, and MLL rearrangements in 4 (1.4%) patients. The earliest deaths occurred at months 1, 2, and 3 after diagnosis in patients with MLL, ETV6-RUNX1, and BCR-ABL1 gene rearrangements, respectively. Gene rearrangements could be related to the aggressiveness of leukemia observed in Mexican children.

Susceptibility to advanced age-related macular degeneration and alleles of complement factor H, complement factor B, complement component 2, complement component 3, and age-related maculopathy susceptibility 2 genes in a Mexican population.

PURPOSE: To investigate the association of age-related macular degeneration (AMD)-high risk alleles of the complement factor H (CFH), complement factor B (CFB), complement component 2 (C2), complement component 3 (C3), and age-related maculopathy susceptibility 2 (ARMS2) genes in a Mexican population for the first time. METHODS: Genotyping was performed for the Y402H variant of CFH, for the L9H, R32Q, and K565E variants of CFB, the E318D variant of C2, the A69S variant of ARMS2, and the R102G variant of C3 in 159 Mexican mestizo patients at advanced stages of AMD, i.e., CARMS (Clinical Age-Related Maculopathy Staging System) grade 4 or 5. The frequency of these variants was also investigated in a group of 152 control subjects without AMD. Genomic DNA was extracted from blood leukocytes, and genotyping was performed using PCR followed by direct sequencing. Allele-specific restriction enzyme digestion was used to detect the R102G polymorphism in C3. RESULTS: There were significant differences in the allelic distribution between the two groups for CFH Y402H (p=1×10(-5)), ARMS A69S (p=4×10(-7)), and CFB R32Q (p=0.01). The odds ratios (95% confidence interval) obtained for the risk alleles of these three variants were 3.8 (2.4-5.9), 3.04 (2.2-4.3), and 2.5 (1.1-5.7), respectively. Haplotype analysis including the two most significantly associated alleles (CFH Y402H and ARMS A69S) indicated that the C-T combination conferred an odds ratio (95% confidence interval) of 6.9 (3.2-14.8). The exposed attributable risk for this particular haplotype was 85.5%. CONCLUSIONS: This is the first case-control investigation of AMD-high risk alleles in a Latino population. Our results support that CFH, ARMS2, and CFB AMD-risk alleles are consistently associated with the disease, even in ethnic groups with a complex admixture of ancestral populations such as Mexican mestizos.

A novel filamin A D203Y mutation in a female patient with otopalatodigital type 1 syndrome and extremely skewed X chromosome inactivation.

Otopalatodigital syndrome type 1 (OPD1) [OMIM 311300] is an X-linked dominant multiple congenital anomalies disease mainly characterized by a generalized skeletal dysplasia, mild mental retardation, hearing loss, cleft palate, and typical facial anomalies. OPD1 belongs to a group of X-linked skeletal dysplasias known as oto-palato-digital syndrome spectrum disorders that also include OPD2, Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). Recently, it has been demonstrated that mutations in the gene encoding the cytoskeletal protein Filamin A (FLNA) are responsible for this group of clinically overlapping human syndromes. We present the phenotypic and molecular data of a sporadic female patient clinically diagnosed with an OPD1 syndrome who carried a novel FLNA point mutation resulting in an Asp203Tyr substitution in the actin-binding domain of the protein. X-inactivation analyses demonstrated an extremely skewed pattern towards her maternal chromosome. Our results add to the molecular spectrum of the oto-palato-digital related syndromes and contribute to the delineation of phenotype-genotype correlation in this group of X-linked skeletal disorders.