An Overall View of the Functional and Structural Characterization of Glucose-6-Phosphate Dehydrogenase Variants in the Mexican Population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.

Re-emergence of mayaro virus and coinfection with chikungunya during an outbreak in the state of Tocantins/Brazil.

OBJECTIVE: To perform a molecular screening to detect infections by the mayaro virus and possible coinfections with Chikungunya during an outbreak in the state of Tocantins/Brazil in 2017. RESULTS: Of a total 102 samples analyzed in this study, 6 cases were identified with simultaneous infection between mayaro and chikungunya viruses (5.88%). In these 6 samples, the mean Cycle threshold (Ct) for CHIKV was 26.87 (SD ± 10.54) and for MAYV was 29.58 (SD ± 6.34). The mayaro sequences generated showed 95-100% identity to other Brazilian sequences of this virus and with other MAYV isolates obtained from human and arthropods in different regions of the world. The remaining samples were detected with CHIKV monoinfection (41 cases), DENV monoinfection (50 cases) and coinfection between CHIKV/DENV (5 cases). We did not detect MAYV monoinfections.

RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact.

Background: Several variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population. Methods: Targeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico. Results: In silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico. Conclusion: This work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

Molecular screening of cattle ticks, tick-borne pathogens and amitraz resistance in ticks of Santo Domingo de los Tsáchilas province in Ecuador.

The province of Santo Domingo de los Tsáchilas in Ecuador is a strategic place for cattle production and trade. The lack of knowledge about tick species, tick control and tick-borne diseases in Ecuador prompted this study with the goal of identifying the cattle-tick species and tick-borne agents present in the area and molecularly determining the potential acaricide resistance to amitraz of the major cattle tick species. Eighty-four cattle farms were visited and in 88 % of them, cattle were infested with ticks. Additionally, 24 historical samples from other surrounding Ecuadorian provinces, were screened as well. Besides morphological keys, PCR-RFLP MspI was used to confirm the presence of the Rhipicephalus ticks. The tick samples were also screened for tick-borne agents using PCR-RFLP BseDI and Hhal tests to identify circulating Babesia sp. and Anaplasma spp. Furthermore, the PCR-RFLP EciI technique was used to identify the amitraz resistance gene in populations of Rhipicephalus microplus in the province. Pooled testing was used to determine prevalence at individual-tick level. The presence of R. microplus and Amblyomma cajennense sensu lato (s.l.) ticks was found in 83 % and 21 % of the cattle farms respectively, showing R. microplus is widespread in the province of Santo Domingo de los Tsáchilas. Regarding tick-borne agents, only Anaplasma marginale was observed in 50 % of the visited farms of the province, while about 27 % of the ticks tested positive according to estimations from the data of the tick pools. The presence of Babesia bigemina was only confirmed in samples collected outside the province. The amitraz resistance allele in R. microplus was found in 62 % of the farms, but the percentage of farms with cattle ticks completely resistant to this acaricide was low (2%). The findings of this study should prompt cattle producers and animal health authorities to monitor control strategies, which address the management of resistant tick populations and the epidemiologically-unstable areas of tick-borne diseases.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Screening of RB1 alterations in Brazilian patients with retinoblastoma and relatives with retinoma: phenotypic and genotypic associations.

PURPOSE: To identify constitutional alterations of the retinoblastoma 1 gene (RB1) in two cohorts of Brazilian patients with retinoblastoma and to analyze genotype-phenotype associations. METHODS: Molecular screening was carried out by direct sequencing of the 27 RB1 exons and flanking regions in blood DNA of 71 patients with retinoblastoma and 4 relatives with retinoma, and with multiplex ligation-dependent probe amplification (MLPA) in 21 patients. The presumed impact of nucleotide substitutions on the structure of the retinoblastoma protein (pRB) was predicted by Polymorphism Phenotyping-2 (PolyPhen-2). Kaplan-Meier and log-rank test were used for estimating 60-month survival rates. RESULTS: One hundred two nucleotide substitutions were detected, 92 substitutions in 59 patients with retinoblastoma and 10 substitutions in 4 individuals with retinoma. Eight substitutions were novel. The majority of substitutions were intronic (86.2%). More than one substitution was present in 37.3% of patients. Twenty-one duplications and 11 deletions were found in 12 patients; some of which with both types of alterations. Duplications/deletions were found in four patients lacking constitutional alterations when analyzed by sequencing, and in eight patients carrying one or more polymorphic intronic substitutions. The global 60-month survival rate in patients was 91.8% (Confidence Interval95% = 85.0 - 99.1). Significant, lower survival rates were found in extraocular presentation (81.0%) versus intraocular tumors (P = 0.014), first enucleation after 1 month following diagnosis (80.9%) versus earlier first enucleation (P = 0.020), and relapse (100.0%) versus absence of relapse (P = 0.0005). CONCLUSIONS: Fifteen substitutions (4 intronic and 11 exonic) were identified as probably or likely pathogenic. Four of these 11 exonic substitutions were novel. Survival rates, however, were not affected by presence of these probably or likely pathogenic alterations, most of which not found in patients with retinoblastoma from other Latin American countries. These differences might be related to the different ethnic composition of the Latin American cohorts. Portuguese Abstract.