Prevalence of molecular and serological tests of the new coronavirus (SARS-CoV-2) in Carlos Chagas laboratory - Sabin group in Cuiabá

SUMMARY BACKGROUND: Coronavirus disease 2019, which is caused by the new severe acute respiratory syndrome coronavirus 2, became a pandemic in 2020 with a mortality rate of 2% and high transmissibility, thus making studies with an epidemiological profile essential. OBJECTIVES: The aim of this study was to characterize the population that performed the severe acute respiratory syndrome coronavirus 2 molecular and serological tests in Carlos Chagas Laboratory - Sabin Group in Cuiabá. METHODS: A retrospective cross-sectional study was carried out with all the samples collected from nasal swab tested by RT-PCR and serological for severe acute respiratory syndrome coronavirus 2 IgM/IgG from the population served between April and December 2020. FINDINGS: In the analysis period, 23,631 PCR-coronavirus disease 2019 examinations were registered. Of this total number of cases, 7,649 (32.37%) tested positive, while 15,982 (66.31%) did not detect viral RNA and 374 of the results as undetermined. The peak of positive RT-PCR performed in July (n=5,878), with 35.65% (n=2,096). A total of 8,884 tests were performed on serological test SOROVID-19, with a peak of 1,169 (57.16%) of the positive tests for severe acute respiratory syndrome coronavirus 2 in July. MAIN CONCLUSIONS: Molecular positivity and serological tests, both peaked in July 2020, were mostly present in women aged 20-59 years, characterizing Cuiabá as the epicenter of the Midwest region in this period due to the high rate of transmissibility of severe acute respiratory syndrome coronavirus 2.

Dificultades en la detección de genomas del nuevo coronavirus 2 (SARS CoV-2) / Pandemic and social inequiality: the popular neighborhoods of the Buenos Aires suburbs in preventive social isolation

RESUMEN El manejo de las infecciones virales respiratorias, tanto a nivel nacional como a nivel mundial, requiere resultados científicos de calidad. La reacción en cadena de la polimerasa de transcriptasa inversa (rRT-PCR, por su sigla en inglés) es considerada el "patrón de oro" para detectar el genoma del nuevo coronavirus 2 (SARS-CoV-2), agente causal de la enfermedad por el nuevo coronavirus (COVID-19) sobre todo en la fase aguda de la infección. Su uso es controvertido fuera de un contexto de exposición viral. El objetivo del presente trabajo es analizar escollos encontrados durante la detección del genoma del SARS-CoV-2 que pueden producir resultados falsos. Los falsos negativos de rRT-PCR pueden deberse al momento y la eficacia de la toma de la muestra, la congelación, el almacenamiento y la descongelación, y a la inactivación térmica de la virulencia. Además, las señales retardadas de los controles internos invalidan la negatividad. Por otra parte, las muestras con escaso material biológico llevan a conclusiones negativas falsas, por lo que determinar un umbral (número mínimo de células epiteliales) contribuirá a reducirlas. Sin embargo, la mayoría de los kits detectan ADN humano, pero no fueron calibrados para cuantificar carga celular. Los ácidos ribonucleicos nucleares (ARN) virales adheridos a guantes, tubos y gorros, -entre otros elementos-, son fuente de falsos positivos. Las farmacopeas sugieren que la contaminación externa se controle en series de 100 muestras con al menos una representatividad del 10%. Si se extrapola esta aproximación al laboratorio de análisis clínicos, en lugar de uno se deberían procesar al menos 10 controles negativos contiguos a 10 positivos cada 100 pruebas. Mejorar la detección por rRT-PCR implica un aumento de al menos 20% en el costo de los reactivos, por lo que se necesitan recursos adicionales.

ABSTRACT Emerging respiratory viral infections like the severe coronavirus disease (CoVID 19) caused by novel coronavirus 2 (SARS-CoV-2) require quality results for science-based responses. The reverse transcriptase polymerase chain reaction (rRT-PCR) is considered the gold standard for detecting SARS-CoV-2 (particularly in the acute phase of infection). The aim of the present work was to analyze pitfalls during the search of viral genomes. False negative conclusions are result of sampling timing, performances of swabbing, storage, and thawing and heat-infectivity inactivation. Samples with low biologic material also lead to false negatives. Qualitative controls to detect the presence of human DNA are available in several kits but they were not calibrated for quantification of human cell loads. Moreover, negativity cannot be reported for samples with delayed signals for the internal control (due to deficiency in extraction and/or retro transcription and/or or to the presence of rRT-PCR inhibitors). The viral RNA that may have stick on gloves, on tubes, caps, etc. may produce false positives. The International Pharmacopoeias recommend for external contamination to test at least 10% of the samples. Couples of 10 negative contiguous to 10 positive controls randomly distributed should be therefore included in each series of 100 rRT-PCR tests. These improvements increase the cost of each determination (at least by 20% only for the reactants) and require additional resources.

Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas

Background: Synovial sarcoma (SS) is an aggressive, but a relatively chemosensitive soft tissue sarcoma, characterized by a specific, t (X;18)(p11;q11) translocation, leading to formation of SS18–SSX chimeric transcript. This translocation can be detected by various techniques, such as fluorescence in-situ hybridization (FISH), reverse transcriptase PCR (RT-PCR) and fragment analysis. Objectives: To compare the results of detection of t (X;18)(p11;q11) translocation, across three different platforms, in order to determine the most optimal and sensitive technique. Methods: Formalin-fixed paraffin embedded (FFPE) tissue sections of 45 soft tissue sarcomas were analyzed, including 16 cases of SS confirmed by histopathology, immunohistochemistry and molecular technique (s)(Group 1); 13 cases, wherein SS was one of the differential diagnosis, preceding molecular testing (Group 2) and 16 cases of various other sarcomas (Group 3). Various immunohistochemical (IHC) markers studied, including INI1/SMARCB1. All cases were tested for t (X;18) translocation, by fragment Analysis, FISH and RT-PCR. Results: There were 23 cases of SS, including 16 of group 1 and 7 of group 2. By fragment analysis, t (X;18)(p11;q11) translocation was detected in 22/23 cases (95.6%). By FISH, SS18 gene rearrangement was detected in 18/22 cases (78.2%), whereas by RT-PCR, SS18-SSX transcripts were detected in 15/23 cases (65.2%). Immunohistochemically, a unique “weak to absent”/reduced INI1 immunostaining pattern was exclusively observed in 12/13 cases of SS (92.3%). Fragment analysis and FISH were relatively more sensitive techniques. Unique “weak to absent”INI1 immunoexpression significantly correlated with positive t (X;18) translocation results (P = 0.0001). Conclusion: The present study constitutes first such study from our subcontinent. Fragment analysis is a promising technique for detection of t (X;18)(p11;q11) translocation. FISH and INI1 immunostaining pattern were also relatively more sensitive, over RT-PCR.

Expression of long non-coding RNA MEG3 and its relationship with the prognosis of human gastric cancer / 中国肿瘤临床

Objective:To investigate the expression of maternally expressed gene 3 (MEG3), a long non-coding RNA gene, in gastric can-cer tissues;determine the relationship of MEG3 with the prognosis of gastric cancer;and explore the relationship between MEG3 and apoptosis-associated protein P53 as well as murine double minute 2 (MDM2). Methods:Fifty-five consecutive patients with gastric cancer admitted to Qingdao Municipal Hospital for surgical treatment from September 2012 to June 2013 were included in this study. Gastric cancer and paired normal tissues were collected. The expression of MEG3 was tested through real-time quantitative poly-merase chain reaction (qRT-PCR). Western blot analysis was used to detect the expression of P53 and MDM2 in gastric cancer and eval-uate their correlations with MEG3. Results:The expression of MEG3 decreased in cancer tissues (7.98±0.19) relative to the correspond-ing normal tissues (9.47±0.18) (P<0.05). P53 and MDM2 showed negative relationships in the gastric cancer and normal tissues. A posi-tive relationship was found between P53 and MEG3 (r=0.591, P<0.05), whereas a negative relationship was found between MDM2 and MEG3 (r=?0.346, P<0.05). The median survival time was significantly prolonged in patients with high MEG3 expression compared with patients with low MEG3 expression. Conclusion:MEG3 exerts an inhibiting effect on the development of gastric cancer. MEG3, P53, and MDM2 may have important relationships in the biological mechanisms of gastric cancer development. Detecting the expression level of MEG3 may be useful for the prognosis of gastric cancer.

Corneal strength change and keratocan expression of corneas during the healing of penetrating injury in rabbits / 中华实验眼科杂志

Background Corneal walleye formation is a result of excessive corneal wound healing.Controlling the corneal trauma or excessive healing reaction after operation of penetrating corneal trauma is a focus in the study of corneal wound healing.Objective This study was to investigate the changes of corneal strength and the expression of keratocan,a marker of corneal stroma,after corneal penetrating injury operation,and to determine the optimal removal time of corneal suture.Methods Full-thickness incisions of 5 mm along the vertical diameter were done at the central cornea on 80 eyes of 80 six-month-old New Zealand rabbits in this study.Then the incisions were interruptedly sutured with 10-0 nylon thread to establish the corneal wound healing models.Each 4 rabbits were sacrificed in 1 week,2,3,4,5,6,7,8 weeks after operation,respectively,and the central corneal stripes were prepared with the size of 7 mm×5 mm.The mean maximal strength of the corneal bands was measured by uniaxial tensile test with electroforce3220-AT biomachanics machine.Then,each 6 whole corneas were obtained at the above time points,and the dynamic changes of keratocan mRNA expression in the specimens were detected by reverse trancription PCR (RT-PCR).The care and use of the animals followed the rules of ARVO.Results The mean maximal strength of corneal stripes was 0,(1.007 ± 0.041),(1.991 ± 0.034),(2.512 ± 0.030),(3.630 ± 0.049) and (4.935 ± 0.004)M Pa in 1 week,2,3,4,5,6 weeks after modeling,and the corneal strength values from 3 weeks through 6 weeks were significantly enhanced in comparison with the value at the adjacent before timepoint (q =6.35,7.54,8.21,5.86,all at P＜0.01).The relative expression levels (absorbance) of keratocan mRNA in the corneas were 0.869±0.015,0.779 ± 0.065,0.621 ± 0.027,0.460 ± 0.018,0.393 ± 0.057 and 0.255 ± 0.045 in 1 week,2,3,4,5,6 weeks after operation,and each value was lower than that of the adjacent before timepoint (q =5.24,5.61,7.49,4.75,5.47,all at P＜0.01).The intensity of corneal stripes and the expression levels of keratocan mRNA in the corneas were stable in 7 weeks and 8 weeks after operation.Conclusions The dynamic change of corneal strength during the repair of penetrating corneal injury is associated with the down-regulation of keratocan in cornea.Rabbit cornea reaches a maximal strength capacity in 6 weeks after penetrating injury,therefore,it is the optimal time to remove suture.

Types of Rotavirus Causing Acute Diarrhea among Children in Western India, their Demographic Pattern and Disease Severity.

Background: Rotavirus infection is a major cause of severe acute gastroenteritis among infants and children all over the world1 with winter out-breaks of diarrhea in temperate and cooler parts almost round the year. However, this varies in different part of India.2-6 Diarrhea is a major cause of under-5 mortality, contributing to approximately over 1,50,000 infant deaths in our country per year.15,16 Different genotypes have been identified and many more are emerging by way of mutation, genetic shift and genetic drifts. Rotavirus are classified antigenically as A (Most common), B, C, D, E by ELISA and genotypically as G (1 through 12) and P (1 through 8) by Reverse Trans criptase PCR, in combinations. Materials and methods: Stool samples of 110 infants and children from 6 to 60 months of age, with suspected viral diarrhea over one year period were studied for serotypes and genotypes; and compared for their respective disease severity. Results: Thirty-four percent were found positive for Rotavirus-A by ELISA. Of the positive, 33.4% were found to be of G9 genotype, much higher than reported from other parts of the country. On the other hand, merely 13.6% of G1 and G4 each were detected, contrary to high prevalence elsewhere. On electro-pherotyping, the long-arm types were associated with more severe disease (64.6% showing moderate to severe dehydration) than their short-arm types (Only 16.6% showed moderate dehydration only) p < 0.009. No difference in incidence of severe dehydration between AD positive for Rotavirus (11.7%) and those found nega tive (11.8%), presumably due to other viruses, after excluding invasive diarrhea. Conclusion: Emergence of diverse strains, i.e. more of G9 and G12 genotypes than earlier reports of G1 and G2 types indicate considerable genetic shift in the region. Such trend could have significant implication on degree of seroconversion from currently used live vaccines, using G1 or bovine reassortant G1-3 strains only, seen in recent studies from Africa and Malayi.29 Contrary to claims that Rotavirus diarrhea usually threatened severe diarrhea, no significant difference in incidence of severe diarrhea was observed between Rotavirus positive and Rotavirus negative acute diarrhea.

Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

BACKGROUND: Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. OBJECTIVE: The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. METHODS: Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. RESULTS: The results of the current study suggest that majority of the patients display expression of lipase RES_0242. CONCLUSION: These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

BACKGROUND: Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. OBJECTIVE: The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. METHODS: Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. RESULTS: The results of the current study suggest that majority of the patients display expression of lipase RES_0242. CONCLUSION: These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

Sybr Green Real-Time PCR is a Useful Method in Screening for High Risk Human Papilloma Virus Types 16 and 18 in Neoplastic Cervical Cancer

Human papillomavirus (HPV) plays an important role in the pathogenesis of cervical cancer. HPV has been found in 99.7% of cervical cancers worldwide. In Malaysia, it is the second most common cancer among women in all major ethnic groups. The main purpose of this study was to establish the method of SyBrGreen Real-Time PCR and apply it for identification of multiple infections of the two high risk HPV subtypes. In this study, 57 positive samples for HPV 16 and HPV 18 were used to establish a simple and sensitive method to detect and identify HPV infection in the cervical neoplasia at different stages of the disease by using real-time ABICycler SyBrGreen 1 technology. The results showed 67 HPV genomes in 57 samples. HPV 16 genome was detected in 55/67 (82%) cases while HPV 18 was detected in 8/67 (12%) cases with 4 cases showing multiple infections of HPV 16 and HPV 18. HPV 16 was the most prevalent followed by HPV 18. Using SyBr Green Real-Time PCR techniques, the results showed that DNA melting curve for HPV 16 had a peak around 80.2ºC and Ct value of 20 cycles whereas the DNA melting curve for HPV 18 around 79.2ºC and Ct INTRODUCTION value of 22 cycles. In conclusion, a SyBr Green Real-Time PCR method has the potential for clinical usage in detection and identification of HPV infection in cervical neoplasia at different stages of the disease.

Clathrin Assembly Lymphoid Myeloid Leukemia-AF10-positive Acute Leukemias: A Report of 2 Cases with a Review of the Literature / 대한진단검사의학회지

The translocation t(10;11)(p13;q14q21) has been found to be recurrent in acute lymphoblastic and myeloid leukemias, and results in the fusion of the clathrin assembly lymphoid myeloid leukemia (CALM) gene with the AF10 gene; these genes are present on chromosomes 11 and 10, respectively. Because the CALM-AF10 rearrangement is a rare chromosomal abnormality, it is not included in routine molecular tests for acute leukemia. Here, we describe the cases of 2 patients with the CALM-AF10 fusion gene. The first patient (case 1) was diagnosed with T-cell ALL, and the second patient (case 2) was diagnosed with AML. Both patient samples showed expression of the homeobox A gene cluster and the histone methyltransferase hDOT1L, which suggests that they mediate leukemic transformation in CALM-AF10-positive and mixed-lineage leukemia-AF10-positive leukemias. Both patients achieved complete remission after induction chemotherapy. The first patient (case 1) relapsed after double-unit cord blood transplantation; there was no evidence of relapse in the second patient (case 2) after allogenic peripheral blood stem cell transplantation. Since CALM-AF10- positive leukemias have been shown to have poor prognosis with conventional therapy, molecular tests for CALM-AF10 rearrangement would be necessary to detect minimal residual disease during follow-up.

Identification and Analysis of Afe0329 Transcripton Relative to Copper Homeostasis in Acidithiobacillus ferrooxidans / 微生物学通报

With reverse transcriptase PCR, the transcripton of copper homeostasis relative gene Afe0329 in Acidithiobacillus ferrooxians standard strain ATCC23270 was investigated. The further analysis of genes in this transcripton was analyzed employed by Vector NTI, Blast, TMHMM Server, PSORTb software and so on. From the DNA of different strains, the transcripton of Afe0329 was amplified using special primer pairs to identify the universality of it in the genome of A.ferrooxidans strains. The results showed that gene Afe0330 and Afe0331 were cotranscribed with Afe0329, and they were in a single transcripton. Gene Afe0329 was supported to express a P1b3-type ATPase which is a heavy metal ion pumping transmembrane protein, protein AFE0330 which expressed by gene Afe0330 was a cytoplasmic protein, no significant ho- mologous sequences of Afe0330 or Afe0331 had been obtained by Blast analysis. And the transcripton of Afe0329 was universal in genome of A. ferrooxidans strains.

Real-time quantitative RT-PCR for measurement of expression of eNOSmRNA and Tie-2mRNA in BMSCs-derived Endothelial Cells / 中国矫形外科杂志

0.05).[Conclusion]BMSCs could be successfully differentiated into endothelial cells in vitro,and the functional genes were stably expressed in BMSCs-derived endothelial cells.They are ideal donor cells of tissue engineering vascularization.

Detection of circulation AFPmRNA and telomerase activity in peripheral blood mononuclear cells of patients with hepatocellular carcinoma / 中国普通外科杂志

Objective To investigate the role of detection of circulation alpha-fetoprotein(AFP) mRNA and telomerase activity in peripheral blood mononuclear cells(PBMC) of patients with hepatocellular carcinoma(HCC).Methods In patients with HCC,benign liver tumors,chronic liver diseases,and healthy volunteers,expression of circulation AFPmRNA and telomerase activity in PBMC were detected using nest reverse transcriptase-PCR(Nest RT-PCR) or PCR-ELISA respectively.Results(1) All the patients with benign liver tumors(n=10),chronic liver diseases(n=30),and healthy volunteers(n=30) had negative expression of circulation AFPmRNA,and the positive rate of circulation AFPmRNA in patients with HCC(73.3%,22/30)(all P