Role of the IL8 rs4073 polymorphism in central nervous system toxicity in patients receiving multidrug-resistant tuberculosis treatment / Papel do polimorfismo rs4073 do gene IL8 na toxicidade do sistema nervoso central em pacientes em tratamento para tuberculose multirresistente

ABSTRACT Objective: To determine the role of the IL8 rs4073 polymorphism in predicting the risk of central nervous system (CNS) toxicity in patients receiving standard pharmacological treatment for multidrug-resistant tuberculosis (MDR-TB). Methods: A cohort of 85 consenting MDR-TB patients receiving treatment with second-line antituberculosis drugs had their blood samples amplified for the IL8 (rs4073) gene and genotyped. All patients were clinically screened for evidence of treatment toxicity and categorized accordingly. Crude and adjusted associations were assessed. Results: The chief complaints fell into the following categories: CNS toxicity; gastrointestinal toxicity; skin toxicity; and eye and ear toxicities. Symptoms of gastrointestinal toxicity were reported by 59% of the patients, and symptoms of CNS toxicity were reported by 42.7%. With regard to the genotypes of IL8 (rs4073), the following were identified: AA, in 64 of the study participants; AT, in 7; and TT, in 11. A significant association was found between the dominant model of inheritance and CNS toxicity for the crude model (p = 0.024; OR = 3.57; 95% CI, 1.18-10.76) and the adjusted model (p = 0.031; OR = 3.92; 95% CI, 1.13-13.58). The AT+TT genotype of IL8 (rs4073) showed a 3.92 times increased risk of CNS toxicity when compared with the AA genotype. Conclusions: The AT+TT genotype has a tendency to be associated with an increased risk of adverse clinical features during MDR-TB treatment.

RESUMO Objetivo: Determinar o papel do polimorfismo rs4073 do gene IL8 na previsão do risco de toxicidade do sistema nervoso central (SNC) em pacientes em tratamento farmacológico padrão para tuberculose multirresistente (TBMR). Métodos: Amostras de sangue de uma coorte de 85 pacientes com TBMR que assinaram um termo de consentimento livre e esclarecido e que estavam recebendo tratamento com medicamentos antituberculosos de segunda linha foram amplificadas para o gene IL8 (rs4073) e genotipadas. Todos os pacientes foram avaliados clinicamente quanto a evidências de toxicidade do tratamento e categorizados de acordo com os achados. Foram avaliadas as associações brutas e ajustadas. Resultados: As principais queixas enquadraram-se nas seguintes categorias: toxicidade do SNC; toxicidade gastrointestinal; toxicidade cutânea; e toxicidade ocular e ototoxicidade. Sintomas de toxicidade gastrointestinal foram relatados por 59% dos pacientes, e sintomas de toxicidade do SNC foram relatados por 42,7%. Foram identificados os seguintes genótipos de IL8 (rs4073): AA, em 64 dos participantes; AT, em 7; TT, em 11. Houve associação significativa entre o modelo dominante de herança e toxicidade do SNC no modelo bruto (p = 0,024; OR = 3,57; IC95%: 1,18-10,76) e no ajustado (p = 0,031; OR = 3,92; IC95%: 1,13-13,58). O genótipo AT+TT do gene IL8 (rs4073) apresentou risco 3,92 vezes maior de toxicidade do SNC que o genótipo AA. Conclusões: O genótipo AT+TT tende a se associar a um maior risco de características clínicas adversas durante o tratamento da TBMR.

Corneal transplantation and concomitant SARS-CoV-2 infection: a case report / Transplante de córnea e infecção concomitante por SARS-CoV-2: relato de caso

ABSTRACT This report was aimed at presenting a case of neurotrophic keratitis and concomitant SARS-CoV-2 infection in a patient who has recently undergone a corneal DALK transplant. One month after corneal transplantation with adequate corneal epithelialization, the patient presented neurotrophic keratitis with a torpid course of the corneal transplant coinciding with a SARS-CoV-2 infection, with an excessive host immune response. In addition, the patient presented a re-positivization of nasopharyngeal polymerase chain reaction of SARS-CoV-2 with past disease after starting treatment with autologous serum eye drops. The implications at the ophthalmological level of SARS-CoV-2 infection may be clarified as the time the illness progresses and we learn more about how it acts. In this case, the disparity of signs and symptoms, the antecedent of corneal surgery, and the possibility of a herpetic infection as a cause of the primary leukoma suggested neurotrophic keratitis. Nonetheless, the involvement of systemic SARS-CoV-2 infection in the process, triggering an excessive host immune response at the corneal level with an increase in inflammatory cytokines must be taken into account. No relationship was found between treatment with autologous serum and re-positivization of nasopharyngeal polymerase chain reaction, presenting the patient a favorable response to treatment.

RESUMO O objetivo deste relato foi apresentar um caso de ceratite neurotrófica e infecção concomitante por SARS-CoV-2 em paciente submetido recentemente a transplante de córnea DALK. Um mês após o transplante de córnea com adequada epitelização da córnea, o paciente apresentou ceratite neurotrófica com curso tórpido do transplante de córnea, coincidindo com infecção por SARS-CoV-2, com resposta imune excessiva do hospedeiro. Além disso, o paciente apresentou repositivização da reação em cadeia da polimerase nasofaríngeo de SARS-CoV-2, com doença pregressa após iniciar tratamento com colírio de soro autólogo. As implicações a nível oftalmológico da infecção por SARS-CoV-2, podem ser esclarecidas à medida que a doença progride e aprendemos mais sobre sua forma de atuação. Neste caso, a disparidade de sinais e sintomas, o antecedente de cirurgia de córnea e a possibilidade de infecção herpética como causa do leucoma primário sugeriram ceratite neurotrófica. No entanto, deve-se levar em consideração o envolvimento da infecção sistêmica por SARS-CoV-2 no processo, desencadeando uma resposta imune excessiva do hospedeiro no nível da córnea, com aumento de citocinas inflamatórias. Não foi encontrada relação entre o tratamento com soro autólogo e a repositivização da reação em cadeia da polimerase nasofaríngea, apresentando ao paciente uma resposta favorável ao tratamento.

SARS-CoV-2 and the ocular surface: test accuracy and viral load / SARS-CoV-2 e a superfície ocular: acurácia dos testes e carga viral

ABSTRACT Purpose: This study aimed to evaluate the pre-sence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the ocular surface of individuals clinically suspected of coronavirus disease 2019 (COVID-19) and determine the accuracy of different approaches of molecular testing on the ocular surface based on the nasopharyngeal positivity status for COVID-19. Methods: A total of 152 individuals with suspected COVID-19 symptoms who simultaneously underwent nasopharyngeal and two different tear film collection techniques for quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) were included. Tears were collected and randomized: one eye had the filter strip for the Schirmer test and the contralateral eye had conjunctival swab/cytology in the inferior fornix. All patients underwent slit lamp biomicroscopy. The accuracy of various ocular surface collection techniques used for the detection of SARS-CoV-2 RNA was determined. Results: Of the 152 patients enrolled in the study, 86 (56.6%) had COVID-19 confirmed by nasopharyngeal PCR. Both tear film collection techniques detected viral particles: the Schirmer test was positive in 16.3% (14/86) and the conjunctival swab/cytology in 17.4% (15/86), with no statistically significant differences. No positive ocular tests were found among those with negative nasopharyngeal PCR tests. The overall agreement of the ocular tests was 92.7%, and in combination, the sensitivity would increase to 23.2%. The mean cycle threshold values in the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests were 18.2 ± 5.3, 35.6 ± 1.4, and 36.4 ± 3.9, respectively. Compared with the nasopharyngeal test, the Schirmer (p=0.001) and conjunctival swab/cytology (p<0.001) tests had significantly different Ct values. Conclusion: The Schirmer (16.3%) and conjunctival swab (17.4%) tests were comparably capable of detecting SARS-CoV-2 RNA in the ocular surface by RT-PCR accurately based on nasopharyngeal status and demonstrated indistinct sensitivity and specificity. Simultaneous specimen sampling and processing from the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests demonstrated significantly lower viral load in both ocular surface approaches than in the nasopharyngeal test. Ocular manifestations detected by slit lamp biomicroscopy were not associated with ocular RT-PCR positivity.

RESUMO Objetivo: Avaliar a presença de RNA de coronavírus 2 causador de síndrome respiratória aguda grave (SARS-CoV-2) na superfície ocular de indivíduos clinicamente suspeitos com COVID-19 e determinar a precisão de diferentes abordagens de testes moleculares na superfície ocular com base no status de positividade do RT-qPCR de nasofaringe para COVID-19. Métodos: 152 indivíduos com sintomas suspeitos para a COVID-19 foram submetidos a coleta de reação em cadeia da polimerase de nasofaringe simultaneamente a duas técnicas diferentes de coleta de filme lacrimal para RT-qPCR: aleatoriamente, um olho com a tira filtro do teste de Schirmer e, o olho contralateral, com citologia (swab) conjuntival no fórnice inferior. Todos os indivíduos foram submetidos à biomicroscopia com lâmpada de fenda. Resultados: Dos 152 pacientes, 86 (56,6%) tiveram a COVID-19 confirmada por PCR de nasofaringe. Ambas as técnicas de coleta detectaram partículas virais: o teste de Schirmer foi positivo em 16,3% (14/86) e a citologia conjuntival em 17,4% (15/86), sem diferenças estatisticamente significativas. Não houve testes oculares positivos entre aqueles com reação em cadeia da polimerase de nasofaringe negativo. A concordância geral dos testes oculares foi de 92,7% e, em combinação, a sensibilidade aumentaria para 23,2%. Os valores médios do limiar de ciclo nos testes de nasofaringe, Schirmer e citologia conjuntival foram 18,2 ± 5,3, 35,6 ± 1,4 e 36,4 ± 3,9, respectivamente. Conclusão: Os testes de Schirmer (16,3%) e swab conjuntival (17,4%) foram igualmente capazes de detectar RNA de SARS-CoV-2 na superfície ocular por RT-PCR e demonstraram sensibilidade e especificidade indistintas. A coleta simultânea de amostras ao processamento dos testes de RT-PCR de nasofaringe, Schirmer e citologia (swab) conjuntival demonstraram carga viral significativamente menor em ambas as abordagens da superfície ocular em comparação com o teste de nasofaringe. As manifestações oculares detectadas pela biomicroscopia com lâmpada de fenda não foram claramente associadas à positividade do RT-PCR ocular.

Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks / Avaliação clínico-patológica do Vírus da Doença de Newcastle em patos

Abstract Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Resumo A doença de Newcastle (DN) é uma doença infecciosa, altamente contagiosa e letal de espécies aviárias. Considera-se que os patos são reservatórios ou portadores naturais do vírus da doença de Newcastle (VDN) e são resistentes a diferentes cepas de VDN. O presente estudo foi desenvolvido para avaliar a patogênese da DN em patos domésticos por meio de histopatologia, imuno-histoquímica (IHQ) e alterações bioquímicas séricas. Para este propósito, 80 patos foram criados por 42 dias e divididos em dois grupos A e B. Os patos do grupo A foram submetidos ao VDN a uma taxa de 0,1 ml de ELD50 (título viral de 107,32 / 100 µl) na segunda semana de idade, enquanto o Grupo B foi controle negativo. Esplenomegalia, atrofia do timo e lesão necrótica no rim foram observadas no 9º dia pós-infecção. Degeneração hepática e infiltração de células mononucleares foram observadas no proventrículo e intestino em patos infectados. Antígeno viral foi detectado em pulmões, intestino, proventrículo e órgãos linfoides de patos infectados por IHQ. Os valores de albumina e proteína total foram significativamente baixos no grupo A infectado em comparação com o grupo B. Os valores de ALT, AST e ALP foram significativamente altos no grupo A. No 5º e no 7º dia após a infecção, os esfregaços orofaríngeos foram negativos para VDN, enquanto os esfregaços cloacais foram positivos para VDN por meio da reação em cadeia da polimerase via transcriptase reversa. Conclui-se que os patos são suscetíveis ao VDN e à cepa virulenta de VDN que causou doenças em patos.

Recent advances in small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase / 药学学报

Influenza virus causes serious threat to human life and health. Due to the inherent high variability of influenza virus, clinically resistant mutant strains of currently approved anti-influenza virus drugs have emerged. Therefore, it is urgent to develop antiviral drugs with new targets or mechanisms of action. RNA-dependent RNA polymerase is directly responsible for viral RNA transcription and replication, and plays key roles in the viral life cycle, which is considered an important target of anti-influenza drug design. From the point of view of medicinal chemistry, this review summarizes current advances in diverse small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase, hoping to provide valuable reference for development of novel antiviral drugs.

Identification of Scolopendra Dispensing Granules by Allele-specific PCR / 中国实验方剂学杂志

ObjectiveTo establish an allele-specific polymerase chain reaction （PCR） method for identifying Scolopendra dispensing granules， so as to ensure the quality and therapeutic effects of Scolopendra and its preparations. MethodThe primer interval suitable for the PCR was selected based on the cytochrome c oxidase subunit 3（COX-3） gene sequence of Scolopendra， and the single nucleotide polymorphism （SNP） loci of Scolopendra and its adulterants were mined from the interval for the design of specific primers. The samples of Scolopendra and its adulterants were collected. The PCR system was established and optimized regarding the annealing temperature， cycles， Taq enzymes， DNA template amount， PCR instruments， and primer concentrations， and the specificity and applicability of this method were evaluated. ResultThe PCR system was composed of 12.5 μL 2×M5 PCR Mix， 0.4 μL forward primer （10 μmol·L-1）， 0.4 μL reverse primer （10 μmol·L-1）， 2.5 μL DNA template， and 9.2 μL sterile double distilled water. PCR parameters： Pre-denaturation at 94 ℃ for 3 min， 30 cycles （94 ℃ for 20 s， 62 ℃ for 20 s， 72 ℃ for 45 s）， and extension at 72 ℃ for 5 min. After PCR amplification with the system and parameters above， the electrophoresis revealed a bright band at about 135 bp for Scolopendra and no band for the adulterants. ConclusionThe established allele-specific PCR method can accurately identify the medicinal materials， decoction pieces， and standard decoction freeze-dried powder of Scolopendra， as well as the intermediates and final products of Scolopendra dispensing granules， which is of great significance for ensuring the quality and clinical efficacy of Scolopendra and its preparations.

PCR-RFLP for Distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method for rapid distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex， so as to avoid the influence of genetic confusion on drug safety. MethodThe DSS-tagged sequences of Periplocae Cortex were obtained from the Chloroplast Genome Information Resource （CGIR） and analyzed to find the enzymatic cleavage sites that were different from those of Acanthopanacis Cortex and Lycii Cortex. The specific enzymatic cleavage site， Cla I， of Periplocae Cortex was selected， on the basis of which the primers for PCR-RFLP were designed. Furthermore， the factors such as annealing temperature， number of cycles， Taq enzyme， PCR instruments， and enzymatic treatment time that may influence PCR-RFLP were studied. The established PCR-RFLP method was applied to the identification of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex samples produced in different regions. ResultThe PCR-RFLP at the annealing temperature of 59 ℃ and with 40 cycles showed clear bands of the samples. When the enzyme digestion time was 30 min. The reaction produced the target bands at about 140 bp and 290 bp for both Periplocae Cortex and its original plant and only a band at about 430 bp for Acanthopanacis Cortex， Lycii Cortex， and their original plants. The method can accurately distinguish Periplocae Cortex from its confounders Acanthopanacis Cortex and Lycii Cortex. ConclusionThe PCR-RFLP method for distinguishing Periplocae Cortex from Acanthopanacis Cortex and Lycii Cortex was established. It has high stability， sensitivity， and applicability， providing a reference for the quality control of Periplocae Cortex， Acanthopanacis Cortex， and Lycii Cortex.

Identification of Original Plants of Lycii Cortex by Multiplex Allele-specific PCR / 中国实验方剂学杂志

ObjectiveTo identify Lycium chinense and L. barbarum as the original plants of Lycii Cortex simply and efficiently by multiple allele-specific polymerase chain reaction （PCR）. MethodThe chloroplast genome sequences of L. chinense and L. barbarum were downloaded from the Chloroplast Genome Information Resource （CGIR）， and then IdenDSS was employed to screen out the specific single nucleotide polymorphism （SNP） sites between the two plants. Primer 5.0 was used to design the specific primers， including primers GQ-F/R for identifying L. chinense and primers NX-F/R for identifying L. barbarum. Furthermore， the primer concentration ratio， annealing temperature， cycles， and Taq enzyme were optimized to establish the optimal PCR system and conditions for plant identification. Finally， the applicability of the established method was examined with the plant samples collected from different regions. ResultThe PCR with GQ-F/R∶NX-F/R concentration ratio of 2∶1 at the annealing temperature at 59 ℃ and for 30 cycles showed specific bands at 183 bp and 295 bp， respectively， for L. chinense and L. barbarum samples from different regions. ConclusionThe established PCR approach can simply， rapidly， and efficiently identify the original plants of Lycii Cortex， serving as a new method for the discrimination between L. chinense and L. barbarum. Moreover， the method provides technical support for the research and development of classic famous prescriptions containing Lycii Cortex.

A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Specific PCR for Identification of Astragalus membranaceus var. mongholicus Seeds， A. membranaceus Seeds， and Adulterants / 中国实验方剂学杂志

ObjectiveTo establish a method based on specific polymerase chain reaction （PCR） that can accurately and rapidly identify Astragalus membranaceus var. mongholicus （AMM） seeds and A. membranaceus （AM） seeds. MethodThe Chloroplast Genome Information Resource （CGIR） and IdenDSS were used to obtain the characteristic DNA fragments of AMM and AM， and the specific single nucleotide polymorphism （SNP） sites of AMM and AM were screened out， on the basis of which the specific primers MG-F/MG-R of AMM and MJ-F/MJ-R of AM were designed. The specific PCR method for identifying AMM and AM was established and optimized， and the specificity and applicability of the method were investigated. ResultThe specific PCR conditions for the identification of AMM were primers MG-F/MG-R， annealing at 62 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the specific band appeared at about 220 bp， with no band for the seeds of AM or adulterants. The specific PCR conditions for identifying the AM were primers MJ-F/MJ-R， annealing at 58 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the band appeared at about 150 bp， with no band of AMM or adulterants. ConclusionThe specific PCR method established in this study can accurately and quickly identify the seeds of AMM and AM， which provides a basis for the classification and accurate identification of Astragalus seeds and adulterants.

Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks

Abstract Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Resumo A doença de Newcastle (DN) é uma doença infecciosa, altamente contagiosa e letal de espécies aviárias. Considera-se que os patos são reservatórios ou portadores naturais do vírus da doença de Newcastle (VDN) e são resistentes a diferentes cepas de VDN. O presente estudo foi desenvolvido para avaliar a patogênese da DN em patos domésticos por meio de histopatologia, imuno-histoquímica (IHQ) e alterações bioquímicas séricas. Para este propósito, 80 patos foram criados por 42 dias e divididos em dois grupos A e B. Os patos do grupo A foram submetidos ao VDN a uma taxa de 0,1 ml de ELD50 (título viral de 107,32 / 100 µl) na segunda semana de idade, enquanto o Grupo B foi controle negativo. Esplenomegalia, atrofia do timo e lesão necrótica no rim foram observadas no 9º dia pós-infecção. Degeneração hepática e infiltração de células mononucleares foram observadas no proventrículo e intestino em patos infectados. Antígeno viral foi detectado em pulmões, intestino, proventrículo e órgãos linfoides de patos infectados por IHQ. Os valores de albumina e proteína total foram significativamente baixos no grupo A infectado em comparação com o grupo B. Os valores de ALT, AST e ALP foram significativamente altos no grupo A. No 5º e no 7º dia após a infecção, os esfregaços orofaríngeos foram negativos para VDN, enquanto os esfregaços cloacais foram positivos para VDN por meio da reação em cadeia da polimerase via transcriptase reversa. Conclui-se que os patos são suscetíveis ao VDN e à cepa virulenta de VDN que causou doenças em patos.

Estandarización de PCR en tiempo real para detección y cuantificación de los oncogenes E7 de seis Virus del Papiloma Humano de Alto Riesgo

El cáncer de cuello uterino es causado por la infección persistente del epitelio cervical con los genotipos de alto riesgo del Virus del Papiloma Humano. Para su detección se realizan pruebas moleculares que detectan el gen L1 del VPH. Este gen puede perderse hasta en el 11 % de los casos durante la integración del ADN viral en el genoma del hospedero originando falsos negativos. Por otra parte, el oncogén E7 se expresa durante todas las etapas de progresión de la enfermedad. El objetivo de este trabajo fue estandarizar una PCR en tiempo real del oncogén E7 (E7-qPCR) para genotipificación y cuantificación de 6 VPH-AR. Los resultados muestran que la E7- qPCR amplificó VPH-16, -18, -31, -33, -35 y -45 con una alta sensibilidad con límites de detección desde 102 copias, eficiencias entre 90 y 110 %, valores R2 > 0,97 y análisis de curva de fusión que revelan productos específicos.

Cervical cancer is caused by persistent infection of the cervical epithelium with the high-risk genotypes of the Human Papilloma Virus (HR-HPV). For its detection, molecular tests are carried out that detect the L1 gene of HPV. This gene can be lost in up to 11 % of cases during the integration of viral DNA into the host genome, causing false negatives. On the other hand, the E7 oncogene is expressed during all stages of disease progression. The aim of this work was to standardize a real-time PCR of the E7 oncogene (E7-qPCR) for genotyping and quantification of 6 HR-HPV. The results show that the E7-qPCR amplified HPV-16, -18, -31, -33, -35 and -45 with high sensitivity with detection limits from 102 copies, efficiencies between 90 and 110 %, R2 values >0,97 and melting curve analysis revealing specific products.

Cycle threshold predicted mortality in a cohort of patients with hematologic malignancies infected with SARS-CoV-2 / El umbral de ciclado predijo la mortalidad en una cohorte de pacientes conneoplasias hematológicas infectados con SARS-CoV-2 / Microbiología clinica y enfermedades infecciosas

Abstract When a SARS-CoV-2 RT-qPCR test is performed, it may determine an indirect measureof viral load called cycle threshold (Ct). Respiratory samples with Ct <25.0 cycles are consideredto contain a high viral load. We aimed to determine whether SARS-CoV-2 Ct at diagnosis couldpredict mortality in patients with hematologic malignancies (lymphomas, leukemias, multiplemyeloma) who contracted COVID-19. We included 35 adults with COVID-19 confirmed by RT-qPCR performed at diagnosis. We evaluated mortality due to COVID-19 rather than mortalitydue to the hematologic neoplasm or all-cause mortality. Twenty-seven (27) patients survivedand 8 died. The global mean Ct was 22.8 cycles with a median of 21.7. Among the survivors,the mean Ct was 24.2, and the median Ct value was 22.9 cycles. In the deceased patients, themean Ct was 18.0 and the median Ct value was 17.0 cycles. Using the Wilcoxon Rank Sum test,we found a significant difference (p = 0.035). SARS-CoV-2 Ct measured in nasal swabs obtainedat diagnosis from patients with hematologic malignancies may be used to predict mortality.

Resumen Cuando se realiza una RT-qPCR para SARS-CoV-2, es posible determinar una medidaindirecta de la carga viral llamada umbral de ciclado (Ct). Las muestras respiratorias con Ct<25,0 ciclos se consideran de alta carga viral. Nos propusimos determinar si el Ct para SARS-CoV-2 al diagnóstico predice la mortalidad en pacientes con neoplasias hematológicas (linfomas,leucemias, mielomas) que contrajeron COVID-19. Incluimos 35 adultos con COVID-19 confirmadopor RT-qPCR al diagnóstico. Evaluamos la mortalidad por COVID-19, no la mortalidad por la neo-plasia hematológica o la mortalidad por cualquier causa. De los 35 pacientes, 27 sobrevivierony 8 fallecieron. El Ct global medio fue 22,8 ciclos con una mediana de 21,7 ciclos. Entre lossobrevivientes, el Ct medio fue 24,2 ciclos con una mediana de 22,9 ciclos. Entre los fallecidos,el Ct medio fue 18,0 y el Ct mediano fue 17,0 ciclos. Empleando la prueba de suma de rangosde Wilcoxon, encontramos una diferencia significative (p = 0,035). En pacientes con neoplasiashematológicas infectados con coronavirus, el Ct de SARS-CoV-2 medido en hisopados nasales almomento del diagnóstico podría ser utilizado para predecir la mortalidad.

Asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en la enfermedad cerebrovascular isquémica

Fundamento: la depresión es una de las complicaciones no neurológicas más frecuentes en la enfermedad cerebrovascular isquémica. Objetivo: determinar la asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en pacientes con enfermedad cerebrovascular isquémica. Métodos: se realizó un estudio analítico, prospectivo de corte transversal en pacientes con enfermedad cerebrovascular isquémica en fase aguda (N=22) y no aguda (N=37); atendidos en el Instituto de Neurología y Neurocirugía y el Hospital Manuel Fajardo, de La Habana, Cuba. Se recogieron variables demográficas, factores de riesgo, etiología y localización del infarto, deficiencia neurológica, discapacidad para las actividades de la vida diaria (índice de Barthel), neuropsicológicas (depresión por inventario de Beck y test de Hamilton). Se determinó proteína C-reactiva, alfa-1-antitripsina, complementos C3 y C4 y microalbuminuria. Resultados: las puntuaciones de las pruebas neuropsicológicas no tuvieron diferencias significativas entre la fase aguda y no aguda, pero hubo un aumento estadístico de la frecuencia de pacientes sin depresión y con ligera depresión en la fase no aguda. En la fase aguda, el complemento C4 y en la fase no aguda el complemento C3, la proteína C-reactiva y el alfa-1-antitripsina se correlacionaron directamente con la puntuación del inventario de Beck. La proteína C-reactiva y C3 se correlacionaron estadísticamente con la puntuación del test de Hamilton. En el análisis multivariado, la proteína C-reactiva mostró asociación independiente con el grado de depresión por el test de Hamilton. Conclusiones: la proteína C-reactiva pudiera estar relacionada con la severidad de la depresión, quizás por asociación con la discapacidad para las actividades de vida diaria.

Foundation: depression in ischemic cerebrovascular disease is one of the most frequent non-neurological complications. Objective: to determine the association of inflammatory markers and endothelial dysfunction with depression in patients with ischemic cerebrovascular disease. Methods: an analytical, prospective, cross-sectional study was carried out in patients with acute (N=22) and non-acute (N=37) ischemic cerebrovascular disease; treated at the Institute of Neurology and Neurosurgery; and the Manuel Fajardo Hospital, in Havana, Cuba. Demographic variables, risk factors, etiology and location of the infarction, neurological deficiency, disability for activities of daily living (Barthel index), neuropsychological (depression by Beck inventory and Hamilton test) were collected. C-reactive protein, alpha-1-antitrypsin, C3 and C4 complements, and microalbuminuria were determined. Results: the scores of the neuropsychological tests did not have significant differences between the acute and non-acute phase, but there was a statistical increase in the frequency of patients without depression and with slight depression in the non-acute phase. In the acute phase, C4, and in the non-acute phase, C3, C-reactive protein and alpha-1-antitrypsin were directly correlated with the Beck inventory score. C-reactive protein and C3 were statistically correlated with the Hamilton test score. In the multivariate analysis, C-reactive protein showed an independent association with the degree of depression by the Hamilton test. Conclusions: C-reactive protein could be related to the severity of depression, perhaps by association with the disability for activities of daily living.

Negativización de carga viral en pacientes positivos persistentes SARS-CoV-2 y uso de plasma de convalecientes / Negative viral load in persistent positive SARS-CoV-2 patients and use of convalescent plasma

No existe medicación específica contra el SARS-CoV-2 y el tratamiento consiste fundamentalmente en medidas de soporte. La transfusión de plasma de convalecientes consiste en administrar pasivamente anticuerpos policlonales, que generan una respuesta inmune inmediata y disminuyen la carga viral. El objetivo del estudio fue describir la estadía hospitalaria y negativización de reacción en cadena de la polimerasa en tiempo real en pacientes positivos persistentes con el uso de plasma de convalecientes. Se realizó un estudio descriptivo transversal, prospectivo, en 8 pacientes positivos persistentes que recibieron dicho plasma, en el Hospital Universitario Clínico Quirúrgico «Cmdte. Manuel Fajardo» de septiembre a noviembre de 2020. El mayor por ciento de casos necesitó una dosis de plasma de convalecientes para negativizar el PCR-TR. La estadía hospitalaria más frecuente fue de 14 a 19 días, el 62,50 % de los pacientes, 24 horas después de administrada la última dosis de este plasma, negativizó el RCP-TR evolutivo.

There is no specific medication against SARS-CoV-2 and the treatment consists mainly of supportive measures. Convalescent plasma transfusion consists of passively administered polyclonal antibodies, which generate an immediate immune response and decrease viral load. The objective of the study was to describe hospital stay and real-time polymerase chain reaction negativization in persistently positive patients with the use of convalescent plasma. A prospective, cross-sectional and descriptive study was carried out in 8 persistently positive patients who received such plasma at "Cmdte. Manuel Fajardo" Clinical and Surgical University Hospital from September to November 2020. The highest percentage of cases required a dose of convalescent plasma to make the RT-PCR negative. The most frequent hospital stay was from 14 to 19 days; 62.50% of the patients had a negative evolutionary RT-PCR, 24 hours after administering the last dose of this plasma.

Validación de la reacción de polimerasa en cadena en tiempo real como herramienta diagnóstica en la aspergilosis asociada a COVID-19 en pacientes hospitalizados en Clínica Dávila de Santiago, Chile, durante los años 2020 y 2021

Introducción: Desde el inicio de la pandemia por COVID-19 se han registrado casos de infecciones de aspergilosis pulmonar asociada a esta infección, la cual tiene características diferentes a la aspergilosis pulmonar clásica y, por lo tanto, han significado un desafío diagnóstico. Objetivo: Validar una reacción de polimerasa en cadena (RPC) en tiempo real (sigla en inglés RT-PCR) comercial, como herramienta diagnóstica alternativa a la técnica de galactomanano (GM) en el diagnóstico de aspergilosis pulmonar asociada a COVID-19 (sigla en inglés CAPA). Pacientes y Método: Se analizaron resultados de RT-PCR de Aspergillus spp y GM en lavado bronco-alveolar (LBA) de 72 pacientes, hospitalizados por COVID-19 de Clínica Dávila entre los años 2020 y 2021. De estos pacientes, 33 presentaron CAPA. Resultados: La RT-PCR de Aspergillus y GM presentaron una correlación positiva (r = 0,6351, valor p < 0,0001). La técnica de RT-PCR presentó una sensibilidad (S), especificidad (E), valor predictor positivo (VPP) y valor predictor negativo (VPN) de 100, 44, 66 y 100%, respectivamente, mientras que en GM fueron de 64, 89, 84 y 73%, respectivamente para LBA. Al utilizar ambas técnicas en combinación se obtuvo una S, E, VPP y VPN de 100, 82, 88 y 100%, respectivamente. Conclusión: Este estudio concluyó que usar una técnica de RT-PCR de Aspergillus y GM en conjunto en LBA mejoraron los parámetros de desempeño de ambas técnicas usadas de manera individual para diagnosticar CAPA. Se requieren más estudios para evaluar el desempeño de técnicas combinadas en otros tipos de aspergilosis.

Background: Since the beginning of the COVID-19 pandemic, there have been cases of pulmonary aspergillosis infections associated with this infection, which has different characteristics from classical pulmonary aspergillosis and therefore, have been diagnostic challenges. Aim: To validate a commercial real-time PCR (RT-PCR) method as an alternative diagnostic tool to the galactomannan (GM) technique in the diagnosis of COVID-19-associated pulmonary aspergillosis (CAPA). Methods: Results of RT-PCR of Aspergillus spp and GM in broncho-alveolar lavage (BAL) of 72 patients hospitalized for COVID-19 at Clínica Dávila between 2020 and 2021 were analyzed. Of these patients, 33 presented CAPA. Results: The RT-PCR for Aspergillus and GM showed a positive correlation (r = 0.6351, p-value < 0.0001). The RT-PCR for Aspergillus technique presented a sensitivity (S), specificity (S), positive predictive value (PPV) and negative predictive value (NPV) of 100, 44, 66 and 100% respectively, while the GM technique presented 64, 89, 84 and 73%, respectively for BAL. Using both techniques in combination a S, E, PPV and NPV of 100, 82, 88 and 100% were obtained respectively. Conclusion: This study concluded that using RT-PCR and GM techniques in combination in BAL improved the performance parameters of both techniques from those used individually to diagnose CAPA. Further studies are required to evaluate the performance of combined techniques in other aspergillosis focus.

The conventional diagnosis challenge: Real-time PCR and nested PCR correlation with the scoring system for individuals at high-risk of Pneumocystis jirovecii pneumonia

Introduction. Pneumocystis jirovecii is an opportunistic fungus that affects mainly people living with HIV (CD4 cell count lower than 200 cells/ml) and other immunosuppressed patients. Since P. jirovecii does not grow on routine mycological media, diagnosis of P. jirovecii pneumonia relies on indirect evidence of its presence in respiratory samples. Objectives. To associate the results of direct immunofluorescence and two molecular methods with a score to predict P. jirovecii pneumonia in patients with AIDS. Materials and methods. A prospective study was conducted with 40 patients. A respiratory sample collected before treatment was subjected to direct immunofluorescence using the Merifluor kit, to nested PCR targeting the mitochondrial large subunit ribosomal RNA, and to the VIASURE real-time PCR kit. Results. These three techniques revealed P. jirovecii in 6, 12, and 15 samples, respectively. All positive samples by direct immunofluorescence were positive by nested PCR, and all positive samples by nested PCR amplified by real-time PCR. There was a statistically significant association between the P. jirovecii pneumonia score and the molecular methods. Two patients were early diagnosed and responded well to treatment. Conclusion. Molecular methods, especially real-time PCR, are recommended for early diagnosis of P. jirovecii pneumonia in AIDS patients.

Introducción. Pneumocystis jirovecii es un hongo oportunista que afecta principalmente a personas con HIV (recuento de CD4 menor de 200 células/ml) y a otros pacientes inmunosuprimidos. Como P. jirovecii no crece en los medios micológicos de rutina, el diagnóstico de neumonía por P. jirovecii se basa en la evidencia presente en muestras respiratorias. Objetivos. Asociar los resultados de la inmunofluorescencia directa y los de dos métodos moleculares con un puntaje para predecir la neumonía causada por P. jirovecii en pacientes con sida. Materiales y métodos. Se realizó un estudio prospectivo de 40 pacientes. Se recolectó una muestra respiratoria antes del inicio de tratamiento y se sometió a una prueba de inmunofluorescencia directa con el kit Merifluor, una PCR anidada para la amplificación de la subunidad larga del ribosoma mitocondrial y una PCR en tiempo real usando el kit VIASURE. Resultados. Estas tres técnicas evidenciaron la presencia de P. jirovecii en 6, 12 y 15 muestras, respectivamente. Todas las muestras positivas por inmunofluorescencia directa fueron positivas en la PCR anidada y todas las muestras positivas en la PCR anidada amplificaron por PCR en tiempo real. Se encontró una asociación estadística entre los valores de la neumonía causada por P. jirovecii y los métodos moleculares. Dos pacientes con diagnóstico temprano respondieron satisfactoriamente al tratamiento. Conclusión. Se recomiendan los métodos moleculares, especialmente la PCR en tiempo real, para el diagnóstico temprano de neumonía causada por P. jirovecii en pacientes con sida.

Hiperplasia pseudoepiteliomatosa: carcinoma escamocelular versus paracoccidioidomicosis oral, un caso con mirada dermatológica / Pseudoepitheliomatous hyperplasia: Squamous cell carcinoma versus oral paracoccidioidomycosis, a case from a dermatological perspective

La paracoccidioidomicosis es una micosis sistémica endémica en Latinoamérica. La presentación más frecuente compromete crónicamente los pulmones, la piel y las mucosas. Al inicio, este paciente presentó, por varios años, una lesión única en la mucosa oral que, en ausencia de otros síntomas, se relacionó con una neoplasia maligna, específicamente con un carcinoma escamocelular. La diferenciación entre los dos diagnósticos se hace mediante un examen directo, un estudio histopatológico y cultivos iniciales y subsecuentes. Sin embargo, tales estudios no fueron concluyentes. Después de varias consultas y pruebas, con los resultados del examen directo, la inmunodifusión y la PCR en tiempo real se confirmó el diagnóstico de paracoccidioidomicosis crónica multifocal. Este caso alerta sobre la ausencia de sospecha clínica de micosis endémicas, dada la presencia de lesiones mucocutaneas que pueden ser producidas por hongos como Paracoccidioides spp, y la importancia de considerarlas entre los diagnósticos diferenciales.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America. The most frequent form involves a chronic compromise of the lungs, skin, and mucosa. The patient started with a single oral lesion that lasted for several years. The absence of other symptoms pointed out a possible malignant neoplasm, specifically a squamous cell carcinoma. Differentiation between both diagnoses-fungal infection and carcinoma-depends on the results of the direct examination, the histopathological study, and the initial and subsequent cultures. However, in this case, those findings were not conclusive. The coexistence of both diagnoses is frequent and increases the diagnostic challenge. After several consultations and tests, direct examination, immunodiffusion and real-time PCR findings the multifocal chronic paracoccidioidomycosis diagnosis was confirmed. This case warns about a systematical absence of clinical suspicion of endemic mycoses before the appereance of mucocutaneous lesions, which can be produced by fungi like Paracoccidioides spp, and the importance of considering those mycoses among the differential diagnoses.

Long-range PCR amplification-based targeted enrichment & next generation sequencing: A cost-effective testing strategy for lysosomal storage disorders

Background & objectives: Lysosomal storage disorders (LSDs) are genetic metabolic disorders which result from deficiency of lysosomal enzymes or defects in other lysosomal components. Molecular genetic testing of LSDs is required for diagnostic confirmation when lysosomal enzyme assays are not available or not feasible to perform, and for the identification of the disease causing genetic variants. The aim of this study was to develop a cost-effective, readily customizable and scalable molecular genetic testing strategy for LSDs. Methods: A testing method was designed based on the in-house creation of selective amplicons through long range PCR amplification for targeted capture and enrichment of different LSD genes of interest, followed by next generation sequencing of pooled samples. Results: In the first phase of the study, standardization and validation of the study protocol were done using 28 samples of affected probands and/or carrier parents (group A) with previously identified variants in seven genes, and in the second phase of the study, 30 samples of enzymatically confirmed or biopsy-proven patients with LSDs and/or their carrier parents who had not undergone any prior mutation analysis (group B) were tested and the sequence variants identified in them through the study method were validated by targeted Sanger sequencing. Interpretation & conclusions: This testing approach was found to be reliable, easily customizable and cost-effective for the molecular genetic evaluation of LSDs. The same strategy may be applicable, especially in resource poor settings, for developing cost-effective multigene panel tests for other conditions with genetic heterogeneity.