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A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

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Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Asunto(s)

Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad

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Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

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Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 µM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Asunto(s)

Brasinoesteroides , Regulación de la Expresión Génica de las Plantas , Esteroides Heterocíclicos , Transcriptoma , Zea mays , Zea mays/genética , Zea mays/metabolismo , Zea mays/efectos de los fármacos , Zea mays/crecimiento & desarrollo , Brasinoesteroides/metabolismo , Brasinoesteroides/farmacología , Esteroides Heterocíclicos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Plantones/genética , Plantones/metabolismo , Plantones/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Perfilación de la Expresión Génica/métodos , Especies Reactivas de Oxígeno/metabolismo , Frío , Estrés Fisiológico , Respuesta al Choque por Frío , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética

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The bovine leukemia virus (BLV) is an important infectious agent transmitted from cattle to humans. It is considered one of the oncogenic viruses in breast cancer, so an accurate detection of this virus is important. The study aimed to design a specific and sensitive method based on TaqMan® real-time polymerase chain reaction (RT-PCR) for BLV detection. Probes and primers were designed using bioinformatics software for a 108 pairs region of the BLV tax gene. Criteria employed for determining analytical sensitivity were prepared using in-vitro RNA transcriptions. The National Center for Biotechnology Information (NCBI), basic local alignment search tool (BLAST) databases various viral panels and genomic samples from healthy individuals (Qom Province, Iran in 2023) were used to verify analytical specificity and clinical specificity, respectively. This method can measure a minimum of 10 copies of DNA and RNA mL-1. Moreover, the assay is linear in the range of 100 - 109 copies mL-1. By testing negative specimens, the method specificity was 100%. The reproducibility results of the reaction were examined at the intra- and inter-assay comparison. In fact, 10 technical replicates of each concentration of the control sample were analyzed in each working reaction. Due to the locally made kit, exact sensitivity and specificity, rapid analysis, and relatively low cost, as compared to commercial kits of other countries, the method introduced in the present study could be suitable for accurate detection of the BLV. Also, the TaqMan® real-time PCR method could be detected in cattle and human and before malignant changes of breast cancer which could reduce infection and breast cancer.

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Objectives: Gonorrhea is a prevalent sexually transmitted infection among men who have sex with men (MSM). In Morocco, the basic laboratory diagnosis of Neisseria gonorrhoeae (NG) is based on microscopy and, in some settings, on culture. However, no nucleic acid amplification test (NAAT) has been implemented for routine diagnosis of gonorrhoeae.The aim of this study is to assess the effectiveness of an in-house real-time PCR test for detecting N. gonorrhoeae DNA in anal swabs samples collected during an Integrated Behavioral and Biological survey. Patients and methods: Samples from 245 MSM, recruited using a Respondent Driven Sampling, were collected and tested for NG infection using GeneXpert CT/NG assay (Cepheid, USA). An In-House real-time PCR technique targeting the pseudo gene porA was developed and used for a parallel investigation of the same infection. The reliability of the in-house RT-PCR was validated through tests of reproducibility, repeatability, limit of detection, and cross-reactivity with other bacteria. The intrinsic performance characteristics of the qRT-PCR were assessed, namely, the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV). The GeneXpert CT/NG assay was adopted as a reference method. Results: For N. gonorrhoeae detection, the in-house real-time PCR assay showed a sensitivity and specificity of 80% and 100%, respectively. The PPV of the assay was 100% and the NPV was 97.3%. Conclusion: The in-house real-time PCR assay has high specificity and sensitivity, and it emerges as a promising approach for detecting N. gonorrhoeae in clinical specimens, particularly in decentralized settings such as regional laboratories.

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Objectives: Our objective was to establish a rapid and precise method for detecting hypervirulent Klebsiella pneumoniae (hvKP) by utilizing a duplex real-time multienzyme isothermal rapid amplification (real-time MIRA) and to evaluate its performance in clinical spiked blood specimens. Methods: The research comprised two phases: an initial pilot study to establish the methodology and a clinical validation study to assess its effectiveness. In the pilot phase, we designed specific primers and probes targeting the hvKP pg344 and incA genes and subsequently developed a duplex real-time MIRA assay to evaluate its detection limits, specificity, and efficiency. In the clinical validation phase, we analyzed thirty-three spiked blood specimens using the duplex real-time MIRA assay. Results: The duplex real-time MIRA assay demonstrated no cross-reactivity with other strains. Sensitivity experiments confirmed that the assay had a detection limit as low as 8 × 102 CFU per reaction for hvKP. The analysis of clinical spiked blood specimens indicated that the sensitivity and specificity of the duplex real-time MIRA assay were on par with those of duplex real-time PCR. Conclusions: These findings confirm that the duplex real-time MIRA assay is a fast, straightforward, and dependable method for detecting hvKP.

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Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Asunto(s)

Anticuerpos Antivirales , COVID-19 , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M , SARS-CoV-2 , Saliva , Humanos , Saliva/inmunología , SARS-CoV-2/inmunología , Inmunoglobulina M/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina A/inmunología , Inmunoglobulina A/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/diagnóstico , COVID-19/virología , Glicoproteína de la Espiga del Coronavirus/inmunología , Pruebas con Sangre Seca/métodos

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BACKGROUND: HTLV-1/2 exhibit a widespread distribution globally and are associated with severe clinical manifestations, necessitating precise viral identification for diagnosis. Currently, there are no official diagnostic guidelines, and a variety of published protocols exists. We introduce an enhanced nested real-time PCR technique followed by high-resolution melting (rtPCR-HRM), designed to offer a cost-effective and straightforward tool for the simultaneous identification of both viruses. METHODS: The technique was tested in a retrospective, blinded study, involving a total panel of 110 samples, of which 47 were positive for HTLV-1, 12 for HTLV-2, and 51 tested negatives. Additionally, we compared the performance of this technique with a line immunoassay (LIA). RESULTS: The results demonstrate 100% sensitivity, specificity, and diagnostic accuracy for both viruses. Sensitivity analysis indicated that at least 1 viral copy of HTLV-1 and 14.4 viral copies of HTLV-2 could be reliably detected. CONCLUSIONS: Our results indicate that rtPCR-HRM is effective in confirming HTLV-1 and HTLV-2 infection, important in Latin American countries where both viruses circulate. Furthermore, the proposed strategy provides a new tool that can be used to resolve indeterminate cases identified by Western blot, with the added advantage of being faster and simpler than n-PCR and more cost-effective than other probe-based RT-PCRs.

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Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX-2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX-2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy.

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Cervical cancer related to high-risk human papillomavirus (HR-HPV) is the second female cancer in Mauritania (Northwest Sahelian Africa). We assessed the distribution of HPV genotypes in Mauritanian women with high-grade cervical intraepithelial neoplasia (CIN2/3) or invasive cervical cancer (ICC). A prospective study was conducted in the Centre Hospitalier National, Nouakchott, Mauritania, to collect cervical biopsies among women suspected of CIN2/3 or cancer. HPV DNA detection and genotyping were carried out from formalin-fixed, paraffin-embedded biopsies using multiplex PCR (Human Papillomavirus Genotyping Real-Time PCR Kit, Bioperfectus Technologies Co., Taizhou, China). Fifty biopsies were included from women (mean age: 56.7 years) suffering from CIN2/3 (28.0%) and ICC (72.0%) which corresponded to 32 (64.0%) squamous cell carcinomas (SCC) and 4 (8.0%) adenocarcinomas (ADC). HPV DNA detection was successful in 47 (94.0%) samples. The most prevalent HR-HPV genotypes were HPV-45 (40.4%), HPV-16 (38.3%), HPV-39 and HPV-52 (23.4%), HPV-33 (17.0%), HPV-18 (14.9%), HPV-35 (4.2%), and HPV-56 (2.1%). The majority (93.6%) of HPV-positive biopsies contained at least one HPV type covered by the 9-valent Gardasil-9® vaccine, and 40.9% were infected by multiple vaccine HPV genotypes. To eradicate cervical cancer in Mauritania, prophylactic HPV vaccination must be combined with primary molecular screening of cervical HR-HPV infection.

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Schistosomiasis, caused by trematodes of genus Schistosoma, is among the most seriously neglected tropical diseases. Although rapid surveillance of risk areas for Schistosoma transmission is vital to control schistosomiasis, the habitat and infection status of this parasite are difficult to assess. Environmental DNA (eDNA) analysis, involving the detection of extra-organismal DNA in water samples, facilitates cost-efficient and sensitive biomonitoring of aquatic environments and is a promising tool to identify Schistosoma habitat and infection risk areas. However, in tropical wetlands, highly turbid water causes filter clogging, thereby decreasing the filtration volume and increasing the risk of false negatives. Therefore, in this study, we aimed to conduct laboratory experiments and field surveys in Lake Victoria, Mbita, to determine the appropriate filter pore size for S. mansoni eDNA collection in terms of particle size and filtration volume. In the laboratory experiment, aquarium water was sequentially filtered using different pore size filters. Targeting >3 µm size fraction was found to be sufficient to capture S. mansoni eDNA particles, regardless of their life cycle stage (egg, miracidia, and cercaria). In the field surveys, GF/D (2.7 µm nominal pore size) filter yielded 2.5-times the filtration volume obtained with a smaller pore size filter and pre-filtration methods under the same time constraints. Moreover, a site-occupancy model was applied to the field detection results to estimate S. mansoni eDNA occurrence and detection probabilities and assess the number of water samples and PCR replicates necessary for efficient eDNA detection. Overall, this study reveals an effective method for S. mansoni eDNA detection in turbid water, facilitating the rapid and sensitive monitoring of its distribution and cost-effective identification of schistosomiasis transmission risk areas.

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BACKGROUND: Malaria is the parasitic disease with the highest morbimortality worldwide. The World Health Organization (WHO) estimates that there were approximately 249 million cases in 2022, of which 3.4% were in Angola. Diagnosis is based on parasite identification by microscopy examination, antigen detection, and/or molecular tests, such as polymerase chain reaction (PCR). This study aimed to evaluate the usefulness of real-time PCR as a diagnostic method for malaria in an endemic area (Cubal, Angola). METHODS: A cross-sectional study was carried out at the Hospital Nossa Senhora da Paz in Cubal, Angola, including 200 patients who consulted for febrile syndrome between May and July 2022. From each patient, a capillary blood sample was obtained by finger prick for malaria field diagnosis [microscopy and rapid diagnostic test (RDT)] and venous blood sample for real-time PCR performed at the Hospital Universitario Vall d'Hebron in Barcelona, Spain. Any participant with a positive result from at least one of these three methods was diagnosed with malaria. RESULTS: Of the 200 participants included, 54% were female and the median age was 7 years. Malaria was diagnosed by at least one of the three techniques (microscopy, RDT, and/or real-time PCR) in 58% of the participants, with RDT having the highest percentage of positivity (49%), followed by real-time PCR (39.5%) and microscopy (33.5%). Of the 61 discordant samples, 4 were only positive by microscopy, 13 by real-time PCR, and 26 by RDT. Plasmodium falciparum was the most frequent species detected (90.63%), followed by P. malariae (17.19%) and P. ovale (9.38%). Coinfections were detected in ten participants (15.63%): six (60%) were caused by P. falciparum and P. malariae, three (30%) by P. falciparum and P. ovale, and one (10%) triple infection with these three species. In addition, it was observed that P. falciparum and P. malariae coinfection significantly increased the parasite density of the latter. CONCLUSIONS: RDT was the technique with the highest positivity rate, followed by real-time PCR and microscopy. The results of the real-time PCR may have been underestimated due to suboptimal storage conditions during the transportation of the DNA eluates. However, real-time PCR techniques have an important role in the surveillance of circulating Plasmodium species, given the epidemiological importance of the increase in non-falciparum species in the country, and can provide an estimate of the intensity of infection.

Asunto(s)

Fiebre , Malaria , Plasmodium , Reacción en Cadena en Tiempo Real de la Polimerasa , Humanos , Angola/epidemiología , Femenino , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Masculino , Estudios Transversales , Malaria/diagnóstico , Malaria/parasitología , Malaria/epidemiología , Niño , Fiebre/parasitología , Preescolar , Plasmodium/aislamiento & purificación , Plasmodium/genética , Plasmodium/clasificación , Adolescente , Adulto , Microscopía/métodos , Adulto Joven , Lactante , Sensibilidad y Especificidad , Persona de Mediana Edad , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Pruebas Diagnósticas de Rutina/métodos

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Background: Parvoviruses, characterized by their tropism for blood cells, can manifest as asymptomatic infections. With their ability to persist in blood, assessing the prevalence of Parvovirus B19 (B19V) and Parvovirus 4 (PARV4) among healthy blood donors is essential for evaluating the potential transmission risks through blood transfusions, emphasizing the need for comprehensive screening protocols. Methods: Four hundred blood donors participated in the study, with their blood specimens subjected to Real-Time PCR analysis for B19V and PARV4 nucleic acids after obtaining informed consent. Additionally, Complete Blood Count (CBC) assessments and determination of anti-B19 V-IgM and anti-B19 V-IgG antibody titers were performed using Enzyme-Linked Immunosorbent Assay (ELISA) for all collected samples. Results: The results reveal that 12 out of 400 individuals (3 %) exhibited positive results for B19V DNA, while 6 out of 400 individuals (1.5 %) tested positive for PARV4 DNA. Additionally, 8 out of 400 individuals (2 %) displayed positive results for anti-B19V IgM, and 306 out of 400 individuals (76.5 %) exhibited positive results for anti-B19 IgG. Notably, one donation from a donor presenting anti-IgM antibodies was subsequently confirmed as B19V DNA-positive through Real-Time PCR. In the analysis of CBC, a significant disparity in platelet levels was observed between B19V-positive donors, PARV4-positive donors, and B19V-negative donors. Conclusions: The study suggests that individuals at high risk, lacking detectable B19V antibodies, should undergo systematic screening and exclusion. This precaution is intended to minimize potential contamination risks within the studied cohort, despite the undefined pathogenesis and clinical implications of PARV4.

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Tick-borne pathogens are significant for human, veterinary, and wildlife health. Coxiella burnetii is an example that is widely distributed across various hosts and can cross species boundaries. In Pakistan, there is a scarcity of data regarding C. burnetii at the intersection of wildlife and livestock. Ticks were collected from ruminants and wildlife from the districts of Kasur, Pakpattan, and Okara in Pakistan. Five tick species totaling 571 ticks were collected, with the following distribution: 56.4% Hyalomma anatolicum, 22.4% Rhipicephalus microplus, 10.5% Hyalomma marginatum, 7.9% Rhipicephalus sanguineus, and 2.8% Rhipicephalus turanicus. Fifty tick pools were screened for C. burnetii to amplify a segment of the IS1111 using real-time PCR assays. Ticks collected from sheep and goats had a greater rate of positivity for C. burnetii (40% and 38%, respectively) compared to Indian long-eared hedgehogs with a prevalence of 2%. Coxiella burnetii was prominent in Rhipicephalus microplus (92.3%) and Hyalomma anatolicum (88.9%), followed by Rhipicephalus turanicus (66.6%), Rhipicephalus sanguineus (33.3%), and Hyalomma marginatum (25.0%). Ticks from Pakpattan district displayed the highest prevalence of C. burnetii (88.9%), whereas the lowest was observed in ticks from Kasur district (77.3%). There was no significant association between tick gender and C. burnetii infection. Female host animals were more likely to harbor ticks containing C. burnetii, with a prevalence rate of 81.8%. The research underscores the urgent need for comprehensive studies on C. burnetii in Pakistan, especially at the interface of wildlife and livestock. The high prevalence rates observed in certain tick species and geographic regions emphasize the importance of targeted public health interventions. Future research should focus on elucidating the transmission dynamics and implementing effective control measures to mitigate the impact of these pathogens on human, veterinary, and wildlife health in the region.

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Animales Salvajes , Coxiella burnetii , Cabras , Ixodidae , Fiebre Q , Infestaciones por Garrapatas , Animales , Coxiella burnetii/aislamiento & purificación , Coxiella burnetii/genética , Pakistán/epidemiología , Infestaciones por Garrapatas/veterinaria , Infestaciones por Garrapatas/epidemiología , Femenino , Fiebre Q/veterinaria , Fiebre Q/epidemiología , Fiebre Q/microbiología , Ixodidae/microbiología , Masculino , Ovinos , Prevalencia , Erizos/microbiología , Erizos/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Ovejas/microbiología , Enfermedades de las Cabras/epidemiología , Enfermedades de las Cabras/microbiología , Animales Domésticos

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Rapid real-time PCR (generally <1 h) has broad prospects. In this study, we synthesized a new type of nanomaterial core-shell tecto-dendrimer coated with Au nanoparticles (Au CSTDs) for research in this field. The experimental results showed that Au CSTDs could significantly shorten the time of real-time PCR (from 72 to 28 min) with different templates, while the detection limit reached 10 copies and the nonspecific amplification was significantly reduced. Furthermore, experimental analyses and theoretical studies using the finite element simulation method confirmed that Au CSTDs function by synergistically enhancing electrostatic adsorption and thermal conductivity. These properties play a key role in improving real-time PCR, especially in particle-particle interactions. This study contributes an advanced method to rapid real-time PCR, which is expected to remarkably improve the efficiency, lower the detection limit, and enhance the specificity of molecular detection.

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The causes of diarrhea after ten years of rotavirus vaccination in Rwanda were investigated in 496 children with and 298 without diarrhea using a real-time PCR. Rotavirus was detected in 11% of children with diarrhea (OR 2.48, P=0.002). Comparison of population attributable fractions (PAF) show that Shigella (PAF=11%) and ETEC-eltB (PAF=12%) have replaced rotavirus as the main causative agents. The PAF for rotavirus had declined from 41% pre-vaccination to 6.5%, indicating that rotavirus has become one among several similarly important causes of childhood diarrhea in Rwanda. A rotavirus genotype shift to G3P[8] points at the importance of continued genotype surveillance.

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Introduction: Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. Methods: This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. Results: The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). Discussion: This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose­d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

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Remdesivir therapy has been declared as efficient in the early stages of Covid-19. Of the 339 patients (males 55.8%, age 71(59;77) years) with a detectable viral load, 140 were treated with remdesivir (of those 103 in the ICU and 57 immunosuppressed) and retrospectively compared with 199 patients (of those 82 in the ICU and 28 immunosuppressed) who were denied therapy due to advanced Covid-19. The viral load was estimated by detecting nucleocapsid antigen in serum (n = 155, median 217(28;1524)pg/ml), antigen in sputum (n = 18, COI 18(4.6;32)), nasopharyngeal antigen (n = 44, COI 17(8;35)) and the real-time PCR (n = 122, Ct 21(18;27)). After adjustment for confounders, patients on remdesivir had better 12-month survival (HR 0.66 (0.44;0.98), p = 0.039), particularly when admitted to the ICU (HR 0.49 (0.29;0.81), p = 0.006). For the immunocompromised patients, the difference did not reach statistical significance (HR 0.55 (0.18;1.69), p = 0.3). The other most significant confounders were age, ICU admission, mechanical ventilation, leukocyte/lymphocyte ratio, admission creatinine and immunosuppression. The impact of monoclonal antibodies or previous vaccinations was not significant. Despite frequent immune suppression including haemato-oncology diseases, lymphopenia, and higher inflammatory markers in the remdesivir group, the results support remdesivir administration with respect to widely available estimates of viral load in patients with high illness severity.

Asunto(s)

Adenosina Monofosfato , Alanina , Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , SARS-CoV-2 , Carga Viral , Humanos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/uso terapéutico , Masculino , Femenino , Carga Viral/efectos de los fármacos , Anciano , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/fisiología , Persona de Mediana Edad , COVID-19/virología , COVID-19/mortalidad , Antivirales/uso terapéutico , Estudios Retrospectivos , Resultado del Tratamiento , Cuidados Críticos , Unidades de Cuidados Intensivos , Índice de Severidad de la Enfermedad

RESUMEN

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16-74.3] for HBV and 82 % [78.09-85.90] for HIV and an overall specificity of 100 % [94.95-100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26-88.73] and >1000 IU/mL for HBV was 100 % [95.51-100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.