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1.
Sci Rep ; 11(1): 4500, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627730

RESUMO

Emerging evidences have shown the utility of saliva for the detection of SARS-CoV-2 by PCR as alternative to nasopharyngeal swab (NPS). However, conflicting results have been reported regarding viral loads between NPS and saliva. We conducted a study to compare the viral loads between NPS and saliva in 42 COVID-19 patients. Viral loads were estimated by the cycle threshold (Ct) values. SARS-CoV-2 was detected in 34 (81%) using NPS with median Ct value of 27.4, and 38 (90%) using saliva with median Ct value of 28.9 (P = 0.79). Kendall's W was 0.82, showing a high degree of agreement, indicating equivalent viral loads in NPS and saliva. After symptom onset, the Ct values of both NPS and saliva continued to increase over time, with no substantial difference. Self-collected saliva has a detection sensitivity comparable to that of NPS and is a useful diagnostic tool with mitigating uncomfortable process and the risk of aerosol transmission to healthcare workers.


Assuntos
/virologia , /genética , Adulto , /métodos , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Reação em Cadeia da Polimerase/métodos , RNA Viral/genética , Saliva/virologia , Manejo de Espécimes/métodos , Carga Viral/métodos
2.
Anal Methods ; 13(2): 169-178, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33399137

RESUMO

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).


Assuntos
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/análise , Carga Viral/métodos , /diagnóstico , Colorimetria/métodos , DNA/análise , DNA/química , Corantes Fluorescentes/química , Humanos , Substâncias Intercalantes/química , Fenolsulfonaftaleína/química , Fosfoproteínas , RNA/química
3.
Expert Rev Mol Diagn ; 21(1): 119-129, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33380245

RESUMO

Introduction: With the ongoing SARS-CoV-2 pandemic, different articles have been published highlighting the superiority of droplet digital PCR (ddPCR) over the gold-standard reverse transcription PCR (RT-PCR) in SARS-CoV-2 detection. However, few studies have been reported on developing multiplex ddPCR assays for SARS-CoV-2 detection and their performance. This study shows steps on how to develop different ddPCR SAR-CoV-2 assays including higher order multiplex assays for SARS-CoV-2 detection and antiviral screening.Methods: Using multiple primer/probe sets, we developed, optimized, and analyzed the performance of simplex (1 target), duplex (2 targets), triplex probe mix (3 targets), and quadruplex (4 targets) SARS-CoV-2 ddPCR assays based on a two-color ddPCR detection system.Results: Results showed that the quadruplex assay had similar limits of detection and accuracy to the lower multiplex assays. Analyzing 94 clinical samples demonstrated that the ddPCR triplex probe mix assay had better sensitivity than the RT-qPCR assay. Additionally, the ddPCR multiplex assay showed that remdesivir could inhibit the growth of SARS-CoV-2 in vitro while another testing drug could not.Conclusion: Our research shows that developing multiplex ddPCR assays is possible by combing probe mix and amplitude-based multiplexing, which will help in developing multiplexed ddPCR assays for different SARS-CoV-2 applications.


Assuntos
/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , /isolamento & purificação , Antivirais/farmacologia , Primers do DNA/genética , Reações Falso-Positivas , Humanos , Limite de Detecção , Pandemias , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , Carga Viral/métodos
4.
Medicine (Baltimore) ; 99(52): e23853, 2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33350777

RESUMO

INTRODUCTION: The association of human immunodeficiency virus (HIV) infection with Burkitt lymphoma is related to the presence of Epstein Barr virus infection and the impact of the HIV antigen on the expansion of B-polyclonal cells. In Southeast Europe, the association is rare, and recognizing this is important in the therapeutic decision to increase patient survival rate. The association of HIV with Burkitt lymphoma and tuberculosis is even more rarely described in the literature. PATIENT CONCERNS: We present the case of a 40-year-old patient who presented with a 3-week history of fever (max. 38.7 °C), painful axillary swelling on the right side, lumbar pain, gait disorders, headache, and night sweats. Clinical manifestations included marked weight loss (about 30 kg in the last 2 months before his admission). DIAGNOSIS: A LyCD4 count of 38/µL and a HIV1 viral load of 384,000/mm3, classified the patient into a C3 stage. A biopsy of the right axillary lymph node was performed for suspected ganglionic tuberculosis due to immunodeficiency. Histopathological examination confirmed the diagnosis of Burkitt lymphoma. Cultures on Löwenstein-Jensen medium from sputum harvested at first admission were positive for Mycobacterium tuberculosis. INTERVENTIONS: Highly active antiretroviral therapy, chemotherapeutic agents for Burkitt lymphoma, anti-tuberculous drug therapy, neurosurgical intervention of spinal cord decompression, and antibiotic therapy of the associated bacterial infection. OUTCOME: Burkitt lymphoma disseminated rapidly, with central nervous system, spinal cord, osteomuscular, adrenal, and spleen involvement. The evolution under treatment was unfavorable, with patient death occurring 6 months after diagnosis. CONCLUSIONS: The association of HIV infection with Burkitt lymphoma and tuberculosis is rare in the highly active antiretroviral therapy (HAART) era, posing prompt and multidisciplinary therapeutic management issues. Similar cases of HIV-TB and Burkitt lymphoma association have been described, but none of the other cases showed the involvement of the central nervous system or of the bilateral adrenal glands.


Assuntos
Antineoplásicos/administração & dosagem , Terapia Antirretroviral de Alta Atividade/métodos , Antituberculosos/administração & dosagem , Encéfalo , Linfoma de Burkitt , Infecções por HIV , Medula Espinal , Tuberculose Pulmonar , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Linfoma de Burkitt/complicações , Linfoma de Burkitt/patologia , Linfoma de Burkitt/fisiopatologia , Linfoma de Burkitt/cirurgia , Contagem de Linfócito CD4/métodos , Deterioração Clínica , Descompressão Cirúrgica/métodos , Evolução Fatal , Infecções por HIV/sangue , Infecções por HIV/complicações , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Procedimentos Neurocirúrgicos/métodos , Medula Espinal/diagnóstico por imagem , Medula Espinal/patologia , Medula Espinal/cirurgia , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/fisiopatologia , Tuberculose Pulmonar/terapia , Carga Viral/métodos
5.
Sci Rep ; 10(1): 22425, 2020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33380736

RESUMO

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100-1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.


Assuntos
/métodos , Hidrogéis/química , Nasofaringe/virologia , RNA Viral/análise , Saliva/virologia , Testes Diagnósticos de Rotina , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes , Carga Viral/métodos
6.
BMC Infect Dis ; 20(1): 836, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176715

RESUMO

BACKGROUND: The KwaZulu-Natal (KZN) province of South Africa has the highest prevalence of HIV infection in the world. Viral load (VL) testing is a crucial tool for clinical and programmatic monitoring. Within uMkhanyakude district, VL suppression rates were 91% among patients with VL data; however, VL performance rates averaged only 38·7%. The objective of this study was to determine if enhanced clinic processes and community outreach could improve VL monitoring within this district. METHODS: A packaged intervention was implemented at three rural clinics in the setting of the KZN HIV AIDS Drug Resistance Surveillance Study. This included file hygiene, outreach, a VL register and documentation revisions. Chart audits were used to assess fidelity. Outcome measures included percentage VL performed and suppressed. Each rural clinic was matched with a peri-urban clinic for comparison before and after the start of each phase of the intervention. Monthly sample proportions were modelled using quasi-likelihood regression methods for over-dispersed binomial data. RESULTS: Mkuze and Jozini clinics increased VL performance overall from 33·9% and 35·3% to 75·8% and 72·4%, respectively which was significantly greater than the increases in the comparison clinics (RR 1·86 and 1·68, p < 0·01). VL suppression rates similarly increased overall by 39·3% and 36·2% (RR 1·84 and 1·70, p < 0·01). The Chart Intervention phase showed significant increases in fidelity 16 months after implementation. CONCLUSIONS: The packaged intervention improved VL performance and suppression rates overall but was significant in Mkuze and Jozini. Larger sustained efforts will be needed to have a similar impact throughout the province.


Assuntos
Síndrome de Imunodeficiência Adquirida/epidemiologia , Monitoramento Epidemiológico , HIV-1/genética , Saúde da População Rural , Carga Viral/métodos , Síndrome de Imunodeficiência Adquirida/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida/virologia , Adulto , Antirretrovirais/uso terapêutico , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , População Rural , África do Sul/epidemiologia , Resposta Viral Sustentada , Carga Viral/efeitos dos fármacos
7.
Indian J Med Microbiol ; 38(3 & 4): 284-287, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154236

RESUMO

The emergence of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), represents a public health emergency of unprecedented proportion. The global containment efforts have been focused on testing, tracing of contacts and treatment (isolation) of those found COVID-19 positive. Since the whole genome sequences of a number of strains of this novel RNA virus were available in the public domain by early January 2020, a number of real-time polymerase chain reaction (RT-PCR) protocols were designed and used for diagnosis of this infection. Most RT-PCRs are designed for qualitative COVID-19 reporting (SARS-CoV-2 detected or not detected), but have been used for semi-quantitative estimation of viral load based on cycle threshold value. Our manuscript discusses the utility of quantitative PCR testing for COVID-19 and its patient management benefits.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Genoma Viral/genética , Humanos , Pandemias , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
8.
Indian J Med Microbiol ; 38(3 & 4): 451-456, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154262

RESUMO

In the current COVID-19 crisis, many national healthcare systems are confronted with a huge demand for mass testing and an acute shortage of diagnostic resources. Considering group testing as a viable solution, this pilot study was carried out to find the maximum number of samples that can be pooled together to accurately detect one positive sample carrying the severe acute respiratory syndrome-coronavirus 2 viral RNA from different pools. We made different pool sizes ranging from 5 to 30 samples. Three positive samples, covering the common range of polymerase chain reaction (PCR) threshold cycle values (an indirect indicator of viral load) observed in our patients, were selected, and different pools were made with known negative samples. The pools underwent real-time qualitative PCR for the determination of effective maximum pool size. It was observed that up to 20-sample pools of all positive samples could accurately be detected in terms of both E gene and RdRp gene, leading to considerable conservation of resources, time and workforce. However, while deciding the optimal pool size, the infection level in that particular geographical area and sensitivity of the test assay used (limit of detection) have to be taken into account.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/métodos , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Humanos , Índia , Pandemias , RNA Viral/genética , Manejo de Espécimes , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
9.
PLoS Pathog ; 16(11): e1008949, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33180882

RESUMO

The COVID-19 has emerged as an epidemic, causing severe pneumonia with a high infection rate globally. To better understand the pathogenesis caused by SARS-CoV-2, we developed a rhesus macaque model to mimic natural infection via the nasal route, resulting in the SARS-CoV-2 virus shedding in the nose and stool up to 27 days. Importantly, we observed the pathological progression of marked interstitial pneumonia in the infected animals on 5-7 dpi, with virus dissemination widely occurring in the lower respiratory tract and lymph nodes, and viral RNA was consistently detected from 5 to 21 dpi. During the infection period, the kinetics response of T cells was revealed to contribute to COVID-19 progression. Our findings implied that the antiviral response of T cells was suppressed after 3 days post infection, which might be related to increases in the Treg cell population in PBMCs. Moreover, two waves of the enhanced production of cytokines (TGF-α, IL-4, IL-6, GM-CSF, IL-10, IL-15, IL-1ß), chemokines (MCP-1/CCL2, IL-8/CXCL8, and MIP-1ß/CCL4) were detected in lung tissue. Our data collected from this model suggested that T cell response and cytokine/chemokine changes in lung should be considered as evaluation parameters for COVID-19 treatment and vaccine development, besides of observation of virus shedding and pathological analysis.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Animais , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Citocinas/imunologia , Modelos Animais de Doenças , Pulmão/imunologia , Pulmão/patologia , Macaca mulatta , Pandemias , Pneumonia Viral/virologia , Carga Viral/métodos , Virulência , Eliminação de Partículas Virais
11.
Trials ; 21(1): 886, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33109246

RESUMO

OBJECTIVES: We will evaluate the efficacy and safety of favipiravir and interferon beta-1a compared to lopinavir/ritonavir and interferon beta-1a in patients with confirmed COVID-19, who are moderately ill. TRIAL DESIGN: This is a phase 3, single-center, randomized, open-label, controlled trial with a parallel-group design carried out at Shahid Mohammadi Hospital, Bandar Abbas, Iran. PARTICIPANTS: All patients with age ≥ 20 years admitted at the Severe Acute Respiratory Syndrome Departments of the Shahid Mohammadi Hospital, Bandar Abbas, Iran, will be screened for the following criteria. INCLUSION CRITERIA: 1. Confirmed diagnosis of infection with SARS-CoV-2 using polymerase chain reaction and/or antibody tests. 2. Moderate COVID-19 pneumonia (via computed tomography and/or X-ray imaging), requiring hospitalization. 3. Hospitalized ≤ 48 h. 4. Signing informed consent and willingness of the participant to accept randomization to any assigned treatment arm. EXCLUSION CRITERIA: 1. Underlying conditions, including chronic hepatitis, cirrhosis, cholestatic liver diseases, cholecystitis, peptic ulcers, acute and chronic renal failure, and peptic ulcers. 2. Severe and critical COVID-19 pneumonia. 3. History of allergy to favipiravir, lopinavir/ritonavir, and interferon beta-1a. 4. Pregnancy and breastfeeding. INTERVENTION AND COMPARATOR: Intervention group: favipiravir (Zhejiang Hisun, China) with interferon beta-1a (CinnaGen, Iran). This group will receive 1600 mg favipiravir twice a day for the first day and 600 mg twice a day for the following 4 days with five doses of 44 mcg interferon beta-1a every other day. CONTROL GROUP: lopinavir/ritonavir (Heterd Company, India) with interferon beta-1a (CinnaGen, Iran). This group will receive 200/50 mg lopinavir/ritonavir twice a day for 7 days with five doses of 44 mcg interferon beta-1a every other day. Other supportive and routine care will be the same in both groups. MAIN OUTCOMES: The primary outcome of the trial is the viral load of SARS-CoV-2 in the nasopharyngeal samples assessed by RT-PCR after 7 days of randomization as well as clinical improvement of fever and O2 saturation within 7 days of randomization. The secondary outcomes are the length of hospital stay and the incidence of serious adverse drug reactions within 7 days of randomization. RANDOMIZATION: Eligible patients will be allocated to one of the study arms using block randomization in a 1:1 ratio (each block consists of 10 patients). A web-based system will be used to generate random numbers for the allocation sequence. Each number relates to one of the study arms. BLINDING (MASKING): This is an open-label trial without blinding and placebo control. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): A total of 60 patients will be randomized into two groups (30 patients in the intervention group and 30 patients in the control group). TRIAL STATUS: The trial protocol is version 1.0, 22 July 2020. Recruitment began on 25 July 2020 and is anticipated to be completed by 25 September 2020. TRIAL REGISTRATION: Iranian Registry of Clinical Trials (IRCT) IRCT20200506047323N3 . Registered on 22 July 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting the dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol.


Assuntos
Amidas , Infecções por Coronavirus , Quimioterapia Combinada/métodos , Interferons , Lopinavir , Pandemias , Pneumonia Viral , Pirazinas , Ritonavir , Adulto , Amidas/administração & dosagem , Amidas/efeitos adversos , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Combinação de Medicamentos , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Interferons/administração & dosagem , Interferons/efeitos adversos , Irã (Geográfico) , Lopinavir/administração & dosagem , Lopinavir/efeitos adversos , Masculino , Pneumonia Viral/diagnóstico , Pneumonia Viral/tratamento farmacológico , Pirazinas/administração & dosagem , Pirazinas/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como Assunto , Ritonavir/administração & dosagem , Ritonavir/efeitos adversos , Índice de Gravidade de Doença , Resultado do Tratamento , Carga Viral/métodos
12.
Trials ; 21(1): 892, 2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-33109252

RESUMO

OBJECTIVES: The primary objectives of this study are to determine efficacy of Siddha medicine, Kabasura kudineer in reduction of SARS-CoV-2 viral load and reducing the onset of symptoms in asymptomatic COVID-19 when compared to Vitamin C and Zinc (CZ) supplementation. In addition, the trial will examine the changes in the immunological markers of the Siddha medicine against control. The secondary objectives of the trial are to evaluate the safety of the Siddha medicine and to document clinical profile of asymptomatic COVID-19 as per principles of Siddha system of Medicine. TRIAL DESIGN: A single centre, open-label, parallel group (1:1 allocation ratio), exploratory randomized controlled trial. PARTICIPANTS: Cases admitted at non-hospital settings designated as COVID Care Centre and managed by the State Government Stanley Medical College, Chennai, Tamil Nadu, India will be recruited. Eligible participants will be those tested positive for COVID-19 by Reverse Transcriptase Polymerase Chain reaction (RT-PCR) aged 18 to 55 years without any symptoms and co-morbidities like diabetes mellitus, hypertension and bronchial asthma. Those pregnant or lactating, with severe respiratory disease, already participating in COVID trials and with severe illness like malignancy will be excluded. INTERVENTION AND COMPARATOR: Adopting traditional methods, decoction of Kabasura kudineer will be prepared by boiling 5g of KSK powder in 240 ml water and reduced to one-fourth (60ml) and filtered. The KSK group will receive a dose of 60ml decoction, orally in the morning and evening after food for 14 days. The control group will receive Vitamin C (60000 IU) and Zinc tablets (100mg) orally in the morning and evening respectively for 14 days. MAIN OUTCOMES: The primary outcomes are the reduction in the SARS-CoV-2 load [as measured by cyclic threshold (CT) value of RT-PCR] from the baseline to that of seventh day of the treatment, prevention of progression of asymptomatic to symptomatic state (clinical symptoms like fever, cough and breathlessness) and changes in the immunity markers [Interleukins (IL) 6, IL10, IL2, Interferon gamma (IFNγ) and Tumor Necrosis Factor (TNF) alpha]. Clinical assessment of COVID-19 as per standard Siddha system of medicine principles and the occurrence of adverse effects will be documented as secondary outcomes. RANDOMISATION: The assignment to the study or control group will be allocated in equal numbers through randomization using random number generation in Microsoft Excel by a statistician who is not involved in the trial. The allocation scheme will be made by an independent statistician using a sealed envelope. The participants will be allocated immediately after the eligibility assessment and informed consent procedures. BLINDING (MASKING): This study is unblinded. The investigators will be blinded to data analysis, which will be carried out by a statistician who is not involved in the trial. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Sample size could not be calculated, as there is no prior trial on KSK. This trial will be a pilot trial. Hence, we intend to recruit 60 participants in total using a 1:1 allocation ratio, with 30 participants randomised into each arm. TRIAL STATUS: Protocol version 2.0 dated 16th May 2020. Recruitment is completed. The trial started recruitment on the 25th May 2020. We anticipate study including data analysis will finish on November 2020. We also stated that protocol was submitted before the end of data collection TRIAL REGISTRATION: The study protocol was registered with clinical trial registry of India (CTRI) with CTRI/2020/05/025215 on 16 May 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).


Assuntos
Ácido Ascórbico , Betacoronavirus , Infecções por Coronavirus , Medicina Ayurvédica/métodos , Pandemias , Pneumonia Viral , Zinco , Adulto , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/efeitos adversos , Infecções Assintomáticas/terapia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Suplementos Nutricionais , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Índia , Masculino , Pneumonia Viral/diagnóstico , Pneumonia Viral/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Carga Viral/métodos , Zinco/administração & dosagem , Zinco/efeitos adversos
13.
Trials ; 21(1): 785, 2020 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-32928313

RESUMO

OBJECTIVES: 1- To compare the effectiveness of 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline and a novel solution Neem extract (Azardirachta indica) in reducing intra-oral viral load in COVID-19 positive patients. 2- To determine the salivary cytokine profiles of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL- 17 among COVID-19 patients subjected to 1% Hydrogen peroxide, 0.2% Povidone-Iodine, 2% hypertonic saline or Neem extract (Azardirachta indica) based gargles. TRIAL DESIGN: This will be a parallel group, quadruple blind-randomised controlled pilot trial with an add on laboratory based study. PARTICIPANTS: A non-probability, purposive sampling technique will be followed to identify participants for this study. The clinical trial will be carried out at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. The viral PCR tests will be done at main AKUH clinical laboratories whereas the immunological tests (cytokine analysis) will be done at the Juma research laboratory of AKUH. The inclusion criteria are laboratory-confirmed COVID-19 positive patients, male or female, in the age range of 18-65 years, with mild to moderate disease, already admitted to the AKUH. Subjects with low Glasgow coma score, with a history of radiotherapy or chemotherapy, who are more than 7 days past the onset of COVID- 19 symptoms, or intubated or edentulous patients will be excluded. Patients who are being treated with any form of oral or parenteral antiviral therapy will be excluded, as well as patients with known pre-existing chronic mucosal lesions such as lichen planus. INTERVENTION AND COMPARATOR: Group A (n=10) patients on 10 ml gargle and nasal lavage using 0.2% Povidone-Iodine (Betadiene® by Aviro Health Inc./ Pyodine® by Brooks Pharma Inc.) for 20-30 seconds, thrice daily for 6 days. Group B (n=10) patients will be subjected to 10 ml gargle and nasal lavage using 1% Hydrogen peroxide (HP® by Karachi Chemicals Products Inc./ ActiveOxy® by Boumatic Inc.) for 20-30 seconds, thrice daily for 6 days. Group C will comprised of (n=10) subjects on 10ml gargle and nasal lavage using Neem extract solution (Azardirachta indica) formulated by Karachi University (chemistry department laboratories) for 20-30 seconds, thrice daily for 6 days. Group D (n=10) patients will use 2% hypertonic saline (Plabottle® by Otsuka Inc.) gargle and nasal lavage for a similar time period. Group E (n=10) will serve as positive controls. These will be given simple distilled water gargles and nasal lavage for 20-30 seconds, thrice daily for six days. For nasal lavage, a special douche syringe will be provided to each participant. Its use will be thoroughly explained by the data collection officer. After each use, the patient is asked not to eat, drink, or rinse their mouth for the next 30 minutes. MAIN OUTCOMES: The primary outcome is the reduction in the intra-oral viral load confirmed with real time quantitative PCR. RANDOMISATION: The assignment to the study group/ allocation will be done using the sealed envelope method under the supervision of Clinical Trial Unit (CTU) of Aga Khan University, Karachi, Pakistan. The patients will be randomised to their respective study group (1:1:1:1:1 allocation ratio) immediately after the eligibility assessment and consent administration is done. BLINDING (MASKING): The study will be quadruple-blinded. Patients, intervention provider, outcome assessor and the data collection officer will be blinded. The groups will be labelled as A, B, C, D or E. The codes of the intervention will be kept in lock & key at the CTU and will only be revealed at the end of study or if the study is terminated prematurely. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): As there is no prior work on this research question, so no assumptions for the sample size calculation could be made. The present study will serve as a pilot trial. We intend to study 50 patients in five study groups with 10 patients in each study group. For details, please refer to Fig. 1 for details. TRIAL STATUS: Protocol version is 7.0, approved by the department and institutional ethics committees and clinical trial unit of the university hospital. Recruitment is planned to start as soon as the funding is sanctioned. The total duration of the study is expected to be 6 months i.e. August 2020-January 2021. TRIAL REGISTRATION: This study protocol was registered at www.clinicaltrials.gov on 10 April 2020 NCT04341688 . FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2). Fig. 1 Flow diagram of study-participants' timeline.


Assuntos
Azadirachta , Betacoronavirus , Infecções por Coronavirus , Peróxido de Hidrogênio/administração & dosagem , Pandemias , Extratos Vegetais/administração & dosagem , Pneumonia Viral , Povidona-Iodo/administração & dosagem , Solução Salina Hipertônica/administração & dosagem , Carga Viral , Adulto , Anti-Infecciosos Locais/administração & dosagem , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Feminino , Hospitalização , Humanos , Masculino , Monitorização Imunológica/métodos , Antissépticos Bucais/administração & dosagem , Lavagem Nasal/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Ensaios Clínicos Controlados Aleatórios como Assunto , Carga Viral/efeitos dos fármacos , Carga Viral/métodos
14.
J Infect Chemother ; 26(12): 1324-1327, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32900659

RESUMO

Most patients with coronavirus disease 2019 (COVID-19) have just only mild symptoms, but about 5% are very severe. Although extracorporeal membranous oxygenation (ECMO) is sometimes used in critically patients with COVID-19, ECMO is only an adjunct, not the main treatment. If the patient's condition deteriorates and it is determined to be irreversible, it is necessary to decide to stop ECMO. A 54-year-old man was admitted on day 6 of onset with a chief complaint of high fever and cough. Computed tomography (CT) showed a ground glass opacity in both lungs, and reverse transcription-polymerase chain reaction (RT-PCR) diagnosed COVID-19. He was admitted to the hospital and started to receive oxygen and favipiravir. After that, his respiratory condition deteriorated, and he was intubated and ventilated on day 9 of onset, and ECMO was introduced on day 12. Two days after the introduction of ECMO, C-reactive protein (CRP) increased, chest X-p showed no improvement in pneumonia, and PaO2/FiO2 decreased again. As D-dimer rose and found a blood clot in the ECMO circuit, we had to decide whether to replace the circuit and continue with ECMO or stop ECMO. At this time, the viral load by RT-PCR was drastically reduced to about 1/1750. We decided to continue ECMO therapy and replaced the circuit. The patient's respiratory status subsequently improved and ECMO was stopped on day 21 of onset. In conclusion, viral load measurement by RT-PCR may be one of the indicators for promoting the treatment of severe COVID-19 patients.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Oxigenação por Membrana Extracorpórea/métodos , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Carga Viral/métodos , Amidas/uso terapêutico , Antivirais/uso terapêutico , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Tomada de Decisões , Hospitalização , Humanos , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Pirazinas/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada por Raios X , Resultado do Tratamento
16.
Int J Food Microbiol ; 333: 108785, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-32717668

RESUMO

Norovirus in oysters is a significant food safety risk. A recent ISO detection method allows for reliable and repeatable estimates of norovirus concentrations in pooled samples, but there is insufficient data to estimate a distribution of copies per animal from this. The spread of norovirus accumulated across individual oysters is useful for risk assessment models. Six sets of thirty individual Crassostrea gigas oysters were tested for norovirus concentration levels by reverse-transcription quantitative PCR (RT-qPCR): three from a commercial harvest site, and three post-depuration. Five sets had norovirus GII means above the limit of quantification (LOQ), and one below the LOQ, but above the limit of detection. No norovirus GI was detected in pooled tests, and individual oysters were not tested for norovirus GI. Depuration was shown to reduce the mean concentration of GII copies, but not to affect the shape of the distribution around the mean. Deconvoluting the uncertainty of the method, the coefficient of variation was stationary (0.45 ±â€¯0.2). The best fit distribution was either a lognormal distribution or a gamma. Multiplying these distributions by the weight of oyster digestive tissues gave an estimate for the count mean. This was used as the parameter λ in three compound Poisson distributions: Poisson-lognormal, Poisson-gamma, and Poisson-K. No model was found to fit better than the others, with advantages for each. All three could be used in future risk assessments. Preliminary validation of sampling uncertainty using repeated testing data from a previous study suggests that these results have predictive power.


Assuntos
Crassostrea/virologia , Norovirus/isolamento & purificação , Frutos do Mar/virologia , Carga Viral/métodos , Animais , Contaminação de Alimentos/análise , Inocuidade dos Alimentos , Norovirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco/métodos
17.
J Infect Dis ; 222(6): 903-909, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32592581

RESUMO

High-throughput molecular testing for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) may be enabled by group testing in which pools of specimens are screened, and individual specimens tested only after a pool tests positive. Several laboratories have recently published examples of pooling strategies applied to SARS-CoV-2 specimens, but overall guidance on efficient pooling strategies is lacking. Therefore we developed a model of the efficiency and accuracy of specimen pooling algorithms based on available data on SAR-CoV-2 viral dynamics. For a fixed number of tests, we estimate that programs using group testing could screen 2-20 times as many specimens compared with individual testing, increase the total number of true positive infections identified, and improve the positive predictive value of results. We compare outcomes that may be expected in different testing situations and provide general recommendations for group testing implementation. A free, publicly-available Web calculator is provided to help inform laboratory decisions on SARS-CoV-2 pooling algorithms.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Manejo de Espécimes/métodos , Algoritmos , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Humanos , Incidência , Pandemias , Pneumonia Viral/diagnóstico , Valor Preditivo dos Testes , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Carga Viral/métodos
18.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
19.
Viruses ; 12(6)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32521706

RESUMO

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Inativação de Vírus/efeitos dos fármacos , Animais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Técnicas de Cultura de Células/métodos , Chlorocebus aethiops , Contenção de Riscos Biológicos/métodos , Contenção de Riscos Biológicos/normas , Humanos , Nasofaringe/virologia , Pandemias , RNA Viral/isolamento & purificação , Manejo de Espécimes/métodos , Células Vero , Carga Viral/métodos
20.
PLoS One ; 15(5): e0232107, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379782

RESUMO

OBJECTIVE: To explore the relationship between the viral load reflected by the Ct value of Cobas 4800 HPV test and cervical lesions, and the effectiveness of the viral load for secondary triage of HPV-positive women. METHODS: The Chinese Multi-Center Screening Trial (CHIMUST) evaluated both self-collected samples and physician-collected samples from women, aged 30 to 59, who were screened for cervical cancer in 6 regions across China. Using physician collected samples, the relationship between the HPV-Ct values of different subtypes and the cervical lesions was analyzed. Then the combined use of the HPV-Ct values with the HPV subtypes was evaluated as a secondary screening algorithm for the women who were HPV positive. RESULTS: The Ct values of HPV16 and 12 other HPV subtypes(12-type pool), tested with Cobas decreased with the progression of cervical lesion (HPV16: r = -0.429, P<0.001; 12 other HR-HPV subtypes: r = -0.099, P<0.01). The HPV18-Ct value was not correlated with cervical lesion(P>0.05). Compared with HPV16/18 and cytology (HPV16/18 positive and 12-type pool plus cytology ≥ ASC-US), the sequential secondary screening using HPV16/18 and the viral load of 12-type pool (cut-point HPV-Ct≤31) had equal sensitivities for CIN2+ and CIN3+ (83.1%vs.80.3%,100%vs.92.6%,P>0.05), with slightly lower specificities (96.2%vs.94.4%,96.5%vs.93.9%,P<0.001) and higher colposcopy referral rate (4.90%vs.6.59%, P<0.05), but required no cytology. CONCLUSION: Type-specific HPV viral load is closely related to cervical lesions severity. It is feasible and efficient to use HPV16/18 and the viral load of 12 other HPV subtypes (with cut-point HPV-Ct≤31) as the secondary screening for HPV positive women. This algorithm may be useful in low resource regions.


Assuntos
Detecção Precoce de Câncer/métodos , Neoplasias do Colo do Útero/diagnóstico , Carga Viral/métodos , Adulto , Grupo com Ancestrais do Continente Asiático , Neoplasia Intraepitelial Cervical/virologia , Colo do Útero/patologia , China/epidemiologia , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade , Sorogrupo , Manejo de Espécimes/métodos , Triagem , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
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