Month: January 2022

Abstract

To determine whether patients with tuberculosis (TB) and nontuberculous mycobacteria (NTM) infection could be distinguished from each other with limited information, we compared patients with pulmonary TB and NTM during 2005-2006. Our finding that age, place of birth, and the presence of chronic obstructive pulmonary disease could differentiate TB and NTM disease could help in TB control efforts.

Keywords:

Bacteria, Mycobacterium tuberculosis, Mycobacterium avium, tuberculosis and other mycobacteria, pulmonary and respiratory infections, non-tuberculous mycobacteria, infection control, diagnosis, clearance.

General description

• Non-tuberculous mycobacteria (NTM) are mycobacteria other than M. tuberculosis (the cause of tuberculosis) and M. leprae (the cause of leprosy). NTMs are also called atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria.
• Although anyone can get an NTM infection, NTMs are opportunistic pathogens that put some groups at higher risk, including those with underlying lung disease or suppressed immune systems. These pathogens are not normally spread from person to person. However, person-to-person transmission of M. abscessus has been reported in patients with cystic fibrosis.
• NTMs are environmental organisms that can be found in soil, dust, and water, including natural water sources (such as lakes, rivers, and streams) and municipal water sources (such as the water that people drink or use). showering). NTM can form difficult-to-remove biofilms, which are collections of microorganisms that stick together and adhere to surfaces in moist environments, such as the inside of building plumbing.

Symptoms

1. NTMs can cause infections in a wide variety of body sites, most commonly the lungs and the following areas:

• Skin and soft tissues (typically after surgery, trauma, injection of drugs or other substances)
• Device-associated infections (eg, central line-associated bloodstream infections, exit-site infections, pacemaker pocket site infections, etc.)
• Lymph nodes (more commonly in children)
• Blood or other generally sterile places in the body (spread) (most commonly in immunocompromised patients, such as those with HIV or AIDS, but can also be due to invasive medical devices or procedures)

2. Symptoms can be vague and nonspecific, such as:

• Fever
• Weightloss
• Night sweats
• Decreased appetite
• power loss

3. Other symptoms depend on the site of infection and may include cough, shortness of breath, blood in the sputum, and skin rashes.

Tuberculosis (TB) is a chronic bacterial disease that infects the lungs, kidneys, spine, and/or brain. TB is an airborne mycobacterium and can be spread from person to person, such as when an infected person coughs or sneezes. It can also cause an infection after a latent period in a person who was infected at an earlier time.

Without treatment, TB can cause serious complications in other parts of the body or even be fatal. Treatment requires months of adherence to various antibiotics, but most cases can be cured. Drug-resistant TB is much more difficult to cure and treatment takes much longer (9 to 18 months). Brigham and Women’s Hospital (BWH) works with the Massachusetts Department of Public Health, which subsidizes all diagnostic testing and treatment.

Surgery is used to diagnose and treat complications of TB. Surgical approaches to lung resection include thoracotomy and video-assisted thoracic surgery (VATS). For surgical treatment to be most effective, it must be preceded and followed by extensive antibacterial therapy.

Non-tuberculous mycobacterial (NTM) disease is a general term for a group of lung infections caused by exposure to mycobacteria found in soil and water. The mycobacteria that cause these diseases exclude those that cause tuberculosis and leprosy. NTM most commonly affects people with weakened immune systems due to other lung diseases, but healthy people with no history of lung disease can also be affected. If left untreated, the disease can become chronic and require ongoing treatment.

Despite medical and surgical advances, TB remains a global epidemic and its presentation requires an aggressive and coordinated approach. The Lung Center at BWH provides high-quality multidisciplinary care and research in the area of ​​TB and NTM treatment. Pulmonologists, thoracic surgeons, and infectious disease physicians who specialize in TB and NTM collaborate with other BWH specialists to provide patients with a highly informed diagnosis and consistent treatment plan.

What are the risk factors for TB and nontuberculous mycobacterial disease?

Tuberculosis and nontuberculous mycobacterial diseases affect people of all ages, genders, races, and income levels. The following groups face a higher risk:

• People who live or work with other people who have TB, including hospital staff
• People from countries where TB is prevalent
• People who abuse alcohol or use intravenous drugs
• people who smoke
• Homeless people
• Young children and the elderly
• People without access to health care.
• People in group settings, such as nursing homes.

Patients with weakened immune systems are particularly susceptible to both TB and nontuberculous mycobacterial disease. Some conditions that weaken the immune system and/or damage the lung structure include:

• bronchiectasis
• Chronic obstructive pulmonary disease (COPD) and emphysema
• Cystic fibrosis
• Human immunodeficiency virus (HIV)
• Occupational and environmental lung diseases
• lung transplant

Allergy tests and your pet

Just like us, pets can also experience allergies. Spring pollen, irritating shampoos, and even food allergies can be a problem for pets. Fortunately, allergy tests can often identify the cause of your pet’s reaction.

How is it done?

Your pet can be screened for a list of environmental allergens with a simple blood test. The sample is sent to a laboratory where tests will determine the cause and severity of your pet’s allergy.

How does it help?

The best treatment for an allergy is to avoid contact with the offending substance. However, this is not always possible. In many cases, intermittent treatment is necessary. Steroids, antibiotics, antihistamines, special shampoos, or supplements may be used to treat allergies. However, if used for too long, steroids can have adverse side effects.

Antibiotics and antihistamines can be a considerable expense. Allergy-positive pets may benefit from desensitizing vaccines specially formulated for your pet. The goal is to control your pet’s allergy and minimize the need for additional medication.

How successful is it?

Dogs and cats, like people, are individuals. No two dogs or cats are exactly alike and neither are their allergies. Therefore, some patients will respond better than others. Allergy shots are not a quick cure for your pet’s allergies. Rather, they are safer and, in many cases, cheaper than alternative treatment methods.

What is the treatment process?

The initial blood test is taken at a normal veterinary appointment. When the treatment vials are tested by a laboratory, an injection regimen is started. If your pet develops clinical symptoms of his allergy during treatment, they can be controlled through the use of antibiotics or shampoos. The use of steroids during desensitization is avoided. Your Greencross veterinarian will administer the injections during scheduled, one-on-one visits. Talk to your local Greencross veterinarians if you are concerned about allergies in your pet.

Allergen:

Something to which the immune system develops an allergy; they are usually proteins in things like pollen, dust mites, and some moulds. Antibody: A specialized protein called immunoglobulin (Ig). There are many subtypes, including those that are responsible for allergic reactions, IgE. Gold standard: For the purpose of our discussion: a diagnostic test that is considered to be the most reliable.

When evaluating the usefulness or reliability of a new or different test, that test is compared to the “gold standard” test. Hyposensitization Injections – Another name for allergy shots or allergy shots. These injections reduce the patient’s sensitivity to allergens. IgE: The allergic antibody; type E immunoglobulin.

Allergy testing is done to determine which allergens may be responsible for a pet’s allergy symptoms. Allergy tests do this by looking for the presence of specific allergy-type antibodies (IgE) to common allergens. Two areas of the body are commonly tested for the presence of specific IgE: the skin and also the blood.

The intradermal allergy test (or allergy skin test) is considered the gold standard of allergy testing. It is very similar to the allergy skin test that is done on humans. In pets, it consists of injecting small amounts of allergens in an organized way into the skin. When a pet produces IgE that is specific to the injected allergen during testing, the IgE produced by the pet causes the skin to redden and swell exactly where the allergen was injected.

A scoring system is used to differentiate positive from negative injection sites. Training and experience are needed to test injections correctly. Skin allergy tests, like blood allergy tests, are reliable only for non-food allergens such as pollen, dust mites, and mould.

Does your dog or cat scratch and groom itself too much?

Dog and cat allergy symptoms are distressing for your pet. Skin allergies are one of the most common skin conditions we see in dogs and cats and can have a negative impact on your pet’s life. Atopic dermatitis is a reaction to environmental aeroallergens such as pollen from grasses, weeds, trees, mould spores, and house dust mites.

What are the common symptoms of cat allergies?

• Belly and sides too trimmed
• excessive hair loss
• Itchy crusty rash
• Itchy, moist, red raised lesions in the groin or armpit region
• Severe scratching of the head, ears, and neck
• Greasy and dishevelled coat
• Changing diet doesn’t make any difference.

What are common dog allergy symptoms?

• Itching, most commonly around the nose, eyes, ear flaps, armpits, groin, and legs (especially if the itching affects the feet, face, ears, buttocks, legs, or areas that come in contact with the ground when the dog lies down)
• Recurring skin or ear infections
• Inflamed skin prone to secondary infections with bacteria and yeast (as a result of chewing, licking, rubbing, and scratching)
• If your dog becomes “smelly” within a few days of being washed
• If symptoms started or get worse in hot weather
• If your dog gets itchy after touching grass
• If your dog is itchy after being washed
• If your dog has persistently oily skin
• If a diet change makes no difference

How do we diagnose atopic skin disease?

We identify allergies by intradermal allergy testing. This involves cutting a strand of hair from one side and pricking the skin with a small amount of purified allergen extract. If the skin swells thirty minutes after the injection, it means that an allergy has been detected. Another way to identify allergens is to perform blood serology tests. Using the results of intradermal and blood tests provides a more accurate allergy profile for your pet and a better outcome with immunotherapy.

*Please note: Intradermal allergy testing is affected by a number of medications, including antihistamines and corticosteroids (cortisone tablets, injections, lotions, eye and ear drops). It is important that your pet is taken off treatment prior to testing. Talk to our technicians about medication withdrawal times for your dog and cat.

How do we treat atopic dermatitis?

The best treatment for allergies is usually immunotherapy (desensitization) or avoiding allergy triggers. Immunotherapy involves creating a vaccine for your pet. Once we have identified what your pet is allergic to, we formulate an allergy vaccine (immunotherapy) that contains the allergens your pet has reacted to. As the vaccine starts to work, there will be fewer allergic signs like infections and itching. We prefer to use immunotherapy over medication, as it minimizes subsequent reactions.

Objectives:

COVID-19 emerged and rapidly spread throughout the world. Testing strategies focussing on patients with COVID-19 require assays that are high-throughput, low-risk of infection, and with small sample volumes. Antigen surveillance can be used to identify exposure to pathogens and measure acute infections.

Methods:

A total of 914 serum samples, collected from 309 currently infected COVID-19 patients, 48 recovered ones, and 410 non-COVID-19 patients, were used to measure N protein antigen levels by a chemiluminescent immunoassay. Diagnostic performances were analyzed in different periods after onset.

• Patients, samples and data collection

Consecutive patients with COVID-19 who were presented or admitted to Zhongnan Hospital, Wuhan University from January 28 to March 10, 2020, were included to test for SARS-COV-2 N-protein antigen and specific antibodies in serum. COVID-19 and recovered patients enrolled in this study were diagnosed according to the diagnostic guideline published by the National Health Commission.

Healthy volunteers and other virus-infected patients who were examined or admitted to Zhongnan Hospital, Wuhan University, from November 18, 2020, to December 6, 2020, were enrolled in the control group. Exclusion criteria for controls were as follows: (a) SARS-COV-2 RNA positive in throat swab, (b) patients with poor basic clinical data. All throat swabs and venous blood samples were collected and processed at Zhongnan Hospital of Wuhan University.

The remaining sera were collected and stored at -80°C for SARS-CoV-2 specific antibody and N-protein antigen testing. Clinical characteristics, laboratory findings, and results were collected from the medical record. electronics. The study was reviewed and approved by the Ethics Committee of Zhongnan Hospital, Wuhan University. All study targets have signed informed consent.

• Real-time RT-PCR assay for SARS-CoV-2 RNA

Throat swabs were collected from COVID-19 patients for SARS-CoV-2 RNA testing. First, total RNAs were extracted from the swab within 3 h using a respiratory sample RNA isolation kit (Zhongzhi, Wuhan, China). Briefly, 40 μL of cell lysis solution was transferred to a collection tube consisting of a swab followed by vortexing for 30 s. After incubation at room temperature for 15 min, the collection tube was centrifuged at 1000 rpm/min for 5 min.

The suspension was used as a template for amplification using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay kits (Daan Gene, Guangzhou, China). Two target genes, SARS-CoV-2 nucleocapsid protein (N) and open reading frame 1ab (ORF1ab), were simultaneously amplified and detected during real-time RT-PCR assay. The real-time RT-PCR reaction system volume was 25 μL, including 2 μL of template, 3 μL of pure water, 17 μL of mix A, and 3 μL of mix B.

Each amplification was performed in an Eppendorf tube. by ABI prism 7500 (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were as follows: transcription at 50°C for 15 min and pre-denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 94°C for 15 s and extension at 55°C for 45 s. Fluorescence was collected at regular intervals during each extension phase. The lower detection limit of the real-time RT-PCR assay for two target genes was 500 copies/mL. According to the manufacturer’s recommendation, a cycle threshold value (Ct) of <40 was defined as positive.

• Chemiluminescence immunoassay to analyze SARS-CoV-2 N serum antigen

Serum SARS-CoV-2 protein N antigen was determined by double-antibody sandwich chemiluminescence immunoassay using an iFlash immunoassay analyzer (Shenzhen Yhlo Biotech Co., Ltd, Shenzhen, China). Paramagnetic carboxylated microparticles (Thermo Scientific) were coated with one of 10 candidate-specific antibodies (Shenzhen YHLO Biotech Co., Ltd, Shenzhen, China) by cross-linking N-ethyl-N’-(3-dimethyl aminopropyl)carbodiimide (Thermo Scientific) for protein N antigen-capture as described above. Another antibody was conjugated with NSP-DMAE-NHS (Maxchemtech) for antigen detection.

SARS-CoV-2 recombination nucleocapsid protein (Shenzhen YHLO Biotech) dissolved in healthy human serum was used as a calibrator. Tests can be run after calibration. In the test, paramagnetic carboxylated microparticles coated with the capture antibody bound N-protein antigens. After washing away unbound material, the antibody N-protein antigen-capture antibody compounds reacted with the acridinium-labelled antibody.

The mixture was kept in a tube under a magnetic field. And then the preactivation and activation solution were added to calculate the N-protein antigen based on the resulting relative light units (RLU) through a 2-point calibration curve.

• Statistic analysis

Statistical analyzes were performed with IBM SPSS version 23.0 software. Normal distribution continuous data were presented as mean ± standard deviation (SD) and skewed distribution continuous data were presented as median and range. The student’s t-test or non-parametric test was used for the comparison of continuous data. The chi-square test was used to analyze categorical data. Diagnostic sensitivity and specificity were calculated using the receiver operating characteristic (ROC) curve and the area under the curve (AUC). p ≤ 0.05 was considered statistically significant.

Results:

There was a high level of N protein antigen in COVID-19 patients (0.56 COI), compared to the recovered patients (0.12 COI) and controls (0.19 COI). In receiver-operating characteristic curve analysis, the area under the curve of serum N protein antigen was 0.911 in the first week after onset. In this period, the Sensitivity and specificity of serologic N protein antigen testing was 76.27 and 98.78%. Diagnosis performance of specific antibodies became better from the third week after onset. Subgroup analysis suggested that severe patients had higher levels of antigens than mild patients.

Conclusions:

A high level of serum antigen suggested early infection and serious illness. Serum N protein antigen testing by chemiluminescence immunoassay is considered a viable assay used to improve diagnostic sensitivity for current patients.

Keywords

Antigen, antigenemia, Blood, COVID-19, Diagnosis, Plasma, SARS-CoV-2, Serum

Product introduction

The dUTP/UDG anti-contamination system is a very effective means of controlling the contamination of PCR amplification products. In PCR detection reagents using DNA as a template, it has gradually become a mandatory standard.

However, the commonly used E. coli UDG still has high activity at the usual reverse transcription temperature (42-55℃), which will degrade the cDNA, thus reducing the sensitivity of One Step RT-PCR/qPCR. The Hiscript II U+ One-Step qRT-PCR Probe Kit contains an optimized ratio of dUTP/dNTP mix and heat-labile UDG derived from psychrophilic marine bacteria.

Heat-labile UDG has high activity at room temperature and can completely degrade U-containing double-stranded DNA during the mixing process of the reaction system. When the reaction system is heated to 50-55°C (the optimal HiScript II reaction temperature), the heat-labile UDG is rapidly and completely inactivated, maintaining cDNA integrity and ensuring that detection sensitivity is not compromised. be affected.

Storage conditions

Store at -20℃

Product Advantages

• Support multiple probe detection;
• UDG anti-contamination system: Heat-labile UDG rapidly degrades U-containing double-stranded DNA contaminants at room temperature. Heat-labile UDG is rapidly and completely inactivated at 50-55°C to maintain cDNA integrity;
• Ultra-high detection sensitivity: single-digit template copies or 0.1 pg of total RNA can be detected
• Ultra-high amplification specificity: AceTaq® DNA polymerase based on a hot chemical start, enzyme activity completely blocked before 95°C, and equipped with a patented specific promoting factor Exactor, which makes the amplification more specific;

Abstract

An epidemic of respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in China and has spread to other countries. Real-time reverse transcriptase-polymerase chain reaction (RRT-PCR ) from nasopharyngeal swabs has generally been used to confirm the clinical diagnosis. However, it is unknown whether the virus can be detected in samples from other sites and thus may be transmitted in ways other than respiratory droplets.

Methods

We investigated the biodistribution of SARS-CoV-2 among different tissues of hospitalized patients with coronavirus disease 2019 (COVID-19) diagnosed based on symptoms and radiology and confirmed by SARS-CoV-2 detection. This study was approved by the ethics committees of the participating hospitals, waiving informed consent.

Patients were included with samples collected according to clinical indications from 3 hospitals in Hubei and Shandong provinces and Beijing, China, from January 1 to February 17, 2020. Pharyngeal swabs were collected from most patients at 1 to 3 days after hospital admission.

Blood, sputum, stool, urine, and nasal samples were collected throughout the illness. Bronchoalveolar lavage fluid and fiberoptic brush biopsy samples were taken from patients with severe disease or undergoing mechanical ventilation. RNA was extracted from clinical samples and determined by RRT-PCR targeting the SARS-CoV-2 open reading frame gene 1ab as described previously.2 RRT-PCR cycle threshold values ​​were used as indicators of the SARS-CoV copy number.

RNA in samples with lower cycle threshold values ​​corresponding to higher viral copy numbers. A cycle threshold value less than 40 is interpreted as positive for SARS-CoV-2 RNA. Four high copy number SARS-CoV-2 positive faecal samples were cultured and then electron microscopy was performed to detect the live virus. Patterns were explored in a subgroup of patients with multiple samples collected during hospitalization.

Results

A total of 1,070 samples were collected from 205 COVID-19 patients who had a mean age of 44 years (range, 5-67 years) and 68% were men. Most of the patients presented fever, dry cough and fatigue; 19% of patients had severe disease. Bronchoalveolar lavage fluid samples showed the highest positive rates (14 of 15; 93%), followed by sputum (72 of 104; 72%), nasal swabs (5 of 8; 63%), fiberoptic brush biopsy (6 of 13; 46%), throat swabs (126 of 398; 32%), stool (44 of 153; 29%), and blood (3 of 307; 1%). None of the 72 urine samples was positive.

Mean cycle threshold values ​​for all sample types were greater than 30 (<2.6 × 104 copies/mL), except for nasal swabs with a mean cycle threshold value of 24.3 (1.4 × 106 copies/mL), indicating higher viral loads.

Twenty patients had 2 to 6 specimens collected simultaneously. Viral RNA was detected in individual samples from 6 patients (respiratory samples, faeces or blood), while 7 patients excreted virus in respiratory tract samples and faeces (n = 5) or blood (n = 2). Live SARS-CoV-2 was observed in the stool sample of 2 patients who did not have diarrhoea.

Discussion

In this study, SARS-CoV-2 was detected in multi-site samples from 205 COVID-19 patients, with lower respiratory tract samples testing positive for the virus more frequently. Importantly, the live virus was detected in faeces, implying that SARS-CoV-2 can be transmitted via the faeces. A small percentage of blood samples had positive results on the PCR test, suggesting that the infection can sometimes be systemic.

Respiratory and extra-respiratory transmission of the virus may help explain the rapid spread of the disease. In addition, multi-site sample testing can improve sensitivity and reduce false-negative results. Two smaller studies reported the presence of SARS-CoV-2 in anal or oral swabs and blood from 16 patients in Hubei province, and the viral load in throat swabs and sputum from 17 confirmed cases.

Limitations of this study include that some patients did not have detailed clinical information available, so the data could not be correlated with symptoms or disease course, and that the number of some sample types was small. Further investigation of patients with detailed temporal and symptom data and consecutively collected samples from different sites is warranted.