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I heard that the scientist in Australia found a way to segregate the 2019 NovCoronovirus from the blood. Does this mean they are one step closer to the cure? Why is virus segregation crucial in finding a cure for a viral infection?
2019-NCoV was first cultured by a Chinese group, as published here on January 24. China has not shared samples, but did share sequencing data in the linked paper and via GISAID. The Australian group at the Doherty Institute hasn't published yet, but did put out a press release today (January 29), and appears to be the first group outside of China to have cultured 2019-NCoV.
Viral isolation, and in particular, culture, is an important, and often difficult step in characterizing a novel virus. Once characterized, viral culture serves as a gold standard for diagnosis. Generally speaking, the ability to grow virus in cell culture allows for a broad range of pre-clinical studies that can lead to clinical advances including prevention, diagnosis, and treatment. Consider how difficult it may be to study a pathogen if you are not able to manipulate it in a living culture. You can read about this in Murray Medical Microbiology, Ch 50. This article, which does not require a subscription, may also be of interest. The article describes a variety of historical and modern methods, but also includes some discussion about the use of viral culture.
Isolating virus particles is important for accurate genomic sequencing, which in turn enables study of mutation rates and determination of hotspots, locations in the genome where mutations occur at higher rates:
“Sorting individual viral particles makes it possible to identify and sequence the genomes of viruses one by one,” states Òscar Fornàs, head of the Flow Cytometry Unit of Pompeu Fabra University and the Centre for Genomic Regulation.
Researchers attempting to create vaccines need to know this information about the viral sequence, in order to make a functional vaccine:
Dr. Corbett and others had studied the spike proteins on SARS and MERS viruses in detail, using them to develop experimental vaccines. The vaccines never made it to market because SARS was successfully contained with public health measures before the vaccine was ready and preliminary human trials for the MERS vaccine showed success last year.
But the scientists had a method for developing vaccines that could help them fast-track production for the new coronavirus. They used the template for the SARS vaccine and swapped in just enough genetic code that would make it work for the new virus. “I call it plug and play,” Dr. Corbett said.
Characterizing how quickly a virus mutates, and where, can decide the effectiveness of a vaccine.
In the United States, for example, this year's batch of influenza vaccine did not match up well with the strain of flu virus that ended up becoming dominant:
An unusual viral strain is dominating flu activity across the United States and may be one reason for the severe infections in children so far, according to a new report from the Centers for Disease Control and Prevention, and this season's influenza vaccine is not a close match for the virus.
There are limited resources to allocate to vaccine development. Faster and more accurate sequencing can help with a more targeted and effective response.
What Are Gram Negative Rods?
Gram-negative rods, or bacilli, are rod-shaped bacteria that give a negative result with Gram staining. These bacteria have a thin peptidoglycan layer sandwiched between the inner and outer cell membrane. This layer is removed when the bacterial cells are washed with an alcohol solution so they do not retain the crystal violet stain first applied. Instead, the cells turn red or pink with the safranin or fuchsine counterstain.
In contrast, gram-positive bacteria have a thick peptidoglycan layer and retain the crystal violet stain, even after washing the cells with an alcohol solution. Some examples of gram-negative rods are the Salmonella, Escherichia, Pseudomonas, Vibrio, Campylobacter and Shigella species. These type of bacteria are known to cause pneumonia, infections in the urinary tract, gastrointestinal tract, bloodstream and other parts of the body. Escherichia coli are commonly found in the gastrointestinal tract of people and cause urinary tract and gastrointestinal tract infections. Salmonellosis is the diarrhea caused by Salmonella bacteria. The common sources of this bacteria are eggs, dairy and poultry products. A particular species, Salmonella typhi, however, does not cause diarrhea but rather enteric fever. Another gram-negative bacilli that are causing a growing concern regarding the incidence of pneumonia are Klebsiella pneumonia. Because of the resistance of these bacteria to nearly all modern antibiotics, they are more difficult to control and are spreading worldwide.
Blood Smear: Method of Preparation and Principle | Blood | Biology
A well prepared blood smear is necessary for microscopic examination of blood. Blood smears are used to determine leukocyte differentials, to evaluate erythrocyte, platelet and leukocyte morphology, and, if necessary, to estimate platelet and leukocyte counts.
Desirable qualities of a blood smear include:
i. Sufficient reading area
ii. Acceptable morphology within the working area
iii. Even distribution of leukocytes
The Clinical Pathology Laboratory uses the wedge technique for preparation of blood smears. This method produces a gradual decrease in thickness of the blood from thick to thin ends with the smear terminating in a feathered edge approximately 2 mm long.
The smear is greater than 25 mm long and the feathered edge stops approximately 10 mm from the end of the slide. The blood film occupies the central portion of the slide and has definite margins on all sides that are accessible to examination by oil immersion.
The thin end of the film becomes thinner gradually and does not have grainy streaks, troughs, ridges, waves or holes – features that can result in an uneven distribution of leukocytes. In preparations from normal patients, the thin section of the smear occupies approximately 1/3 of the total area and, within that area erythrocytes are distributed in a monolayer.
The thickness of the spread is influenced by the angle of spreader slide (the greater the angle, the thicker and shorter the blood smear), the size of the drop of blood and the speed of spreading. Glass cover slips are mounted on all blood smears to prevent damage to smear during examination, cleaning, handling and storage.
Preparation of Blood Smear:
2. E.D.T.A. blood (within 1 hr. of collection)
Preparation of Blood Film:
The slide should be clean. Place a small drop of blood, or one side about 1-2 cm from one end. Without delay place a spreader at an angle of 45° from the slide and move it back to make contact with the drop. The drop should spread out quickly along the line of contact of spreader with the slide.
The moment this occurs, spread the film by rapid smooth forward movement of the spreader. The film should be 3-4 cm in length. The ideal thickness is such that there is some overlap of R.B.C. throughout most of its length with proper separation and lack of distortion of RBC’s. the end from where the spread had ended is called tail end.
The ideal zone to examine the blood film is the areas between tail and body. If the film is made too thin or if a rough edged spread is used many leucocytes accumulate in edges and at tail. DLC should not be attempt on such a slide.
Characteristics of an Ideal Blood Smear:
1. It should be in the central 2/3 of slide.
2. It should have straight lateral border and short tongue shaped tail.
Precautions to be taken during preparation:
1. Angle should be maintained at 45°.
2. Blood drop should be of proper size.
3. Spreader’s edges should be smooth and it should be smaller than the slide on which smear is being made.
4. Pressure applied should be proper.
5. Drop should be pulled with spreader not pushed with it.
6. Preparation should be in one single stroke.
Staining of Blood Film:
Process of Staining:
The slide is covered with leishman stain for 2 mins. This much time is required for fixation. After 2 mins it is diluted with double the volume of buffer water. On adding buffer water a metallic shin will be formed, if the stain is dry. Allow this to stand for 15 min. after min, flood the slide with water to remove stain. Then wash under tap water wiping the back of slide with finger or cotton. Dry in air.
Precautions Suring Staining:
1. Time: Initial time 2 minutes, is important. After dilution increase of 1-2 minutes, does not alter staining.
2. Never let the stain dry on the slide otherwise stain deposits will make it impossible to count leucocytes (DLC).
3. Staining should be deposit free.
4. For washing the smear – let the water stream replace the stain. Don not throws the stain first.
Technique of Differential Leucocyte Counting:
D.L.C. is done is oil immersion, with wide open diaphragm and high up condenser. D.L.C. is best done in area where RBC morphology is good. There tends to be somewhat unequal distribution of WBC is normal blood film.
The smaller cells (like small lymphocytes) being in greater relative number in central thicker portions and the larger cells (monocytes eosinophils) being in greater relative number- along the edges and the tail. So the D.L.C. on a smear depends upon the area of the slide used for counting. To overcome this difficulty both area are included in counting.
Two methods are employed in counting:
1. Going back and forth lengthwise including both body and tail.
2. Going back and forth sidewise including both edges and centre. Usually 100 cells are counted.
It is used for malaria and microfilaria.
Sample is taken at or just after height of fever with shivering.
Sample is collected at midnight. Drop of blood is taken on slide which is spread evenly, air dried and then de-hemoglobinised by placing film in distilled water for 5-10 min. Leishman’s stain is mostly used for malaria.
A buffy coat preparation facilitates the search for immature and abnormal white blood cells, particularly when the peripheral blood count is low, and in identification of bacteria or yeast in septic and immune-compromised or immunosuppressed patients.
Micro-hematocrit centrifugation of specimen. Concentration of WBC’s Wright-Giemsa Stain.
Specimen Requirements: Collect:
Routine venipuncture, EDTA (Lavender-top tube)
Stable at Room Temperature for 24 hours.
Stable Refrigerated for 36 hours.
Clotted, QNS, old specimen, identification errors, labeling errors, inappropriate anticoagulants
What are viruses made of?
At the core of a virus particle is the genome, the long molecule made of DNA or RNA that contains the genetic instructions for reproducing the virus. This is wrapped up in a coat made of protein molecules called a capsid, which protects the genetic material.
Some viruses also have an outer envelope made of lipids, which are fatty organic molecules. The coronavirus that causes COVID-19 is one of these these “enveloped” viruses. Soap can dissolve this fatty envelope, leading to the destruction of the whole virus particle. That’s one reason washing your hands with soap is so effective!
Quality control of Blood Agar
- The pH of the blood agar range from 7.2 to 7.6 at room temperature.
- Inoculate the plates with 5-hour broth cultures of Streptococcus pyogenes and S. pneumoniae.Inoculate also a plate with H. influenzae and streak with S. aureus (i.e. Satellitism Test).
- Incubate the plates in a carbon dioxide enriched atmosphere at 35-37°C overnight.
- Check for the growth characteristics of each species
- S. pyogenes: Beta-hemolysis
- S. pneumoniae: Alpha-hemolysis
Optimizing Epidemiology–Laboratory Collaborations
Alexander Langmuir was quoted in the early 1960s instructing incoming Epidemic Intelligence Service (EIS) officers that the only need for the laboratory in an outbreak investigation was to &ldquoprove their conclusions were right.&rdquo
Although these isolated quotes make Louis Pasteur and Alexander Langmuir seem to have opposing ideas about the role of the laboratory in outbreak investigations, each quote has some validity. In the late nineteenth century, Pasteur&rsquos laboratory data supporting the &ldquogerm theory&rdquo of disease not only led to pasteurization and vaccination (1), but also provided the evidence that swayed the court of scientific opinion toward accepting the theory that microorganisms are a cause of disease. This shift in scientific focus shed light on the then relatively unknown work of Dr. James Lind, Dr. Ignaz Semmelweiss, and Dr. John Snow, whose elegant maps and statistics introduced epidemiology as a science. Nearly a century later, at the time of Dr. Langmuir&rsquos comment (2), a field investigation team, armed only with epidemiology tools, arrived in Pontiac, Michigan, and determined the chain of infection in a point-source outbreak associated with an unknown microbial agent growing in a new reservoir with a new mode of transmission (3). Importantly, the epidemiology results were used to halt the Pontiac fever outbreak&mdash8 years before the laboratory of the Center for Disease Control (later Centers for Disease Control and Prevention [CDC]) identified Legionella spp. as the causative agent (4). These examples demonstrate that comparing the relative importance of epidemiology and laboratory science to a field investigation is similar to the age-old debate about the order of appearance of the chicken or the egg&mdashit is circular and obscures more fundamental truths. What both Pasteur and Langmuir believed&mdashand what history has shown&mdashis that both epidemiologists and laboratory scientists can make independent discoveries that have significant scientific impact, but collaboration across these disciplines has a synergistic effect, yielding public health data that are stronger than either discipline can provide alone (2).
Modern Role of Public Health Laboratories in Field Investigations
During the early 1980s, revolutionary developments in molecular biology and computer science began to be reflected in technologic advances in laboratory science. As a result, investigators can now accomplish sample collection, electronic sample receipt and tracking, sample processing, patient diagnostics, chemical identification and quantification, microbial identification, and antimicrobial susceptibility testing more rapidly, safely, and accurately than ever before. Laboratory technology&mdashincluding whole-genome sequencing, bioinformatics, and other technologies associated with advanced molecular detection&mdashcontinues to develop at a phenomenal pace.
As laboratory technology has expanded over time, so has the laboratory role in field investigations. In addition to the traditional roles of providing causative agent and point-source identification and case confirmation, current laboratory results can be leveraged to inform all aspects of an outbreak investigation. Critical field investigation goals that have been informed by laboratory data include
- Etiologic agent identification and characterization (5,6).
- Case determination and detection (7,8).
- Point-source identification (9).
- Clinical care guidance and case management (10&ndash12).
- Root cause analysis and intervention design (13).
- Outbreak control (14&ndash16).
- Chain of infection definition (17,18).
Improving a multidisciplinary understanding of epidemiologic needs and requirements, as well as ways to best leverage advanced diagnostic laboratory capabilities, will continue to strengthen the performance of future outbreak investigation teams.
This chapter provides general guidance and recommendations for the field investigation team. While the chapter&rsquos primary focus is infectious disease outbreaks, laboratory-related information for nonbiological exposures also is included. These instructions, although not necessarily universal, are intended to help the team consider how to integrate local, state, regional, national, and international laboratory expertise and capacity throughout the outbreak investigation process. Online resource links for the specific laboratory-related tasks involved in a field investigation are included to provide access to the most recently updated information.
These recommendations represent a best-case scenario. Adjustments to laboratory-related plans often are required because of resource or time constraints, emergencies, and local considerations. Local considerations can include policies, regulations, environment, climate, culture, infrastructure, and socioeconomics. In many cases, the field investigation team needs to use judgment to adapt these recommendations to the unique local circumstances&mdashbut only after careful consideration of their effects on safety, patient care, and outbreak control. This analysis should involve collaboration with field epidemiologists, laboratory experts, and local stakeholders.
Step 1. Involve the Laboratory Early and Often
Because new technologies are regularly added to the arsenal of available laboratory tests, listing them is impractical. This evolving catalog of tests means that optimal sample types also can change, thus dictating changes in collection protocols. Therefore, it is vital that outbreak investigators contact the relevant laboratories as soon as possible, preferably before deploying to the field investigation site. Local health department, state public health, and contract laboratories can perform some testing, whereas CDC subject-matter experts and laboratories can support investigations as needed through consultation, testing, and collaboration.
The general types of laboratory activities performed by CDC include
- Outbreak investigation.
- Emergency response.
- Population health studies.
- Laboratory quality improvement.
- Culture-based pathogen detection and characterization.
- Molecular-based pathogen detection and characterization.
- Detection of high-consequence pathogens.
- Genetic studies.
- Vaccine development.
- Pathogen discovery.
- Newborn screening.
- Occupational health.
- Chemical exposure testing.
- Assessment of environmental exposure.
A public health professional conducting an outbreak investigation should use the following checklist to enhance the effectiveness of these early communications with the laboratory receiving samples. This early communication is especially important in outbreaks of unknown etiology to ensure that samples are collected in a manner that enables rapid laboratory testing of a variety of potential etiologic hypotheses. It is also important to remain aware that etiologic hypotheses based on clinical signs and symptoms should include consideration of multiple factors (both biological and abiological) (19).
- If the investigation involves a suspected pathogen (e.g., bacteria, viruses, parasites, fungi) or biological toxin as an etiologic agent, then consult local laboratory resources and appropriate clinical and laboratory guidance (20). Pathogen-specific tests and contacts for CDC resources also can be found in the CDC Laboratory Test Directory (https://www.cdc.gov/laboratory/specimen-submission/list.html).
- If the investigation involves a suspected chemical or radiologic agent as an etiologic agent or if the outbreak involves a noninfectious disease or if the event might have been caused by biological, chemical, or radiologic terrorism, then contact the local or state public health department. CDC&rsquos National Center for Environmental Health, Division of Laboratory Services (NCEH-DLS) (https://www.cdc.gov/nceh/dls/) also can offer guidance on options for testing and epidemiologic data, local laboratory resources, and relevant studies (21&ndash25) and should guide testing during the investigation.
- Consult test descriptions relevant for the suspected agent and collaborate with the appropriate laboratory to determine the tests to perform based on the needs of the field investigation.
- Inform the laboratory of the purpose of the samples so that resources can be prioritized. The various purposes of sample submittal can include management of patient care, outbreak source identification, surveillance, research, law enforcement, and definition of cases.
- Gather information about the turnaround time for the selected test to manage expectations during the field investigation. Some tests can take up to 2 months to yield data.
Special Consideration: Test Selection
Test selection is one of the most important tasks to complete as early in the investigation as possible. A large number of tests are available to identify and characterize microorganisms, and serologic and antimicrobial susceptibility tests are available to provide a better understanding of potential exposures and inform treatment and infection control decisions. Local, state, and federal public health laboratories, as well as private hospital and contract laboratories, are available testing resources. However, it is important that the field investigator inquire about the accreditations or quality management programs of the laboratories to ensure use of the best resources for each field investigation. CDC laboratories can perform more than 300 infectious disease tests for a wide variety of microorganisms the current list of available tests can be found at https://www.cdc.gov/laboratory/specimen-submission/list.html. This list can be used to select the appropriate tests for the suspected organism during a field investigation or to find CDC contact information for subject-matter experts on a particular organism.
Not all field investigations involve infectious microorganisms. For those investigations of noninfectious sources, some local and state health departments and NCEH-DLS perform testing in the areas of chronic disease markers, chemical and radiologic threat agents, environmental chemicals, newborn screening, and nutritional markers of disease. NCEH-DLS also can provide the field investigation team with appropriate sampling containers and protocols to minimize sample contamination from extraneous sources, as well as personnel to assist the field team. Contact NCEH-DLS directly through its website (https://www.cdc.gov/nceh/dls/).
To ensure the most rapid turnaround of accurate test samples, laboratories need lead time to order surge supplies, rearrange workloads, implement or modify protocols, and train additional staff. Laboratory experts can also help with some of the more difficult aspects of test selection. For example, specific symptoms (e.g., atypical pneumonia) can be associated with exposure to multiple biological and abiological etiologic agents similar organisms (e.g., Chlamydia spp.) can cause different symptoms. In addition, multiple types of tests are available for most organisms, and not all of them give results that can be interpreted in the same way (i.e., a positive reverse-transcription polymerase chain reaction [RT-PCR] is not the same as a positive culture because RT-PCR tests for presence of nucleic acids and culture tests for viable organisms). Endemic diseases at the field investigation site also can affect testing for specific organisms (e.g., dengue, chikungunya, and Zika viruses), and some infections might require testing across multiple labs with different sample collection and submission requirements (e.g., healthcare-associated fungal infections might need testing by an environmental laboratory and a mycology laboratory). The setting (e.g., hospital), geography (e.g., Old World vs. New World hantaviruses), disease (e.g., genital ulcer disease), circumstances (e.g., biodefense), agent grouping (e.g., respiratory agents), sample type (e.g., whole blood), or purpose (e.g., surveillance) of the samples also can form the basis of test selection instead of, or in addition to, the suspected organism. Finally, some tests (especially chemical, radiologic, and molecular) are often exquisitely sensitive, and great care must be taken when selecting these tests to ensure appropriate sampling.
Special Consideration: Classic Versus Molecular Tests
Advanced molecular technologies backed by bioinformatics provide powerful new molecular detection systems that enable public health agencies to conduct surveillance, identify pathogens, recognize outbreaks, track transmission of a pathogen, detect antimicrobial resistance, and identify better ways to prevent disease. Genomics is central to many advanced molecular detection systems, and proteomics and transcriptomics are becoming important tools for public health. Molecular testing often can yield results more quickly than tests based on classical culture-based microbiology or serology/immunology, making them attractive alternatives during a field investigation. These tests play an increasingly important role in the general trend toward culture-independent diagnostic tests, which have the advantage of quickly and simultaneously testing for multiple pathogens within one sample. However, classical tests remain critical for completely characterizing disease-causing organisms and host responses and identifying new pathogens and new methods of antimicrobial resistance because detecting the presence of organism DNA/RNA by PCR is not the same as detecting a viable organism. Thus, investigation planning should include sample collection protocols suitable for the tests deemed most effective after consultation with the receiving laboratory.
Rapid molecular tests, such as real-time PCR, can yield definitive results within hours of sample receipt in the laboratory, but these use primers and probes designed to detect likely pathogens, which means these assays might miss an unsuspected or new pathogen. Technologies based on mass spectrometry (e.g., matrix-assisted laser desorption ionization&ndashtime of flight) are similarly limited in that the signal is compared with a reference library of likely pathogens. In contrast, untargeted nucleic acid sequencing (e.g., microbiome sequence analysis) can theoretically identify all organisms in a sample but present formidable analytic challenges because of the computational resources required to filter out irrelevant signals from host genome and benign commensal organisms. Even when a rapid molecular test identifies a pathogen, additional characterization using classical protocols often is needed to present a complete picture of the epidemiologic situation. For example, real-time PCR might identify the causative pathogen in an outbreak of meningitis as Neisseria meningitides, but the picture would be incomplete without also determining the serotype, which requires classical culture techniques.
Deciding on the tests to be performed on the samples before embarking on an investigation is necessary. The types of samples suitable for culture-independent diagnostic tests, which focus on the genotype (sequencing or PCR methods) or physical characteristics (mass spectroscopy) of the organism, can be different from those sample types needed for classical culture methods, which focus on the phenotype of the organism through methods such as serotyping and antimicrobial susceptibility testing. Protocols for culture-independent diagnostic tests often destroy the sample during nucleic acid or other target extraction, leaving no viable organisms to culture. Ideally, multiple samples should be collected, enabling both molecular and classical testing. However, if samples are in short supply or packaging and shipping limitations are a factor, testing and sample collection decisions should be made in advance in collaboration with the laboratory to determine whether molecular or classic tests would generate the most useful information.
For all tests, consult with the laboratory as early in the investigation as possible to address the following criteria for appropriate test selection:
- Supplemental information.
- Supplemental forms.
- Sample types.
- Acceptable samples.
- Minimum volumes required.
- Storage and preservation of samples.
- Sample transport medium.
- Sample labeling.
- Shipping instructions.
- Sample handling requirements.
- Testing methods.
- Testing turnaround time.
- Test interferences and limitations.
- Additional information.
- Laboratory points of contact.
Step 2. Collaborate on the Planning and Execution of Field Sample Collection
Collaborate with the laboratory before collecting samples to ensure samples are collected safely and are acceptable for the selected test. Accidental exposures during sample collection could result in severe illness, and unacceptable sample collection can result in missed opportunities for testing. For example, collecting blood from 20 people with potential exposures to an unknown pathogen and using the wrong anticoagulant can result in delayed microbial identification and delayed treatment, which could have serious consequences. The laboratory scientist can also provide ecology, growth, transmission, and pathogenesis expertise about microorganisms, as well as chemical and radiologic expertise. This expertise can support the investigation in many ways, some of which include
- A risk assessment of the planned sampling activities to create a safe environment for the work. At a minimum, the risk assessment should identify the potential biological, chemical, radiologic, and physical hazards and plan appropriate mitigations, including the use of personal protective equipment (PPE), to minimize exposure to hazards.
- A sampling plan, which includes preliminary hypotheses and ways the laboratory can assist in testing those hypotheses through targeted sampling.
- Sample collection methods, which need to be appropriate and sufficient for the specific tests (e.g., immunologic assays require specific timing of sample collection for diagnostic testing to be performed).
- Sample collection training, including assessing whether all the field investigators have adequate sampling experience and training (including training in PPE use) or whether laboratory personnel should deploy to the field to collect samples.
- Sample transport. Ideally, sampling activities should be planned to ensure that shipments arrive at the receiving laboratory on a weekday. Some shipments cannot be accepted on weekends.
A public health professional conducting an outbreak investigation should follow these generalized recommendations during sampling:
- Identify and obtain appropriate PPE in sufficient quantities before deploying to the field and double-check it for completeness at the sample collection location. Ensure the team has been trained in donning, doffing, cleaning/disinfection, storage, and proper disposal of the designated PPE.
- Review all relevant safety, infection control, and patient management guidelines before, and adhere to them during, sample collection. For instance, identify and maintain a specific area for donning and doffing the designated PPE and have a plan for managing sample collection waste.
- Collect an appropriate volume of sample.
- Label each clinical sample with at least two identifiers that link the sample to the patient, with the expectation that personal identifying information will not be used.
- Label each nonclinical sample (e.g., environmental, animal) with at least two identifiers that enable linking of the sample with the most pertinent organism, place, or thing (e.g., OrganismID and LocationID, SampleTypeID and MedicalDeviceID).
- Coordinate with local and state labs and clinicians to obtain samples. Do not collect samples without specific training in the collection procedure because the generalized guidance in this chapter might not be appropriate for a specific requested test.
- Review the special considerations discussed later and contact the laboratory for specific guidance before sampling.
Special Consideration: Risk Assessment
A risk is the possibility that an undesired event will occur (i.e., a function of the likelihood and consequences of a particular undesired event). Before conducting a field investigation or any laboratory activity, assess the risks associated with that activity. The epidemiologists and the laboratory scientists should conduct the risk assessment for a field investigation jointly, with assistance from other subject-matter experts including local and state public health laboratory scientists and epidemiologists, clinicians, appropriate facility staff, and appropriate emergency response planners and responders. Share the results of the risk assessment among the investigation team members so that everyone understands the risks involved. Use the results of the risk assessment as the basis for determining how to mitigate those risks. During the investigation, routinely monitor and reassess operations to identify and mitigate additional risks and to account for any new information or circumstance associated with particular activities. After the field investigation, review operations to identify ways to improve future field investigation risk assessment.
The principles of risk governance (26,27) articulate that a risk assessment should follow three general steps:
- Define the situation: What work will occur?
- Define the risks in that situation: What can go wrong?
- Characterize each of the risks: How likely is each risk to occur? What would be the consequences of each risk?
Start the risk assessment process by thoroughly defining the situation and the activity. Particularly important is where the work will take place, who will conduct it (including their knowledge, skills, and abilities), what equipment they will use (including sampling and PPE), and what hazards they will encounter. A hazard is something that has the potential to cause harm, such as a sharp object or a biological agent. Considerations of risk should address the most obvious agent-related hazards of a field investigation (e.g., unintended exposure, physical injury). However, risk assessments should also encompass the less obvious hazards that could result in negative consequences. For each field investigation, in addition to agent-related hazards, also consider any investigation activities&ndash related hazards that might result in negative outcomes (e.g., poor sample collection yielding incorrect or ambiguous laboratory results shipping hazard resulting in delayed or incorrect case management data management issues with patient privacy or consent inadvertent violation of facility rules or local, state, or federal regulations miscommunications that might erode collaborative relationships).
In a field investigation, the hazards are multifactorial, diverse, unique, and potentially of major consequence. To mitigate the risks, everyone involved in an investigation must consider everything that might go wrong during the various stages of that activity and then evaluate each risk from the perspective of its likelihood of occurrence and the consequences. After prioritizing those risks from highest to lowest, review the use of specific mitigations measures to reduce those identified risks. Before work begins, agree that the mitigated risks are acceptable. If the risks cannot be adequately mitigated and remain unacceptable, do not undertake the work.
Special Consideration: Etiologic Agents of Disease Syndromes
The same disease syndrome (e.g., respiratory) can be caused by one or more of many pathogens (e.g., influenza virus, Legionella spp., or hantaviruses) or by an abiological chemical or radiation exposure (e.g., chemical-induced acute respiratory distress syndrome). It is also possible for clinical presentations to be atypical, which can complicate field investigations (28&ndash30). To maximize the effectiveness of investigations of outbreaks of unknown etiology, investigators need to collaborate with laboratory and epidemiology subject matter experts representing a diverse range of potential etiologic agents. Table 9.1 can be used as a tool in these larger discussions to help formulate hypotheses about the etiologic agents of various disease syndromes and to determine appropriate samples to collect for testing. Additional online resources for investigations of outbreaks of unknown etiology based on disease syndrome are also identified.
Special Consideration: Sample Collection
Table 9.2 shows sample collection supplies, a basic collection procedure, and sampling considerations for various clinical sample types for infectious disease testing. For similar information for environmental toxicant testing, see Table 9.3. These guidelines are presented to help in planning for sample collection in the field but should always be discussed with the laboratory before sample collection takes place.
Infectious disease a syndromes and types of clinical samples
Syndrome and online resource Some possible etiologies Sample type Suspected agent Dermatologic Chickenpox, monkeypox, variola, vaccinia, measles, cutaneous anthrax, herpes, Vesicular fluid, scab, serum, vesicular exudate Viruses, bacteria, Diarrheal (http://cifor.us/products/guidelines) Watery (cholera), dysentery (shigellosis), febrile gastroenteritis (typhoid fever), vomiting (norovirus, bacterial intoxications) Feces, blood, emesis Bacteria, viruses, parasites, toxins, chemicals Hemorrhagic fever Arboviral (dengue fever), arenaviral (Lassa fever), filoviral (Ebola virus disease), malaria Blood, blood smear, serum, postmortem tissue biopsy Viruses, parasites Jaundice Hepatitis A&ndashE, spirochetal (leptospirosis), yellow fever Serum, postmortem liver biopsy, blood culture, urine Viruses, bacteria Neurologic Guillain-Barré syndrome, polio, meningoencephalitis, rabies, meningitis Stool, cerebrospinal fluid, blood, blood smear, serum, throat swab, postmortem samples Viruses, bacteria Ophthalmologic Trachoma, keratoconjunctivitis, conjunctivitis Conjunctival swab/smear, serum, throat swab Viruses, bacteria Respiratory (https://www.cdc. gov/urdo/index. html) Influenza, hantavirus, pertussis, legionellosis, pneumonia, tuberculosis, severe acute respiratory syndrome coronavirus Throat swab, serum, nasopharyngeal swab, blood, sputum, urine Viruses, bacteria, toxins Systemic Varied and often caused by same agent as other syndromes Postmortem tissue biopsy, serum, cerebrospinal fluid, urine, blood culture, aspirate, blood smear Viruses, parasites, bacteria
a For definition and review of syndromes for chemical or radiologic agents, see the CDC Emergency Preparedness and Response website for chemical (https://emergency.cdc.gov/chemical/) and radiologic (https://emergency.cdc.gov/radiation/index.asp) emergencies.
Sample collection for suspected infectious agent exposures
Sample type Supplies Considerations Blood Sterile collection tubes Needles and syringes Patient age and other demographics may be useful for laboratory to select reference ranges. Blood Tourniquet Collect
Sterile transport tubes Sterile lancet or needle
Syringe and wide-bore needle
Viral and bacterial samples might have different storage and transport conditions.
- Two 10 mL tubes without anticoagulant
- One 5-mL tube without anticoagulant
- 50 mL
- 10&ndash20 mL
- Whole blood (Heparin)
- One 7 mL tube or three 4 mL tubes or four 3 mL tubes
- Two 3 mL tubes
- 50 mL
- 10&ndash20 mL
- Whole blood (EDTA)
- One 3 mL tube
- One 3 mL tube
- One 7 mL trace metals-free tube
- One 7 mL trace metals-free tube
- Two 10 mL tubes without anticoagulant
- One 5 mL tube without anticoagulant
- 50 mL
- 10&ndash20 mL
- Whole blood (EDTA)
- One 2 mL tube
- One 2 mL tube
- Whole blood (Heparin)
- One 7 mL tube or three 4 mL tubes or four 3 mL tubes
- Two 3 mL tubes
Special Consideration: Potential Sampling Pitfalls
One reason it is important to contact the laboratory before sampling is because of sampling pitfalls. The pitfalls can include issues with sampling tools (e.g., inhibition of tests by certain swab materials), sample collection technique (e.g., hemolysis of blood samples), sample storage (e.g., degradation of RNA), or sample timing (e.g., matched sera). Table 9.4 describes some specific pitfalls to avoid, but it is not a comprehensive list, so consult with the laboratory prior to collecting samples.
Special Consideration: Personal Protective Equipment
PPE is specialized clothing or equipment used to protect against exposure to hazards that can cause serious injury or illness. Exposures can result from contact with chemical, radiologic, physical, electrical, mechanical, or other hazards. PPE may include items such as gloves, safety glasses and shoes, earplugs or muffs, hard hats, respirators, or coveralls, vests, and full-body suits. PPE selection should be tailored to the specific risks associated with each individual field investigation. The Occupational Safety and Health Administration has created multiple online resources that can be used to help select appropriate PPE and identify additional safety-related information for specific hazards (https://www.osha.gov/dts/osta/oshasoft/index.html external icon ). Additional Occupational Safety and Health Administration (https://www.osha.gov/Publications/osha3151.pdf pdf icon external icon ) and National Institute for Occupational Safety and Health (https://www.cdc.gov/niosh/docs/2005100/pdfs/2005-100.pdf pdf icon ) guidance is also available.
In general, there are three major questions to consider when selecting PPE. First, what is the anticipated exposure type (splash, spill, spray), volume (large, small), and source (chemical, biological, or radiologic agent)? Second, what PPE is durable enough and appropriate for the task (protect against fluids, powders, gases)? Third, how will the PPE affect movement and work (appropriate size, not too hot)? Some PPE considerations are shown in Table 9.5, but investigation teams should consult with appropriate laboratory scientists on the types of PPE that are most effective for any particular field investigation as biological, chemical, and radiologic hazards each require specialized PPE.
Specific sampling pitfalls, by laboratory test type
Test type Consideration Antimicrobial susceptibility testing Store at conditions best suited to maintaining viability of culture.
Low viral loads and genetic variance can affect assays.
Include treatment history of patient.
Preservatives (formalin or alcohol) can affect organism viability.
Disinfectants (chlorine, Lysol, alcohol) can affect tests.
Cultures of some organisms might require stricter shipping rules.
Storing blood at temperatures other than 2&ndash8°C can affect tests.
Multiple freezes&ndashthaws (especially >3) can affect test performance.
Heparin can interfere with molecular assays.
Insufficient volume of sample can invalidate tests.
Therapeutic agents can affect the detection of organisms.
Co-infections or contaminations can affect test results.
Calcium alginate swabs or swabs with wooden sticks can contain substances that inhibit some molecular assays.
Not separating serum from cells in blood samples can result in RNA degradation.
Multiple freezes&ndashthaws (especially >3) can affect test performance.
Decomposition of tissues can affect test performance.
Less than 1:10 ratio of tissue to 10% formalin can prevent fixation.
Contamination can interfere with testing.
Bilirubin, lipids, and hemoglobin can interfere with serologic assays.
Not separating and freezing serum from cells in blood samples can result in antibody degradation.
Failure to use plastic tubes can prevent shipment and may result in samples not being accepted at the lab.
Pooled samples can cause difficulties in interpretation of lab results.
Step 3. Collaborate with Laboratory for Storage and Shipment of Samples
The sender is responsible for ensuring that samples are stored and transported to the laboratory under appropriate conditions. Shipping requirements for infectious or potentially harmful samples submitted for diagnostic or investigational purposes must be packaged and shipped in compliance with appropriate regulations. Before shipping any samples from the field investigation, consult with the receiving laboratory and appropriate shipping experts to ensure adherence to relevant regulations and best practices. The information and links given here provide a starting point to navigating the storage, submission, and shipping requirements you may need to address.
Personal protective equipment (PPE) considerations for collecting potentially infectious samples a
Examples of PPE When to wear Considerations Apron, lab coat, gown, coveralls To protect skin and clothing from splash hazards Recommend clean, disposable, fluid-resistant isolation gown that covers torso, fits comfortably, and has long sleeves that are snug at wrists. Consider coverage (i.e., apron instead of gown for limited potential contamination), cleaning (i.e., laundering and reuse of gown), permeability to fluids, and patient risk (i.e., sterile gown for invasive procedure). Latex or nitrile gloves To protect hands from touch hazards Recommend single pair of nonsterile, disposable vinyl, latex, or nitrile gloves changed between patients and samples or when torn or soiled. When selecting or using gloves, consider fit of gloves, duration of task, &ldquowetness&rdquo of task, potential for transmission from gloves to patient, potential for touching environmental surfaces. Surgical masks To protect mouth and nose from splash, spray hazards Recommend masks that cover nose and mouth and prevent fluid penetration with flexible nose piece and elastic straps to secure fit. If aerosols are a concern, wear a respirator instead. N95 respirator, elastomeric respirator, PAPR To protect respiratory tract from inhalable particulate hazards Recommend N95 to protect from inhalable particles <5 &mu in diameter. For an invasive procedure that might result in large droplets or copious aerosols, consider a higher level respirator (e.g., PAPR). Respirator use requires medical evaluation, fit testing, training, and fit checking before each use. Goggles, safety glasses, face shield To protect eyes from splash hazards Recommend snug-fitting, antifog goggles or safety glasses or face shield that covers forehead and below chin and wraps around the side of the face. Personal glasses are not a substitute for goggles. Wear goggles that fit over prescription lenses.
a For definition and review of PPE for chemical or radiologic agents, please see the CDC Emergency Preparedness and Response website for chemical (https://emergency.cdc.gov/chemical/) and radiologic (https://emergency.cdc.gov/radiation/index.asp) emergencies. PAPR, powered air purifying respirator PPE, personal protective equipment.
- Samples to be sent to CDC for infectious disease testing:
- Storage requirements: See the specific storage requirements for each test at https://www.cdc.gov/laboratory/specimen-submission/list.html
- Submission forms: https://www.cdc.gov/laboratory/specimen-submission/form.html
- Shipping instructions: https://www.cdc.gov/laboratory/specimen-submission/shipping-packing.html
- Radiologic: https://emergency.cdc.gov/radiation/labinfo.asp
- Chemical: https://emergency.cdc.gov/chemical/lab.asp
- Samples to be sent to a state, regional, or local laboratory
- Work with the state, regional, local, or facility representative to contact the appropriate laboratory.
- In the event of an emergency in the field, contact the state public health laboratory. A list of emergency contacts for state public health laboratories can be found at http://www.aphl.org/programs/preparedness/Crisis-Management/Pages/Emergency-Lab-Contacts.aspx external icon
The following checklist provides some practical advice about packaging and shipping samples.
- Double-check that all sample containers are closed and intact.
- Disinfect the outside of the sample container before transport, storage, or shipment. Be sure to maintain the integrity of the labels.
- Transport samples in their primary container (e.g., tube, bottle, sample container) placed into a sealed and leak-proof secondary container (plastic bag, plastic container) prior to placing in the outer container (shipping envelope, shipping box).
- Ensure samples are cushioned to prevent breakage.
- Create a line-list of the samples with all appropriate information (e.g., sample site, sample type, patient identifier, device identifier, environment location, suspected source of sample).
- In some investigations (e.g., when criminal activity is suspected), a formal chain of custody form may be needed. Consult your local public health officials, shipping experts, and the receiving laboratory to obtain appropriate forms.
- Determine the specific shipping requirements of the samples (https://www.iata.org/whatwedo/cargo/dgr/Documents/infectious-substance-classification-DGR56-en.pdf pdf icon external icon ).
- Provide the tracking number of the shipment to the laboratory.
Step 4. Collaborate on the Interpretation of Laboratory Test Results
Laboratory tests are more complicated than ever to interpret, and the subsequent conclusions from&mdashand uses of&mdashlaboratory data from any field investigation are most effective and reliable when collaboration is strong among the following:
- Laboratory scientists, who can explain the language of the laboratory report and any specifics of the test or organism.
- Epidemiologists, who interpret the tests in the context of the field investigation.
- Clinicians, who interpret the tests in the context of patient management.
Because each of these specialties has different knowledge and serves different purposes, laboratory data are most effective when interpretation occurs in collaboration. This ensures that data limitations and strengths are understood and that the potential occurrence and consequences of false positives or negatives, undetermined results due to improper sample collection or insufficient sample volume, and nonreportable results due to values below the limit of detection or other confounders found during testing are minimized. Issues to consider when interpreting laboratory results may include the following:
- Laboratory tests for field investigations should be interpreted in the context of properly framed epidemiologic hypotheses. Investigators should always consider why the test was chosen and what was being asked.
- Interpretation of test results depends on sensitivity and specificity of the selected test. Consider how the prevalence of disease will affect the predictive value of the test.
- Consider what population the test characteristics are derived from and how that might differ from the population being tested.
- If a sample is negative on nonspecific media, the sample cannot be interpreted as negative unless it is also negative on a suspected agent-specific medium.
- In field investigations that involve emerging pathogens, Koch&rsquos postulates form the basis of proof that an emergent agent is the etiologic agent. Therefore, the interpretation should consider the successful fulfillment of each of Koch&rsquos postulates. Just because an agent is found does not necessarily mean it caused the disease.
- Consider the status of the patient because pathogens behave differently in different hosts. Patient factors to consider include immunologic status, treatments administered, age, physiologic status, sex, and race.
- Molecular typing and other relatedness studies can confirm the relatedness of isolates to support the epidemiologic hypotheses generated by the field investigation. Interpretation of relatedness is specific for the typing assay used.
- Reference ranges are not precise and can vary by laboratory, depend on the test, and are typically selected to contain 95% of healthy persons. However, correlation between out-of-range values and illness is not always clear. Consider the sample size used to collect the values that established the reference range, the demographics of the population, and the reference range sample population.
Interpreting test results in collaboration with the laboratory is a standard best practice. Table 9.6 shows some considerations for interpretation of laboratory results these can be used as a guide to facilitate collaborations with clinicians, epidemiologists, and laboratory scientists.
Interpretation guidelines for types of laboratory tests
Test type True positive False positive True negative False negative Molecular Presence of suspected organism in sample: &ldquopositive&rdquo Nonspecific binding of primers or probe Cross-contamination of samples Absence of suspected organism in sample: &ldquonegative&rdquo Failure of amplification reaction Failure of primer or probe binding Culture Presence of suspected organism in sample: &ldquopositive&rdquo Contamination during collection Cross-contamination with another sample in lab Absence of suspected organism in sample: &ldquonegative&rdquo Sample collected after antimicrobial treatment began Media used for growth does not support suspect organism Source of infection was removed Serology (39) Presence of antibodies specific for suspected agent: &ldquoexposure&rdquo Cross-reactivity Nonspecific inhibitors and agglutinins Absence of antibodies specific for suspected agent: &ldquono exposure&rdquo Tolerance Improper timing of sample Nonspecific inhibitors Toxic substances Antibiotic-induced suppression incomplete or blocking antibody Antimicrobial resistance testing (40) Microorganism is inhibited by normal doses of antimicrobial agent(s): &ldquosusceptible&rdquo Wrong assay selected for organism or drug being tested Media not sufficient for growth of organism being tested Insufficient number of organisms added Wrong standard tables used Microorganism is not inhibited by normal doses of antimicrobial agents: &ldquoresistant&rdquo Wrong assay selected for organism or drug being tested Insufficient antimicrobial added Wrong standard tables used Pathology (41) Presence of suspected organism in pathology sample: &ldquopositive&rdquo Cross-reactivity Nonspecific interactions Absence of suspected organism in pathology sample: &ldquonegative&rdquo Wrong assay selected for sample type or organism Failure of stain or specific antibodies Wrong sample type
Step 5. Continue Laboratory Collaboration Through Publication of Findings
Any field investigation could lead to discovery of a new pandemic pathogen (31), identification of a product or device hazard (32), discovery of an old pathogen in a new place (33), identification of a new risk to public health (34), or even arrest of a criminal (35). Given the significance of field investigations, accuracy and completeness are critical. History has shown that when public health recommendations must (for political reasons or in emergency situations) be based solely on epidemiologic data, unintended consequences can occur (36). Similarly, poor laboratory diagnostic capabilities also can create unnecessary difficulties in patient care (37). Therefore, the most effective and reliable field investigation teams are built on a strong collaboration between epidemiology and laboratory science. Sustaining that cooperation through all stages of the scientific process, including analysis of the data, formulation of conclusions, and presentation or publication of results, is also necessary. Several general considerations can help inform that collaboration:
Scientists: 'Exactly zero' evidence COVID-19 came from a lab
Since the COVID-19 pandemic began, the Internet has been teeming with provocative conspiracy theories that the novel coronavirus was (1) created in a Wuhan, China, lab and deployed as a bioweapon or (2) derived from bats, grown on tissue culture, intentionally or accidentally transmitted to a researcher, and released into the community.
Politicians have touted these theories in an attempt to blame China for the pandemic, and a discredited US scientist recently released a book and now-banned video claiming that wealthy people deliberately spread COVID-19 to boost vaccination rates. And late last week, an unsubstantiated NBC News report on cell phone location data suggested that the Wuhan lab temporarily shut down after a "hazardous event" in October.
Even Kristian Andersen, PhD, a professor in the Department of Immunology and Microbiology at Scripps Research Institute in La Jolla, California, and lead author of a research letter published Mar 17 in Nature Medicine on the origins of the virus, first thought that COVID-19 was just as likely to have been accidentally released from a lab as it was to have come from nature.
But that was before he learned more about COVID-19 and related coronaviruses, which have features already seen in nature. "There are lots of data and lots of evidence, as well as previous examples of this coming from nature," he said. "We have exactly zero evidence or data of this having any connection to a lab."
And while Andersen, like other prominent virologists, says that he can't completely rule out the possibility that the virus came from a lab, the odds of that happening are very small. He says the new coronavirus clearly originated in nature, "no question about it by now."
Conspiracy theory #1: Chinese bioweapon
COVID-19 is sufficiently unlike other viruses to have been created from them, and making a virus in the lab from scratch would be "virtually impossible," said Stanley Perlman, MD, PhD, professor of microbiology and immunology and pediatric infectious diseases at the University of Iowa in Iowa City. "I don't think we know enough about coronaviruses—or any virus—to be able to deliberately make a virus for release," he said.
James Le Duc, PhD, professor of microbiology and immunology and director of the Galveston National Laboratory at the University of Texas Medical Branch in Galveston, said that engineering COVID-19 "would have taken an incredible amount of ingenuity. People's imaginations are running wild."
Andersen said that the virus's receptor binding domain, which makes it an efficient human pathogen, is also found in coronaviruses in pangolins, scaly anteaters proposed as an intermediary host between bats and humans. "It's something that's fully natural, so it's not something that happens in tissue culture," he said.
Angela Rasmussen, PhD, an associate research scientist in the Center for Infection and Immunity at Columbia University in New York City, said computer modeling suggests that the receptor-binding domain of the spike protein in SARS-CoV-2, the virus that causes COVID-19, is suboptimal, "meaning that someone designing an optimal receptor-binding domain sequence probably would not 'engineer' the sequence that evolved in SARS-CoV-2," she said.
"Furthermore, there are no genetic similarities with other virus backbones used in any of the known reverse genetics systems for betacoronaviruses. This suggests that this virus was not engineered."
Another COVID-19 feature, its furin cleavage site, which allows the virus to infect human cells, diminishes in tissue culture, Andersen said. "I think it could probably still infect people, I just think much less efficiently," he added.
Furthermore, he questions why anyone would go through the work of creating a new virus when they could simply take an existing virulent pathogen like the SARS (severe acute respiratory syndrome) or MERS (Middle East respiratory syndrome) coronaviruses and make them even worse, as all bioweapons programs so far have done.
"It doesn't make any sense to make a new virus that you don't know can cause disease in humans and try to create a bioweapon out of it," Andersen said. "That would be a really bad bioweapons person."
Conspiracy theory #2: Lab release of natural virus
For many years around the world, scientists have studied bat coronaviruses by capturing bats in caves and isolating and growing in tissue culture the coronaviruses that they carry to see if they can infect human cells.
Called gain-of-function research, these studies improve a pathogen's ability to cause disease so that researchers can characterize its interactions with humans, allowing evaluation of its potential to cause a pandemic and informing public health, preparedness, and development of potential therapeutics and vaccines.
Shi Zhengli, PhD, director of the Center for Emerging Infectious Diseases at the Wuhan Institute of Virology, a biosafety level 4 (BSL-4) lab in China relatively close (25 to 35 kilometers [15 to 22 miles]) to the Wuhan live-animal market at the epicenter of China's outbreak, has extensively published the genetic sequences of isolates from the bat coronaviruses she studies.
None of them match those of COVID-19, Andersen said, something Shi herself confirmed in a recent interview in Scientific American. "If she would have published a sequence for the virus and then this pops up, then we would have known it came from the lab," Andersen said. "There's no evidence for this, but there is plenty of evidence against it."
Le Duc said that Shi's work on bat coronaviruses has shown that "these viruses exist in nature, and some of them have characteristics that would allow them to be transmissible among humans. The fact that we're seeing it today is not a surprise to folks that have been working in this field."
And although "certainly, accidents happen in laboratories," the high level of biocontainment at Shi's lab makes it unlikely, he said. BSL-4 labs have the most stringent biosafety protocols, which may include airflow systems, sealed containers, positive-pressure personal protective equipment (PPE), extensive training, and highly controlled access to the building.
Having attended conferences at which Shi has spoken about her work, Le Duc said she is a highly reputable scientist. "She's always been extremely open, transparent, and collaborative, and I have no reason to doubt that she's telling the truth," he said.
Plus, Andersen said the likelihood of a researcher becoming unknowingly infected with the coronavirus while wearing full PPE and then going to the Wuhan market is "fleeting compared to the alternative hypothesis, which is that we as humans, because we live amongst animals carrying these viruses—bats, but also many other intermediate hosts—and, of course, we don't go around wearing PPE, we naturally get into contact with these viruses all the time."
Yet a virus with pandemic potential is exceedingly rare, he said. "If these viruses were really frequent, we would all be dead by coronaviruses by now," Andersen said. "We would have coronavirus pandemics all the time. We don't, but they do pop up every 10 years or so, on average."
Rasmussen said that the most plausible scenario is a "natural zoonotic spillover," adding that serology studies have shown that some people in China living near bat caves have antibodies against bat SARS-like coronaviruses in their blood, "suggesting that people are exposed to related viruses in the course of their daily lives, so it’s not implausible that SARS-CoV-2 emerged in humans through a chance encounter between a human and a wild bat or some other animal."
Doubts about ever pinpointing origin
Perlman said scientists may never be able to track down the origins of the virus or its intermediary host. "I think it's really an important issue, figuring out where the virus comes from, but it's just a hard business finding it," he said.
It's human nature, he said, to want to blame someone for a natural but catastrophic event like the pandemic. "Something bad happens, and someone has to be responsible for it," he said.
Even so, Perlman decried the use of unproven origin theories to push agendas. "I think it's been used way too much as a political issue," he said. "This politicization of this is very unfortunate."
But Andersen said he thinks theories do deserve exploration, even if to ultimately refute them. "It's important that we don't dismiss them out of hand," he said. "We need to look at the data and say 'what does the data tell us?' And the data in this case are very strong."
—This story was updated an hour after it was posted on May 12 to add quotes from Angela Rasmussen, PhD.
—On May 13 information was added to clarify the relative distance of the BSL-4 lab to the live-animal market in Wuhan.
Molecular testing & its impact on patient care
Molecular diagnostics is a term used to describe the testing platforms that detect specific sequences in DNA or RNA. By analyzing these biological markers in an individual&rsquos genetic code (genome) and how their cells express their genes as proteins (proteome), molecular diagnostics are increasingly used to guide patient management, from diagnosis to treatment, particularly in the fields of cancer, infectious disease and congenital abnormalities. The increased demand for genetic and genomic information has led to the rapid expansion of molecular diagnostics options across the healthcare spectrum.
From a practical viewpoint, the relevance of molecular testing is nothing short of a revolution in providing lab diagnostic information for certain disease states, including infectious diseases. Speed of results (tests that once took days can now be completed in hours or less) and accuracy (how sensitive and specific test results can be) offer benefits including antibiotic stewardship and reduction of empirical antibiotic prescription. Some molecular tests can offer sensitivities that are 10 times greater 1 than legacy methods or more, and are also specific for the substance being tested.
Acquisition of a good throat, wound or nasopharyngeal swab has been a concern for microbiology testing, particularly for rapid lateral flow antigen detection methods. A significant advantage of molecular testing for infectious agents is the amplification of the substance being tested for, which is inherent in molecular tests. This reduces the concerns about swab sample collection techniques and reduces the number of false negatives inherent in rapid diagnostic methods needing enough specimen to react with the antibody and generate a result.
Molecular testing is a growing market
Molecular testing offers significant advantages in laboratory testing by improving prospects for accurate, timely diagnosis of diseases, which has become a major reason for their adoption in infectious disease testing. Infectious disease tests, including molecular assays for respiratory diseases and enteric pathogens, are growing rapidly and represent about a $3 billion market 2 in the US today.
Molecular tests for cancer diagnosis and treatment monitoring are the next fastest growing test segment and include new tests to guide immunotherapy decisions. 2 Molecular testing use in understanding personal genome mutations that lead to a pre-disposition for certain diseases (Factor V Leiden, colorectal cancer and others) or that point to the most effective cancer treatments is also driving adoption of current molecular tests, as well as research and development directed at pushing back the frontiers of personalized medicine further and further. Today, the market for molecular tests exceeds $6.5 billion and is growing at a predicted rate of 9.1% worldwide. 2 The US market for infectious disease alone is greater than $3 billion.
The future of molecular testing: New tests, new entrants
All signs point to increasing adoption of molecular tests for detection of known disease markers, including influenza and enteric pathogen detection. New molecular diagnostic platforms now have a very wide range of tests for common respiratory and enteric pathogens that deliver fast, accurate results to enable proper antibiotic prescription as necessary. Some manufacturers have a range of methods including tests that can determine whether the patient&rsquos bacteria are resistant to typical antibiotics and that help target the most appropriate antibiotic therapy.
Even more importantly, molecular assays are creating a frontier of new tests for tumor markers and disease pre-disposition (BRCA1 and BRCA2, for example) that promise earlier, more accurate diagnosis, especially for cancer and inherited diseases. Some of these same tests also hold promise to guide more effective treatment of disease, based on an understanding of the underlying relationship between the patient&rsquos metabolism and the disease state itself that is, they can help guide decisions regarding the use of specific chemotherapeutic agents or immunotherapy. Emerging companies like Epigenomics are exploring new markers for colorectal, lung and other cancers.
From the perspective of ease-of-use and market adoption, molecular tests are becoming easier to use, with friendly test formats and sophisticated software and hardware that brings this sophisticated technology into more and more laboratory sites. Some test methods are even CLIA-waived, including Alere I and Roche LIAT respiratory tests. Many other tests are CLIA moderate. Suppliers are competing for increased ease-of-use, more available tests and faster turn-around times, all factors that show promise for wider adoption of these tests and the opportunity for better patient care.
DEFINED VS UNDEFINED MEDIA
This is an important distinction between growth media types. A defined medium will have known quantities of all ingredients. For microorganisms, it provides trace elements and vitamins required by the microbe and especially a defined carbon and nitrogen source. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity &ndash some microorganisms have never been cultured on defined media.
There are many different types of media that can be used to grow specific microbes, and even promote certain cellular processes such as wort, the medium which is the growth media for the yeast that makes beer. Without wort in certain conditions, fermentation cannot occur and the beer will not contain alcohol or be carbonated (bubbly).
Determination of Blood Group | RBC | Blood Cells | Biology
In this article we will discuss about the determination of blood group.
Blood type (also called blood group) is a classification of blood based on the presence or absence of inherited antigenic substances (antigens) on the surface of red blood cells. If the red blood cell had only “A” antigen on it, that blood was called type A.
If the red blood cell had only “B” antigen on it, that blood was called type B. If the red blood cell had a mixture of both antigens, that blood was called type AB. If the red blood cell had neither antigen, that blood was called type O.
In biological terms, a foreign chemical causes the creation of antibodies against it. So in type A blood, there anti-B antibodies because the immune system recognizes type B red blood cells as foreign. In type B blood, there anti-A antibodies. In type AB there are no antibodies against either type A or B antigens. In type O blood, there are antibodies against both type A-and B antigens
1. Prepare a 10% suspension of red blood cells in normal saline (preparation method as given below):
i. Mix 5 drops (0.05ml each) of sediment red cells with 2ml of normal saline.
ii. Centrifuge at 1,500 RPM for 1 to 2 minutes. Discard supernatant.
iii. Add 2 ml of normal saline to the sediment red cells. Mix well. This gives a 10% suspension of red cells.
2. On one half of a glass slide, place I drop of Anti-A blood groping serum.
3. On the other half of the slide, place I drop of Anti-B blood grouping serum.
4. Using a Pasteur pipette add 1 drop of the cell suspension to each half of the slid.
5. With separate applicator sticks, mix each cell-serum mixture well.
6. Tilt the slide back and forth and observe for agglutination.
a. Test that show no agglutination within two minutes are considered negative.
b. Do not interpret peripheral drying or fibrin stands as agglutination.
Blood obtained by finger puncture may be test directly by the slide method. To avoid clotting, the collected blood (on the slide) should be moved quickly with the antisera.
Qualitative Test for ABO Grouping by Tube Method:
1. Test tunes ( 10 × 75mm or 12 × 75mm)
1. Prepare a 5% suspension of red blood cells in isotonic saline.
i. Mix 5 drops (0.05ml) of sedimentary cells with 2 ml of normal saline,
ii. Centrifuge at 1, 500 RPM for 1 to 2 minutes. Discard supernatant.
iii. Add 4ml of normal saline to the sediment red cells, mix well.
2. To a small test tube, add one drop of Anti-A blood grouping serum.
3. To a second test tune add one drop of Anti-B blood grouping serum.
4. Using a Pasteur pipette, ad on drop of 5%, cell suspension to each of the two test tunes
5. Mix well and centrifuge both the tubes at 1,500 RPM for one minute.
6. Responds the cells by gentle agitation.
7. Observe for agglutination. Use microscope if necessary.
1. Anti AB serum can be used to confirm the result of slide and tube tests.
The expected observations are as follows:
O – No agglutination + Agglutination
Only group ‘O’ blood will not agglutinate with Anti-AB.
1. Suggested centrifuged speed and duration
Over centrifugation, causes the cells to adhere to the bottom of the test tube. This requires vigorous agitation before the cells can be re-suspended. During such agitation weak agglutination may be dispersed leading to false negative reaction.
2. In the case of positive reaction for group A. use anti serum to determine the sub group A.
Rh Blood Grouping:
Rh typing is next important Blood grouping system. ABO blood grouping presence of Rh antigen (or D antigen) all the red cell is determined by the haem agglutination reaction of the RBC after reacting the letter with Anti-D Anti-serum at the body temperature. The technique is similar to ABO grouping and hence Rh tying is done with ABO blood grouping.
Rh blood typing refers to the determination of the presence or absence of D- antigen all RBCs when reacted with anti-D serum. Rh positive cells shows agglutination with anti-D.
Anti-D follows manufacture instruction in the use of anti-D Rh-negative blood group cell suspension.
Prepare a 4% red cell suspension for the tube method and cell suspension for slide method is 45%.
I. Place a slide on a drying and allow it to warm up.
II. Add 2 drops 40-45% of cell suspension or whole blood on the slide.
III. Add 1 drop of anti-D serum.
IV. Mix the cell with the anti-serum
V. Continue mixing while tilting the slide back and forth
VI. Look for agglutination which is recognized by the clumping of red-cell do not observed longer than 2 minutes.
VII. A control must be run once a day in order to option reliable result.
1. A 4% suspension in saline of washed cell or used diluted whole blood with saline. Take 2 applicator stick together and transfer sufficient amount of cells from the clotted or whole blood specimen to a test tube with saline.
Make concentration of cell suspension to about 4%. Place small test tube on a rack and label tham. S- specimen, +ve Rh positive control and -ve for Rh negative., S.A. for albumin control.
2. Add drop of anti-Rh in to the first 3 test tube. And the albumin in the tube mark- S.A.
3. Add 2 drops of cells suspension in S. tube and S.A. add 2 drops of control reagent cell in the tube marked as positive-and negative mix all the tube while gently shaking.
4. Incubate all the tube add 37°C for 30min
5. Centrifuge the tube 1500 rpm for 1min.
6. Examine the agglutination reaction in each test tube while dislodging the button gently.
7. Agglutination will be recognize by the formation of small clumps in pear liquid this will be marked as Rh-positive. If the RBC re-suspend homogenously with no visible clumps. It is negative reaction.
Source or Error:
1. Always run Rh controls at least once a day.
2. Check the temp of the water-bath and slide box frequently.
3. Follow the manufactures instruction.
4. A fresh specimen is always desirable, specimen that are several days old may not yield desired and accurate result.
1. False Positive Result:
Bring on the slide, use of infected sera, contaminated specimen, presence of unexpected antibody and roulex formation are some of the causes of false result.
2. False Negative Result:
The serum may be in active it may have been omitted from the tube. The red cells are not in good condition or the technique may have been wrongly performed. Reliability of antiserum is extremely important for optioning correct blood grouping. Avoid exposing the anti-sera to room temp when not in use. Check the anti-sera with occasionally with known cell. Discard out dated contaminated or weak anti-sera which may give false result.
3. Sometimes the red cells are not agglutinated but are piled upon each other like a stack of coins exhibiting rouleaux formation. This gives the appearance, of false agglutination. In that case add 2 drops of isotonic sodium chloride solution to the drop of the red cell-serum mixture and examine under microscope.
i. In false agglutination rouleaux disappears.
ii. In true agglutination the clumps remain.
50 Contaminated anti-sera should not be used. These sera appear cloudy whitish and give a foul smell.
4. After use, store the antisera at 2-8 °C in the refrigerator.
5. Source material used in the preparation of antisera is tested for hepatitis-B virus and HIV (AIDS) antibodies. But since no test method can offer complete assurance that HIV, hepatitis virus or other infectious agents are absent the antisera should be handled carefully as recommended for any other specimen.
6. Every antiserum is carefully tested for specificity, potency, avidity and freedom from rouleaux formation properties Donor’s blood collected for the preparation of different antisera should be free from hepatitis-B surface antigen and HIV (AIDS) antibodies 0. If marine monoclonal Anti-A and Anti-B reagents are used, these are very safe and free from these viruses, besides being very, potent and specific.
7. Anti-D serum can be obtained from Rh negative mothers, who have recently delivered infants suffering from hemolytic disease of the new born or by immunization of Rh-negative volunteers with D-positive red blood cells. It is suitable blended with bovine serum albumin.
Watch the video: Ciclo de Conferencias - Instituto de Genética 2021 II (October 2022).