Question

Influenza: What can be seen with the naked eye? If it is a bacteria that can be cultured, what does the culture look like? Which type of microscope is needed to see the organism? What stains are required to visualize or help classify the organism? List 2 diagnostic tests that can confirm an infection with your pathogen.

Answer 1

1.Researchers have discovered a way to make influenza visible to the naked eye by engineering fluorescent dye molecules to target a specific enzyme of the virus. The team was able to develop a prototype test kit that detects influenza in samples using a handheld lamp or blue laser pointer, and that even helps determine if a given antiviral therapy will likely be effective.

The scientists, from University of Notre Dame, created a new method to detect neuraminidase, which is located on the surface of the virus. They began by designing a dye molecule to emit red fluorescent light when it interacts with the neuraminidase. They then used test samples that mimicked that of an infected patient, and spiked the samples with neuraminidase that had been purified from the virus. Emission of red fluorescent light from a sample is a positive indication of the influenza virus, blue signals a negative result.

2. Culture :-

During outbreaks of respiratory illness when influenza is suspected, some respiratory samples should be tested by molecular assays and both rapid influenza diagnostic tests and by viral culture. The collection of some respiratory samples for viral culture is essential for determining the influenza A virus subtypes and influenza A and B virus strains causing illness, and for surveillance of new virus strains that may need to be included in the next year’s influenza vaccine. During outbreaks of influenza-like illness, viral culture also can help identify other causes of illness.

During outbreaks of respiratory illness when influenza is suspected, some respiratory samples should be tested by molecular assays and viral culture. The collection of some respiratory samples for viral culture is essential for determining the influenza A virus subtypes and influenza A and B virus strains causing illness, and for surveillance of new virus strains that may need to be included in the next year’s influenza vaccine. During outbreaks of influenza-like illness, viral culture also can help identify other causes of illness.

3. Electron microscopy (EM) has long been used in the discovery and description of viruses.Only optical fluoresce microscopes can see inside a virus, and then only indirectly, using dye, which cannot actually penetrate a virus.

4.digitally-colorized negative-stained transmission electron micograph ( TEM) shows a number of influenza A viruses.

Gram staining also used for this organism.

5.

RIDTs :-

Commercial rapid influenza diagnostic tests (RIDTs) are antigen detection assays that can detect influenza viruses within 15 minutes with low to moderate sensitivity and high specificity. Some tests are CLIA-waived and approved for use in any outpatient setting, whereas others must be used in a moderately complex clinical laboratory. These rapid influenza diagnostic tests differ in the types of influenza viruses they can detect and whether they can distinguish between influenza virus types. Different tests can detect 1) only influenza A viruses; 2) both influenza A and B viruses, but not distinguish between the two types; or 3) both influenza A and B viruses and distinguish between the two. Some RIDTs utilize an analyzer reader device to standardize results to and improve sensitivity.

None of the rapid influenza diagnostic tests provide any information about influenza A virus subtypes. The types of specimens acceptable for use (i.e., throat, nasopharyngeal, or nasal aspirates, swabs, or washes) also vary by test. The specificity and, in particular, the sensitivity of rapid influenza diagnostic tests are lower than for viral culture and RT-PCR and vary by test. Because of the lower sensitivity of the rapid influenza diagnostic tests, physicians should consider confirming negative test results with RT-PCR, viral culture or other means, especially in hospitalized patients or during suspected institutional influenza outbreaks because of the possibility of false-negative RIDT results, especially during periods of peak community influenza activity. In contrast, false-positive RIDT results are less likely, but can occur during periods of low influenza activity. Therefore, when interpreting results of a rapid influenza diagnostic test, physicians should consider the positive and negative predictive values of the test in the context of the level of influenza activity in their community. Package inserts and the laboratory performing the test should be consulted for more details regarding use of rapid influenza diagnostic tests.

Immunofluorescence

Immunofluorescence assays are antigen detection assays that generally require use of a fluorescent microscope to produce results in approximately 2-4 hours with moderate sensitivity and high specificity. Both direct (DFA) and indirect fluorescent antibody (IFA) staining assays are available to detect influenza A and B viral antigens in respiratory tract specimens. Subtyping or further identification of influenza A viruses is not possible by immunofluorescent assays. One rapid immunofluorescence assay is an RIDT and utilizes an analyzer device to produce results in approximately 15 minutes.

Note:- Influenza is a virus not bacteria.

Haemophilus influenzae (formerly called Pfeiffer's bacillus or Bacillus influenzae) is a Gram-negative, coccobacillary, facultatively anaerobic pathogenic bacterium belonging to the Pasteurellaceae family. H. influenzae was first described in 1892 by Richard Pfeiffer during an influenza pandemic.

The bacterium was mistakenly considered to be the cause of influenza until 1933, when the viral cause of influenza became apparent, and is still colloquially known as bacterial influenza. H. influenzae is responsible for a wide range of localized and invasive infections. This species was the first free-living organism to have its entire genome sequenced.

Culture

Bacterial culture of H. influenzae is performed on agar plates, the preferable one being chocolate agar, with added X (hemin) and V (nicotinamide adenine dinucleotide) factors at 37 °C in a CO2-enriched incubator.Blood agar growth is only achieved as a satellite phenomenon around other bacteria. Colonies of H. influenzae appear as convex, smooth, pale, grey, or transparent colonies.

Gram stained and microscopic observation of a specimen of H. influenzae will show Gram-negative coccobacillus. The cultured organism can be further characterized using catalase and oxidase tests, both of which should be positive. Further serological testing is necessary to distinguish the capsular polysaccharide and differentiate between H. influenzae b and nonencapsulated species.

Although highly specific, bacterial culture of H. influenzae lacks sensitivity. Use of antibiotics prior to sample collection greatly reduces the isolation rate by killing the bacteria before identification is possible.Beyond this, H. influenzae is a finicky bacterium to culture, and any modification of culture procedures can greatly reduce isolation rates. Poor quality of laboratories in developing countries has resulted in poor isolation rates of H. influenzae.

H. influenzae will grow in the hemolytic zone of Staphylococcus aureus on blood agar plates; the hemolysis of cells by S. aureus releases factor V which is needed for its growth. H. influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of nutrients such as factor V in these areas. Fildes agar is best for isolation. In Levinthal medium, capsulated strains show distinctive iridescence.