Biology Immunity Paper

Case Study Report: Diagnosis of Mild and Severe Respiratory Illness

Introduction

An outbreak of respiratory illness has infected workers in Vietnam on a duck farm. To identify what pathogenic organism is responsible for the mild and severe symptoms seen in the workers, a variety of tests were conducted, both for viral and bacterial presence. Whilst many respiratory infections prove to be of viral etiology, common complications can cause secondary bacterial infections due to the host s weakened immune system, therefore it was necessary to screen for bacteria and well as viruses.

With understanding that the outbreak took place on a duck farm, it could be suspected that Avian Influenza could be the cause of the symptoms. The pathogen may have transmitted from the ducks to the workers and become progressively more pathogenic, mutations may explain the severe symptoms (Burimuah et al., 2016). Although Avian Influenza does not pass easily to humans, and even rarer human to human transmission, recent studies show that humans are becoming more susceptible to the zoonotic pathogen (Lewis, 2006).

Hard labour on the farm and constant interaction with other humans may have potentially lead to an outbreak of Rhinovirus. Rhinovirus is often associated with the common cold - which is what many of the workers suffered from with a selective few going on to experience more severe symptoms. Rhinovirus infections very rarely extends on to cause severe pneumonia and organ failure in immunocompetent individuals however can be a concern for immunocompromised hosts (Kim et al., 2014), this may explain the different between workers suffering from mild symptoms and those suffering severe symptoms.

It is the aim of this experiment to discover the pathogen responsible for the respiratory illness outbreak amongst the workers, causing both mild and severe cases of the outbreak. It should also hopefully be uncovered as to whether the pathogen has resulted in a secondary bacterial infection in both the mild and severe cases.

Method and Materials

Methods were performed according to Appendix A, found at the end of this report.

Results

By observing what isolations of bacteria were present it could be determined what was causing illness in the workers. Tests were undertaken to identify an aetiological agent and it s potency to drugs. Throat swabs from both mild and severe cases were taken and the cultures were isolated on blood agar plates, allowing fastidious organisms to grow. It is evident from the results that fastidious organisms thrived from the blood agar plates as small, white, cocci colonies were detected in the mild cultures and larger cocci colonies were detected in the severe cultures.

Gram stained smears of bacterial colonies demonstrated gram positive results for both the mild and severe cultures. After observation, it was noticed that the mild case grew in clusters, this is highly characteristic of Staphylococci (Barenfanger and Drake, 2001). The severe cultures grew in chains which is likely evidence of Streptococci presence.

A Staphylococcus Latex Agglutination Test was carried out on the bacteria colonies from the mild case. The mild case showed negative results for the presence of bound coagulase or clumping factor. Coagulase negative staphylococci is a commensal bacteria, which can be harmful when found in the bloodstream, an example of coagulase negative

staphylococci is the negative control and the mild case. The positive control evidently shows a reaction took place with the latex particles forming clumps.

The bacterial colonies isolated from the severe case were subject to a Bacitracin sensitivity test. Bacitracin being an antibacterial, inhibits the growth of bacteria should the bacteria be sensitive and not resistant. Sensitivity is shown through a dark halo around the bacitracin on the blood agar plate where the bacteria growth has been inhibited, this is shown in figure 4.

To identify whether respiratory viruses were present in the throat swabs, a Polymerase Chain Reaction (qRT-PCR) was set up. Test tubes 2,3,6 and 7 were all over the threshold, showing positive reactions where Fluorescence indicates the presence of Influenza and Rhinovirus. The cycle threshold (CT) showed strong positive reactions of 2,3 and 7, indicating an abundance of target nucleic acids in the samples (Real Time PCR CT values, 2019). Samples with viruses present contained Influenza (3 and 7) and Rhinovirus (2 and 6), suggesting the respiratory illness is one of these viruses.

To determine which virus the DNA sequence matches, it needs to be compared to known nucleotides of DNA, this can be done using BLAST. Purification of DNA from a PCR reaction is first necessary due to contaminants in the PCR reaction, such as buffer components and primer. Both mild and severe nucleotide sequences returned as Influenza (H5N1). To further support findings from the BLAST data, figure 6 shows a virus under a transmission electron microscope. It can be identified as a virus due to the size, as bacteria is bigger than viruses. Exampled in figure 6 is the known irregular morphology of the influenza virion. There is the appearance of spherical virions and filamentous, exampling a pleomorphic characteristics.

The results from the drug potency test indicated viral growth inhibition in Mild OT, Mild AM and Severe AM. Yellow wells are an indication that viral growth has occurred and therefore the virus is resistant to the antiviral present in the wells. Severe OT and +VE show viral growth through the yellow colouring.

Discussion

Colony size, morphology and hemolysis are crucial evidence in the identification of the organisms, therefore the use of Sheep s blood agar is routinely used due to the rich nutrients and growth factors that permit the maximum cultivation without interfering with the haemolytic reactions (Unknown, 2019). However it should be noted, that although both the blood agar plates and gram staining, in these circumstances, concluded already suspected results, they were only conducted once. To support the findings, multiple tests should have conducted and a variation of blood agar plates could have been used. Other organisms such as the oral presence of normal flora in the respiratory tract may have been misidentified, therefore compromising differentiation between bacteria. Beta-haemolysis is evident from figure 1 (severe) however the use of only one sheep s blood agar plate allows for no comparison, whereas if both horse and sheep blood agar plates were used for the cultivation of the isolations, it would allow for a better understanding for what allows fastidious organisms optimal growth, such as the impact of the age of red blood cells in the blood agar plate.

Whilst the severe and mild cases suffer similar symptoms, it is evident from the blood agar plates and gram staining that there is a variation in the bacteria type. The colony sizes and morphology vary from that present in the severe case to that of the mild. Beta-Haemolytic Streptococcus Pyogenes are gram positive bacteria that grow in chains of variable length. S. Pyogenes appear transparent and round when cultivated using sheep s blood agar (Pichichero, 1998), figure 1 and 2 (severe) demonstrate suspected growth of Streptococci. S. Pyogenes is further distinguished with the use of Bacitracin, as beta haemolytic streptococci such as Streptococcus Pyogenes are susceptible to antibiotics, this is proven through the dark surrounding area around the bacitracin, where the antibiotics have killed the pathogen.

The smaller, cocci clusters in the mild case suggest a Staphylococci infection. To further identify the subtype of Staphylococcus, tests were conducted to indicate coagulase presence. The Staphylococcus Latex Agglutination Test utilises latex particles coated in plasma (Myrick and Ellner, 1981) which reacts with the enzyme bound coagulase this results

in agglutination if the enzyme is present in the Staphylococcus type, which is what can be seen from the positive control. The mild case shows no agglutination, therefore coagulase negative, a characteristic of the gram positive bacteria, Staphylococcus Epidermidis. As part of the human epithelial microflora, the commensal microorganism has a benign relationship, living without harm on the human skin, however, is an opportunistic pathogen, allowing a gateway for infection should the host s immunity weakens. This could explain the symptoms of the mild cases, it could be suggested that labour on the farm has caused skin irritations and small cuts, allowing entry to the bacteria. However, with the use of the qRT-PCR and BLAST data it was determined a virus was present in both the mild and severe cases. The qRT-PRC screened for respiratory viruses using extracted Nucleic Acids from the throat swabs of both mild and severe cases. Extracted DNA was then amplified so the sequence can be used to determine the identity of the virus. BLAST was used to compare the extracted sequence to known data and provided an accurate match. It is evident many workers obtained Influenza with a small minority going on to experience severe symptoms, therefore it is more likely that in mild cases, complications arose and S. Epidermidis may have taken the opportunity to cause a secondary bacterial infection.

The majority of cases showed sensitivity to Neuraminidase Inhibitor Oseltamivir however for the severe cases it did not improve symptoms which suggests resistance. Evidence that the Influenza virion mutated in the severe cases is also shown through the results of the drug potency test. Mild cases proved sensitive to the OT drug with no colouration in the wells, indicating no viral growth . However severe cases showed resistance through yellow colouration, indicating viral presence. Where mild Influenza is susceptible to OT, it can be a successful treatment. The mutated virions causing illness in the severe cases, being resistant to OT could alternatively be treated with AM, where it is susceptible.

References

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Unknown (2019). Oxoid - Product Detail. [online] Oxoid.com. Available at: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0854cat=c=UKlang=EN [Accessed 29 Oct. 2019].

Burimuah, V., Ampofo, W., Awumbila, B., Yebuah, N., Emikpe, B., Tasiame, W. and Folitse, R. (2016). THE EVALUATION OF DOMESTIC DUCKS AS POTENTIAL RESERVOIR OF AVIAN INFLUENZA VIRUS IN POST HPAI H5N1 OUTBREAK AREA, SUNYANI MUNICIPALITY, BRONG AHAFO REGION OF GHANA. African Journal of Infectious Diseases, [online] 10(2), pp.134-145. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411989/ [Accessed 31 Oct. 2019].

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Barenfanger, J. and Drake, C. (2001). Interpretation of Gram Stains for the Nonmicrobiologist. Laboratory Medicine, [online] 32(7), pp.368-375. Available at: https://watermark.silverchair.com/labmed32-0368.pdf [Accessed 3 Nov. 2019].

Russell, F., Biribo, S., Selvaraj, G., Oppedisano, F., Warren, S., Seduadua, A., Mulholland, E. and Carapetis, J. (2006). As a Bacterial Culture Medium, Citrated Sheep Blood Agar Is a Practical Alternative to Citrated Human Blood Agar in Laboratories of Developing Countries. Journal of Clinical Microbiology, [online] 44(9), pp.3346-3351. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1594681/ [Accessed 3 Nov. 2019].