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Legionnaire's Disease at Peel Park Hospital 1 MPHM Assignment 2: Infection Control Scenario (Legionnaire's Disease at Peel Park Hospital) By (Student Name) Course Name and Number Institution Professor Date

Legionnaire's Disease at Peel Park Hospital 2 MPHM Assignment 2: Infection Control Scenario (Legionnaire's Disease at Peel Park Hospital) Question 1 Culture-based methods and genetic testing through whole genome sequencing are the diagnostic methods used to diagnose Legionnaire's disease in this scenario. For the culture-based method, a sputum sample was collected from the patients through direct inoculation onto a buffered yeast agar extract. The sample was then incubated for 48 hours after which L. pneumophilia isolates were identified based on colonial morphology. Gram staining through a specific latex agglutination test confirmed the presence of L. pneumophilia . This method allowed for the isolation and identification of L. pneumophilia in all six patients. However, it did not detect the bacteria in staff and patients with mild flu-like symptoms. Charcoal-buffered yeast agar extract is the primary isolate for L. pneumophilia and adding this composition to a bacteriological medium provides rapid growth of the bacteria (Feeley et al., 1979). Whole genome sequencing was conducted in Patient 1 and Patient 5 for selected clinical and environmental isolates. This method enabled single nucleotide polymorphisms (SNPs) identification in the core genome of L. pneumophilia . Thus procedure revealed the presence of the IpeAB gene in Patient 5 while the gene was absent in Patient 1. This finding provided useful insight into the genetic composition of the isolates and probable virulence factors. An alternative diagnostic method for L. Pneumophilia is PCR-based assays that target specific antigens or genes of the bacteria. This is a molecular method that is widely used for disease diagnosis by detecting DNA of bacteria in sputum samples, loop-mediated isothermal amplification (LAMP), and direct fluorescent antibody (DFA) (Feeley et al., 1979). Molecular methods can be used to detect the DNA or antigens associated with L. pneumophilia directly

Legionnaire's Disease at Peel Park Hospital 3 from samples collected from the patient, eliminating the need for incubation and reducing diagnosis time (EUCAST, 2017). The culture-based diagnostic method is very effective in the isolation and identification of L. pneumophilia. However, the use of this method is limited since the sample must have viable organisms to isolate, and it requires highly specialized laboratory facilities and technical expertise to perform (Arias, 2004). Moreover, the process is time-consuming as the time required to incubate the sample might delay diagnosis and commencement of the necessary medication (Chiaraviglio and Kirby, 2015). For instance, four of the six patients in this scenario responded rapidly to erythromycin, an indicator of effective diagnosis and drug prescription. On the other hand, non-culture-based methods are expensive and require specialized reagents to detect bacterium DNA or antigen. Nevertheless, combining molecular and culture-based diagnosis would enable the detection of L. pneumophilia in staff and patients with mild flu-like symptoms hence improving patient management (Mayo Clinic, 2018). Question 2 According to the information provided, Shower 2 (for patients) is the most probable environmental origin of the outbreak. This conclusion is based on the following reasons: i. Likelihood of each source from a practical perspective: By focusing on a practical perspective, MLST type (21) and serotype (1) of clinical isolates collected from pneumonia patients matched with Shower 2, making it the most likely practical origin of the outbreak. Showers generally produce aerosols that can aid L. pneumophilia transmission and the presence of matching serotypes and MLST type in the air conditioning system and Shower 3 (for staff) are clear evidence of contamination. However, the

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Legionnaire's Disease at Peel Park Hospital 4 absence of clinical isolates from non-pneumonia patients and staff diminishes their connectedness with the outbreak. ii. Serotyping, MLST, and whole genome SNP analysis: a) Serotyping: This is the process by which the specific surface antigens of bacteria are identified and a preliminary connection between isolates from different sources is established (Ewig, Tuschy, and Fätkenheuer, 2002). For instance, the environmental and clinical isolates from the air conditioning system and Showers 2 and 3 (for patients and staff respectively) had the same serotype (1). b) Multi-locus Sequence Typing (MLST): This procedure helps in analyzing the genetic characteristics of various housekeeping genes to get a detailed insight into the characterization of genes from various isolates (Ewig, Tuschy, and Fätkenheuer, 2002). In this case, environmental and clinical isolates from Showers 2 and 3 and the air conditioning system have similar MLST types (21), indicating a strong connection between these surfaces. c) Whole genome SNP analysis: Whole genome sequencing enables one to identify the single nucleotide polymorphisms (SNPs) in the core genome of target bacteria and establish the connection between isolates from various sources (Swanson and Hammer, 2000). SNP analysis in this scenario indicated that the most probable source of the outbreak was Shower 2 since it had the closest genetic relatedness to the clinical isolates. iii. Contribution of each method towards fingerprints: In this scenario, serotyping contributed towards fingerprints by identifying common antigens that confirmed preliminary relatedness. MLST analyzed the genetic characterizations of various housekeeping genes thus providing greater resolution. Whole genome SNP analysis

Legionnaire's Disease at Peel Park Hospital 5 identified SNPs and generated a dendrogram that confirmed the relatedness between the isolates beyond scientific doubt. Question 3 The British Standards Institute (BS7592) provides guidelines for sample collection, processing, and testing that were applied in this scenario as summarized below: a) Sample Collection: Sterilized sampling devices were used to collect water samples from the hospital’s birthing pool, water fountain, showers, and air conditioning system. b) Sample processing: The collected samples were subjected to centrifugation, filtration, and other sample processing procedures outlined in BS7592 to concentrate any bacteria present (British Standards Institute, 2023). c) Inoculation: The concentrated samples were inoculated in buffered yeast extract agar to provide the optimum environmental conditions and nutritional requirements for the rapid multiplication of L. pneumophilia. d) Bacteria selection and identification: The bacteria colonies were identified using selective morphology and confirmed using specific latex agglutination to be L. pneumophilia , the bacteria responsible for the pneumonia outbreak at the facility (British Standards Institute, 2023). The publication INDG458 by the Health and Safety Executive and several other regulations bestow on hospitals the legal obligation to control Legionella disease. To fulfill this obligation, hospitals must perform the following activities: i. They must perform regular risk assessments to identify and assess the risks of growth and transmission of Legionella bacteria within their premises.

Legionnaire's Disease at Peel Park Hospital 6 ii. They must implement appropriate control measures such as disinfection and temperature control to eliminate the risk of bacterial contamination. iii. They should monitor their infrastructure, including water and air conditioning systems, and make prompt repairs where necessary. iv. They must provide frequent training to staff and occupants about Legionella control measures, risk awareness, and reporting procedures. Question 4 The British guidelines for treating legionellosis and various studies on L. pneumophilia antibiotic susceptibility and antimicrobial resistance (AMR) can be used to explain antibiotic failure and success in this scenario. The antibiotic susceptibility test results indicated that L. pneumophilia clinical isolates were susceptible to erythromycin, rifampicin, and doxycycline hence these drugs would be effective in treating the infection. However, the bacteria resisted ciprofloxacin, indicating that it would be ineffective in treating the infection. Of the six patients, four responded rapidly to erythromycin prescriptions. This treatment outcome is consistent with the susceptibility tests, indicating that the isolates from these patients were susceptible to erythromycin. However, treatment failure was reported in two patients as their conditions worsened, forcing the hospital to put them on life support. The hospital prescribed levofloxacin to one of the two resistant patients, resulting in rapid recovery. This observation denotes a probability that the patients had a strain of L. pneumophilia that was resistant to erythromycin. Whole gene sequencing on Patients 1 and 5 discovered the presence of the IpeAB gene in Patient 5 only, indicating the potential difference between antibiotic resistance mechanisms or virulence factors, and genetic variations of the isolates. By cross-referencing with UK guidelines for treating

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Legionnaire's Disease at Peel Park Hospital 7 legionellosis and studies on antibiotic susceptibility by L. pneumophilia, it is evident the antibiotic susceptibility results recorded from the case align with other reputable studies (HSE.gov.uk, 2012).

Legionnaire's Disease at Peel Park Hospital 8 References Arias, M. (2004). [Psychogenic disorders: concepts, terminology and classification]. Neurologia (Barcelona, Spain) , [online] 19(7), pp.377–385. Available at: https://pubmed.ncbi.nlm.nih.gov/15273885/. British Standards Institute (2023). BS 7592 & BS 8580-2 . [online] www.bsigroup.com. Available at:https://www.bsigroup.com/en-GB/standards/water-quality/#:~:text=BS %207592%3A2022%20is%20the [Accessed 17 Apr. 2023]. Chiaraviglio, L. and Kirby, J.E. (2015). High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila. Antimicrobial Agents and Chemotherapy , 59(12), pp.7517–7529. doi:https://doi.org/10.1128/aac.01248-15. EUCAST (2017). EUCAST guidance document on Legionella Antimicrobial susceptibility testing of Legionella pneumophila . [online] Available at: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/General_documents/Le gionella_guidance_document_20171208.pdf. Ewig, S., Tuschy, P. and Fätkenheuer, G. (2002). [Diagnosis and treatment of legionella pneumonia]. Pneumologie (Stuttgart, Germany) , [online] 56(11), pp.695–703. doi: https://doi.org/10.1055/s-2002-35548. Feeley, J.C., Gibson, R.J., Gorman, G.W., Langford, N.C., Rasheed, J.K., Mackel, D.C. and Baine, W.B. (1979). Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. Journal of Clinical Microbiology , 10(4), pp.437–441. doi:https://doi.org/10.1128/jcm.10.4.437-441.1979. HSE.gov.uk (2012). Legionnaires’ disease: A brief guide for dutyholders . [online] www.hse.gov.uk. Available at: https://www.hse.gov.uk/pubns/indg458.htm.

Legionnaire's Disease at Peel Park Hospital 9 Mayo Clinic (2018). Legionnaires’ disease - Symptoms and causes . [online] Mayo Clinic. Available at: https://www.mayoclinic.org/diseases-conditions/legionnaires- disease/symptoms-causes/syc-20351747. Swanson, M.S. and Hammer, B.K. (2000). Legionella PneumophilaPathogenesis: A Fateful Journey from Amoebae to Macrophages. Annual Review of Microbiology , 54(1), pp.567– 613. doi: https://doi.org/10.1146/annurev.micro.54.1.567.

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