NASAL CARRIAGE OF VIRULENT STAPHYLOCOCCUS AUREUS: A POSSIBLE CO-MORBIDITY OF COVID-19

BACKGROUND Coronavirus diseases-19 (COVID-19), caused by a coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a respiratory disease. The disease was first reported in China in December 2019, and has currently spread to most part of the world resulting to a global pandemic. There are currently no vaccine and specific antiviral drug recommended for COVID-19 treatment; however, the disease can be successfully managed by treating specific symptoms such as the use of fluids to reduce dehydration risk, medication to reduce fever, and oxygen therapy for respiratory failure [1,2]. Most people with this disease exhibit mild to moderate symptoms and fully recover without special medications; however, elderly people and people with other co-morbidities like cardiovascular diseases, asthma, diabetes, chronic respiratory disease, and the immunocompromised develop more severe illness leading to fatality [1-3]. Though these conditions are the most commonly reported conditions influencing COVID-19 severity, bacterial superinfection may also affect the severity of disease. In the SARS-CoV epidemic of 2003, an increased rate of methicillin-resistant Staphylococcus aureus (MRSA) was reported from the pre-SARS to the SARS period [2]. In the current COVID-19 pandemic, a case-fatality of a French man in his thirties with no underlying medical conditions has been reported [4]. Investigations into the cause of the severe symptoms seen in this patient showed the presence of a cytotoxin producing methicillin-susceptible Staphylococcus aureus which was inducing necrotizing pneumonia in the patient, leading to his death [4]. Considering the similarities already reported between the 2003 coronavirus infection and this SARS-CoV-2 infection [5], there is a need to assess the presence of Staphylococcus aureus in COVID-19 patients, and its treatment be considered to reduce risk of superinfection and ultimately the severity of disease in patients with COVID-19. AIMS Staphylococcus aureus is a human commensal that causes community-acquired and nosocomial infection. The increase in the resistance of this bacterium to antibiotics makes it of great clinical importance. Nasal carriage of S. aureus are reported to increase the risk of acquiring both community and hospital-acquired infections, and are particularly implicated in bronchopulmonary infection, causing both upper and lower respiratory tract infection [6, 7]. The prevalence of nasal carriage of staphylococcus is high in Nigeria even in apparently healthy people [8-9]. There is currently a paucity of data on the different comorbidities that might vary in different populations with COVID-19. Hence, the aim of this study is to evaluate the association of nasal carriage of S. aureus and its association with COVID-19. The objectives of this study are: 1. To determine the nasal carriage rate of S. aureus among suspected patients presented for SARS-CoV-2 screening 2. To evaluate antibiotics resistance patterns in the identified S. aureus strains. 3. To examine the association between nasal S. aureus carriage and SARS-COV-2 infection. 4. To assess the prevalence and distribution of antibiotic resistance and virulence genes among the S. aureus strains. 5. To type isolates into the different S. aureus protein A types STUDY DESIGN The study will be a quantitative study using a cross-sectional research design. The target population for this study will include individuals who present for SARS-CoV-2 testing at the Nigerian Institute of Medical Research drive through COVID-19 testing centre. The inclusion criterion for this target population is that they are willing to participate in the study. The sample size for this study was estimated using the formula for single proportion. With Z set at p<0.05 = 1.96, d at 0.05, and the expected proportion in the population based on a previous studies was set at 57% [9]. The estimated sample size was 380 based on this calculation. Convenience sampling will be used to recruit participants into the study. The two anterior nares of the participant will be swabbed using a sterile swab moistened with normal saline following all safety guidelines. The samples will immediately be plated on mannitol salt agar and nutrient agar and incubated within two hours of collection at 37ºC for 18 - 24 hours. The swab stick will also be transferred into a lysis buffer containing guanidine isothiocyanate for viral inactivation and transported to the laboratory for direct molecular detection of S. aureus. Isolates will be identified using colony morphology, gram staining, slide agglutination and tube coagulase test, and molecular amplification of the 16s rRNA gene specific to S. aureus. Pure isolates will be subjected to antimicrobial susceptibility testing using the disk diffusion method as recommended by Kirby-Bauer method according to the Clinical and Laboratory Standard Institute (CLSI) criteria with the following antibiotics: Gentamicin (10µg), Augmentin (30µg), Streptomycin (10µg), Tetracycline (10µg), Cotrimoxazole (25µg), Cloxacillin (5µg), Chloramphenicol (10µg), Erythromycin (5µg), Imipenem (10µg), Cefuroxime (30µg), Amoxycillin (10µg), Rifampicin (5µg), Cefoxitin (30µg), Methicillin (5µg), ciprofloxacin (5 μg), Ofloxacin (5µg), ceftriaxone (30μg) ceftazidime (30μg), vancomycin (30µg) and azithromycin (15µg). Sensitivity will be read after 24 hours incubation at 35º-37º C. Isolates will be regarded as sensitive or resistant according to the CLSI criteria [10]. DNA will be isolated from all identified isolates and directly from the swab samples using a commercial DNA extraction kit. The mecA and the SCCmec typing will be done using a multiplex polymerase chain reaction (multiplex PCR) method as previously described [11]. The panton valentine leucocidin (pvl) toxin has been associated with necrotizing pneumonia in children [12, 13]. The virulence of the S. aureus will be evaluated with regards to pvl using PCR methods as previously described [13]. The isolates will also be typed into the different S. aureus protein A (spa) types using methods as previously described [14]. Statistical analysis will be done using SPSS version 20.0. Chi square analysis will be carried out to determine if there is a significant association between the presence of S. aureus and SARS-CoV-2 detection and also if there is an association between the presence of virulent S. aureus strains and COVID-19 severity. Ethical considerations Ethical approval will be obtained from the Institutional Review Board of the Nigerian Institute of Medical Research. Apart from the nasal swabs, demographic information such as age and gender of the participants will be collected. Though no identifying information will be collected from the participants, the result of the COVID-19 screening and severity of the diseases in the participant will also be required. MAXIMISING THE IMPACT OF RESEARCH OUTPUTS Collaborations will be made with relevant experts to improve the research output of this project. This will involve collaborating with laboratory technicians, microbiologists and virologists involved in COVID-19 research. The result of this study will be sent to the head of the Nigerian Institute of Medical Research so as to influence health policies by the federal ministry of health. Additionally, the result of this study will be published in open access, peer-reviewed journals, and will also be presented in local and international conferences EXPECTED OUTCOMES Considering the increase in the incidence of COVID-19 in Nigeria, the fact that Nigeria already has a high prevalence of nasal carriage of S. aureus even in apparently healthy people, it is expected that the nasal carriage of virulent S. aureus may influence the severity of COVID-19. The result of this study may help highlight the need to assess the presence of nasal carriage of S. aureus as comorbidity in COVID-19 patients, and consider the treatment of S. aureus co-infection in COVID-19 patients as a means to reduce the severity of disease in COVID-19 patients. ROLE IN PROJECT - Principal Investigator Some Selected References 1.. Lupia T, Scabini S, Pinna SM, Di Perri G, De Rosa FG, Corcione S. 2019 novel coronavirus (2019-nCoV) outbreak: A new challenge. JGAR. 2020 Jun; 21:22 2. Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention. JAMA. 2020 Feb; 323(13):1239 3. World Health Organization. Coronavirus [Internet]. WHO; 2020 [Cited April 19, 2020]. Available from https://www.who.int/health-topics/coronavirus#tab=tab_1 4. Duployez C, Le Guern R, Tinez C, Lejeune A-L, Robriquet L, Six S, et al. Panton-Valentine leukocidin-secreting Staphylococcus aureus pneumonia complicating COVID-19. Emerg Infect Dis. 2020 Aug; 26(8). 5. Ceccarelli M, Berretta M, Venanzi Rullo E, Nunnari G, Cacopardo B. Differences and similarities between Severe Acute Respiratory Syndrome (SARS)-CoronaVirus (CoV) and SARS-CoV-2. Would a rose by another name smell as sweet? Eur Rev Med Pharmacol Sci. 2020 Mar; 24(5):2781 6. Watanabe H, Masaki H, Asoh N, Watanabe K, Oishi K, Kobayashi S, et al. 2000. Molecular analysis of methicillin-resistant Staphylococcus aureus as a causative agent of bronchopulmonary infection: relation to colonization in the upper respiratory tract. J. Clin. Microbiol. 2000 Oct; 38(10):3867 7. Corne P, Marchandin H, Jonquet O, Campos J, Bañuls AL. Molecular evidence that nasal carriage of Staphylococcus aureus plays a role in respiratory tract infections of critically ill patients. J Clin Microbiol. 2005 Jul; 43(7):3491 8. Tuta KE, Okesola AO, Umeokonkwo CD. The Prevalence and Risk Factors Associated with Nasal Methicillin-Resistant Staphylococcus Aureus Colonization among Children in a Tertiary Hospital in Nigeria. Ethiop J Health Sci. 2019 Jul; 29(4):487