Proposed Research

Section A:

Study Title: Prevalence, antibiotic resistance profile, and genetic characterization of Staphylococcus aureus colonizing adults and children in Botswana and risk factors for colonization

Section B:

Principal Investigator: Rebecca S. Bryson, MPH, PhD Candidate Faculty Mentor: Eric Brown, PhD	Botswana Contact Details Dr Andrew Steenhoff Or Dr Michael Reid
Postal Address: UTHSC School of Public Health, Center for Infectious Diseases RAS-735 1200 Herman Pressler Dr Houston, Texas 77004 USA	Postal Address: PO Box AC 157 ACH Riverwalk Gaborone Botswana
Name of affiliate Institution/Organization: The University of Texas Health Science Center (UTHSC)	Name of Institution/Organization: Botswana-UPenn Partnership

Section C: Description of Research

1. Brief Description of Study The University of Texas School of Public Health (UTSPH) at The University of Texas Health Science Center at Houston respectfully requests the opportunity to conduct ground-breaking research in collaboration with the Botswana-UPenn Partnership (BUP) and the University of Botswana (UB) in Gaborone, Botswana.  The proposed research investigates the epidemiology and characteristics of the bacteria Staphylococcus aureus (S. aureus), a leading cause of severe pneumonia and bacteremia in developing countries, especially among children and among persons of all age who have been infected with human immunodeficiency virus (HIV) 1–4.  Individuals with HIV are at higher risk of developing staphylococcal infections, such as pneumonia, than those who are immunocompetent, and their diseases more frequently lead to hospitalization, treatment failure, and death 1.  In fact, pneumonia remains the primary cause of hospitalizations and of mortality among children with HIV in Africa, even though highly active antiretroviral therapy (HAART) is an available and effective treatment for HIV and antibiotics that combat S. aureus exist 1,4,5.   Understanding the epidemiology of organisms that pose significant health risks to vulnerable populations will lead to targeted prevention and treatment strategies, thereby reducing morbidity and mortality and lowering costs associated with treatment and hospitalizations 6.  S. aureus is normal human bacterial flora, but also one of the most common causes of modern bacterial infections 7.  Staphylococcal infections, which result from S. aureus introduction into the body (for example, through skin disruption, respiratory tract, or ingestion) vary from skin and soft tissue abscesses to serious, often lethal, invasive disease like endocarditis, osteomyelitis, pneumonia, arthritis and sepsis.  The number of S. aureus infections is on the rise worldwide, as is the number of individuals who it colonizes, owing to its increasing occurrence as normal flora, increased acquisition of virulence-determining factors, spread within the community to individuals not traditionally at-risk, and the emergence and spread of antibiotic-resistance (e.g. methicillin-resistant S. aureus (MRSA)) between strains 8–20.  Antibiotic resistance is accelerated by the widespread use of antibiotics that may not be universally appropriate in all clinical settings, among all members of a population, or with all presentations of staphylococcal disease. It is well established that a major risk factor for staphylococcal infection is nasal colonization with S. aureus.  Humans are colonized either persistently, intermittently, or are never colonized.  It is important to distinguish carriage from disease, as carriers may never suffer S. aureus-related disease.  An individual’s carriage status, the nature of their immune response, and the specific infecting bacterial strain affects both one’s likelihood of developing disease and the severity of the disease 13,21–24.  Persistent carriers are most likely to develop disease, while non-carriers, though less prone to disease, either because of lower exposure to the bacteria or efficient host immune defenses, suffer more severe disease 13,21,25–27.  Assessing colonization and infection in the context of host factors is a useful means of helping to define factors underlying clinical consequences.  Given that antibiotic-resistance is becoming more widespread, knowing such characteristics may prove useful for prevention and early identification and treatment of infection, especially in resource-constrained settings 20,27–30.  Individuals with HIV (even when asymptomatic) are at increased risk for both colonization and disease and are more likely to harbor antibiotic resistant strains.  Disease in this group is often more severe 20,25,28,29,31–35.  Low CD4+ count, high viral load, or taking HAART add additional risk of colonization, persistence of colonization (i.e. carriage), and of infection recurrence 19,25,31,35–42.  Children have recently established as a high-risk group for colonization and more often manifest with severe disease 4,26,39,43,44.   Therefore, it is likely that the rate of S. aureus colonization is highest among HIV-infected children and that these children are at particularly high risk of infection and of progressing to severe and life-threatening disease. At the genetic level, S. aureus isolates can be classified using molecular methods for genotyping. Specifically, multilocus sequence typing (MLST) has made it possible to study the evolutionary history of this pathogen, in addition to facilitating epidemiologic studies. As of June, 2012, at least 2,133 MLST types (referred to as sequence types or STs) have been identified, and their distribution worldwide varies between geographic location and human populations24,45,46.  In addition, certain STs are associated with different types of infections, possess different virulence determinants, and vary in their antibiotic resistance profiles 24,46–49.  Knowing which STs are circulating within a region or population can aid in predicting what disease manifestations and clinical outcomes are likely and in identifying how strains are spreading to and within them. The goal of this study is to describe the prevalence of and risk factors for S. aureus colonization among children and adults in Botswana, and compare carriage rates and strain types for individuals living with HIV to those who are not.  Specifically, study aims are to assess the prevalence of nasal colonization among patients of Princess Marina Hospital (PMH) in Gaborone, Botswana, to describe the relationship between colonization and host factors (including HIV parameters), and to describe the antibiotic resistance profiles and genetic profiles of isolated bacterial specimens.  Identification of host risk factors and bacterial strain characteristics in this vulnerable and at-risk population, in which clinic consequences are often severe, can inform targeted prevention strategies and judicious treatment algorithms in this part of the world 30,50,51.

2. Rationale/Justification (Why the need to carry out this study in Botswana): The goal of this study is to describe the prevalence of and risk factors for S. aureus colonization among HIV-infected individuals in Botswana by studying patients at Princess Marina Hospital (PMH) in Gaborone, the largest referral hospital in Botswana and one of the largest HIV treatment centers in the world. S. aureus is emerging as a major pathogen in Africa, where it has become a leading cause of bacteremia, pneumonia, osteomyelitis, and abscesses 1,4,11,16,26,36,49,52,53.  Staphylococcal infection and colonization across the continent are on the rise, antibiotic resistance is rapidly becoming a primary concern, and strains isolated from infections across the continent include those from highly virulent lineages 13,16,26,50,54,55.  S. aureus is a leading cause of morbidity and mortality in Africa, particularly among children4. Colonization and infection remain poorly described in the developing world, including countries of sub-Saharan Africa.  There is limited understanding of risk factors for colonization there, and the impact of social and behavioral factors (eg. hygiene, crowding, poverty, or sharing of personal items) are unclear in this context.  No studies were identified that estimate the prevalence of S. aureus colonization in Botswana, and very few studies have described this among HIV-infected children in Africa or elsewhere. It is important to understand the epidemiology of carriage and infection in this part of the world, since prevalence is known to vary across ethnic groups and geographic locations 49.  The identification of key risk factors in this especially high-risk population in adjacent Botswana is important and may inform targeted prevention strategies (eg. decolonization and transmission interruption) and life-saving treatment protocols (eg. early intervention and more speedy and judicious antibiotic delivery). There is recent evidence that the incidence of S. aureus infections, particularly MRSA infections, is increasing in Gaborone, and there are reports that this is especially the case among HIV-positive children there 53,56,57; however, prevalence of infection and colonization among both adults and children there remains unknown.  In neighboring South Africa, studies show that S. aureus is responsible for severe invasive infections among pediatric HIV hospitalizations, and MRSA is more often responsible for fatalities than MSSA 58,59. Moreover, genetic typing of strains causing infections there reiterate that virulent and antibiotic resistant strains that have been emerging are spreading rapidly 55,60,61.   Nasal carriage of S. aureus there is prevalent among both children and adults, particularly among those with HIV, especially MRSA, and HIV-infected adults with tuberculosis may present with additional risk 33,37.  These findings underscore the need for a greater understanding of the epidemiologic scenario in Botswana. In Botswana, an estimated 25% of the adult population has HIV, many of whom are young adults, and an approximate 15,000 children are HIV-positive 62.  As well, nearly 80% of persons needing HAART are receiving it, about 10% of whom are children62.  The intersection of the HIV and S. aureus epidemics, therefore, requires prompt attention in Botswana.  As of yet, prevalence of S. aureus infections and colonization rates remain unknown.  The population of Botswana, and in particular the patients of PMH, may benefit from this research, as preventive measures and treatment algorithms could be refined to meet their needs 53,56.  In addition, this research, which seeks to apply validated methods to assess bacterial colonization status of individuals in Botswana in order to improve health and to reduce healthcare costs and utilization (e.g. spending on hospitalizations and antibiotics that may be ineffective or costly), will be the first of its scope in this part of the world and will provide important information with the potential to affect health of HIV-infected individuals worldwide.  Furthermore, an understanding of the molecular types of S. aureus circulating in this region will provide important baseline characteristics from which to monitor its changing epidemiology over time.

3. Study Objectives ( Both General and Specific): This study will be the first to describe the epidemiology S. aureus carriage by adults and children in Botswana and to define the bacterial strains affecting that population; in particular, it will describe this in the context of HIV infection status and clinical indicators, as well as antiretroviral treatment regimens, antibiotic use, concurrent carriage of other bacteria, and specific host characteristics. The specific aims of this study are to: 1.      Establish the prevalence of S. aureus colonization among HIV-infected adults and children of the Princess marina Hospital in Gaborone, Botswana; 2.      Describe antimicrobial susceptibility/resistance profiles of isolated S. aureus specimens; and 3.      Establish correlations between carriage status and participant HIV infection status and with CD4+ counts, viral load, anti-retroviral therapies, concurrent S. pneumonia and H. influenza colonization, concomitant infections, and other risk factors for colonization (e.g. age, recent hospitalization, household size, and sharing of personal items and towels). Additional exploratory objectives of this study, which aim to produce pilot data, are: 4.      Describe the persistence of S. aureus carriage over time by estimating the prevalence of persistent, intermittent and never carriers and correlations with factors described in Aim 3; and 5.      Describe genetic lineages (multi-locus sequence typing [MLST]) of isolated S. aureus strains.

4. Expected Results (Both Primary and Secondary endpoints): The primary endpoint of the study is prevalence of and risk factors for colonization by S. aureus. Secondary endpoints are antibiotic resistance patterns and genetic identification of strain lineage and virulence determinants among locally colonizing strains of S. aureus. Specifically, anticipated results are: 1.      Proportion of the HIV-positive study population who are colonized with S. aureus in the anterior nares; 2.      Proportion of strains that are susceptible/resistant to selected antibiotics which are commonly used in treatment of staphylococcal infections in Africa; and 3.      Identification of host factors and microbial factors associated with risk of colonization. The secondary endpoint results, which are anticipated to be exploratory, are: 4.      Proportion of the study population who are persistent carriers, intermittent carriers, and non-carriers among over repeated samples and exploration into risk factors for each; and 5.      Identification of strain types (MLST) of isolates by sequence type lineages and identification of virulence-determining genes and their prevalence in colonized individuals.

Section D. Methodology

1. Study Design This study employs a cross-sectional design.  Adult and pediatric patients will be invited to participate while at regular outpatient well-visits, where the purpose and procedures of the study will be explained and they will be invited to consent to participate and have the opportunity to ask questions.  All participants will be asked to return in 4 weeks in order to capture persistence of colonization.  Participants will be enrolled throughout a 3-month study period until the desired sample size is met.

2. Study sites (Districts, Towns, Villages, Health facilities, Schools etc) : The Princess Marina Referral Hospital (PMH) in Gaborone, Botswana is the main study site, as it is the largest hospital in Botswana and offers inpatient and outpatient adult services.  Specifically, the clinics at PMH from which participants will be recruited are the general OPD and the IDCC.  HIV negative patients will undergo the same evaluation as HIV infected patients Pediatric cases will also be recruited from Balemette Lutheran Hospital in Ramostwa.  BLH has a sizeable population of HIV-infected children.  Pediatric HIV-negative controls will be recruited from the general pediatric outpatient department at BLH.

3. Subject Population(s) (Clinical condition, Gender, age, and other relevant Characteristics): HIV-infected adults will be sourced from the PMH, the public hospital which is overseen by the Botswana Ministry of Health and provides healthcare to persons in and around Gaborone. HIV-infected children will be recruited from the IDCC at Balemette Lutheran Hospital in Ramostwa.  All HIV-infected patients  are seen in the clinic approximately every 1 to 3 months 63.  About 80% of individuals who meet the Botswana National HIV Treatment Guidelines (2008) criteria for anti-retroviral therapy are on one of three lines of HAART (see Table 2) 62,64. About 70% of those on HAART are on first-line HAART, about 25% are on second line HAART, and the remaining approximately 5% are on third line HAART 57. HIV-negative participants will be recruited from the general outpatient facilities at PMH (adults) and BLH (pediatric) respectively.  These patients will be recruited from the general outpatient clinics at these two facilities.  .  We will not confirm their HIV status as part of the assessment.  However, we will record adult participants as HIV-negative if there was an HIV test performed in the last 6 months.  Patients with an unknown HIV status (last test >6 months ago) will all be encouraged to get HIV tested.

4. Sample size(The number of subjects to be involved in the study and how these subjects will be selected from the population): To meet study Aim 1 through Aim 3 among individuals who attend PMH, we propose to enroll a total of 600 adults and children from that setting into this study, to include both adults and children who have a documented HIV infection status.  Specifically, a total of 400 HIV-positive patients of PMH will be enrolled.  In order to further meet Aim 3, an additional 200 HIV-negative pediatric and adult patients will be recruited for comparison. Study participants will be systematically recruited from each of these categories during a 3-month study period or until enough participants have completed study procedures to meet this desired sample size.  The sample size calculation is powered for Aims 1through 3.  The sample size and power estimations were performed using the StatCalc (OpenEpi) module of Epi Info™ (Centers for Disease Control and Prevention (CDC), Atlanta).

5. Subject Recruitment/Sampling Methods (Explain all procedures in detail): Subjects will be recruited systematically at PMH during clinic hours at clinic visits.  Sampling will be based on the daily patient roster of the clinic.  During systematically selected enrollment days, all eligible patients identified on the roster by study staff will be recruited for participation. A short screening will be used by study staff to determine eligibility to participate in the study.  Selection of participants will be stratified according to age; both children (age 6 months to 13 years) and adults (>13 years) will be recruited.  Participants will be invited by a research nurse during their scheduled visits to the PMH clinics.  Recruitment of Adults: HIV-infected adults will be recruited from the IDCC and HIV-negative adults from the general OPD at PMH. Eligible participants will be individuals of at least 13 years of age who assent and whose (if <18 years of age) parent or guardian consents to participate by signed agreement and who have a documented HIV infection status.  Agreement to participate will include willingness to by submit to nasal swab, interview, medical record data abstraction, and to return to a second sampling 4 weeks later.  Recruitment of Children: HIV-infected children will be recruited from the IDCC and HIV-negative children from the pediatric OPD at BLH.  Eligible participants will be patients of at least 6 months of age whose accompanying parent or guardian consents and who (if age 7+ years) assents to participate by giving written agreement and who have a documented HIV infection status.  Agreement to participate will include willingness to by submit to nasal swab, interview, medical record data abstraction, and to return to a second sampling 4 weeks later.  Eligible individuals will be given a description of the study in Setswana and will be offered the opportunity to take part or to decline participation.  Study documents will be available to recruited patients in both English and Setswana.  Participants will be asked to sign a consent form, and children will be asked to give their assent.  Recruited individuals will be offered educational material on prevention and care of staphylococcal infections, regardless of their decision to participate in the study.

6. Data Collection Methods (Explain all procedures in detail) After the purpose and procedures of the study are explained, the study staff will ask the participant (and/or guardian) to give written, signed consent to participate and designate a Study ID to the participant, taken from a listing of identification numbers generated prior to the enrollment phase.  Subsequently, participants will be asked to complete a short interviewer-administered questionnaire.  Figure 1 shows the flow of a participant through the study procedures. Figure 1. Study participant involvement Consents and interviews will be conducted by study staff in Setswana, or alternatively in English or Tswana, if preferred by participant, and will take approximately 10-15 minutes to complete.  The questionnaire consists of approximately 15 open-ended items and 15 closed-ended items and asks about participant sociodemographics and practices that are known or suspected risk factors for S. aureus colonization or disease, staphylococcal infection history, and relevant clinical history. An additional approximately 10 closed-ended items will be abstracted from the medical record and include clinical parameters (HIV infection status, viral load, CD4+ cell count), current/recent infection, pneumococcal vaccination status, current/recent antibiotic therapies, history of cotrimoxizole prophylaxis, and history of HAART therapies. A sample will be collected from the anterior nares of each participant at each visit by study staff by rolling a sterile swabette in a 360 degree arc around the inside of one nostril.  Because carriage is based on the presence or absence of S. aureus in nasal samples collected over time , patients will be asked to return for follow-up two weeks later (Multiple samplings allows for classification of participants as persistent (positive for two samples), intermittent (one negative sample) or never carriers .)  The swabs will be transported to the National Health Laboratory (NHL) in Gaborone for processing. Labwork to be conducted in Gaborone Nasal swab samples will be processed as outlined in Figure 2.  First, each swab will be streaked in duplicate on Manitol Salt Agar (MSA), a S. aureus selective media, and on Gentamicin, to detect presence of S. pneumonia.  Plates will be cultured in favorable conditions for bacterial growth (37ºC incubator for 48-72hrs (MSA) or 36-48hrs (Genta). Swabs may be stored at 4°C for up to 4 days prior to streaking.  In order to detect presence of H. influenza in samples, DNA extraction and qPCR will be performed on nasal specimens.  If present in relatively high load, then MLST may be performed on H. influenza and S. pneumonia.  Presumptive S. aureus and S. pneumonia colonies will be used to inoculate, in duplicate, 1-ml tubes containing tryptic soy (TSB) agar and stored at 4°C.  One set of these TSB tubes will be shipped to UTSPH for further characterization, while a duplicate of all isolates will remain with BUP in Botswana. Figure 2. Laboratory processing of nasal swab samples Labwork to be conducted in Houston Upon receipt of samples from Botswana, UTSPH will conduct confirmation that the sample is S. aureus, as outlined in Figure 3.  Specifically, they will be streaked on blood agar (BA) and TSB agar and incubated at 37°C for 24h.  Following incubations, colonies will then be subjected to catalase and coagulase tests.  Positive reactivity to both tests is considered diagnostic for S. aureus. Isolates will be tested to determine sensitivity to methicillin using the E-test and classified as either MRSA or MSSA.  Samples of confirmed S. aureus isolates will be stored at -80°C for multilocus sequence typing analysis (MLST) analysis, which will establish the genetic profiles and lineage (clonal complexes) of the isolated S. aureus strains.  Figure 3. Flowchart of laboratory testing protocol for S. aureus identification (MSA: Manitol Salt Agar; TSB: Tryptic Soy Broth; MHA: Mueller Hinton Agar; E-Test: Etest® Minimum Inhibitory Concentration of antibiotics test strip)

10. Approximate Date Study Recruitment will begin: September 15, 2012

11. Estimated Duration of entire study: 12 months to complete all elements of the proposed research study (3 months for data collection; 6 months of MLST analysis; 3 months additional for data analysis, report preparation and dissemination

Section E: Subject Information

1. Inclusion Criteria Participants will meet inclusion criteria if they are residents of Botswana who are current patients at the PHM whose gives consent to participate (or for children, whose caregiver consents to have the child participate) in the study.  In addition, children of 7 years of age and older will assent to participate in the study in order to be included.  Participants will be asked to agree to return for 4-week follow up for sample collection.  Subjects who are recruited but who do not meet the inclusion criteria will not be enrolled. This project will enroll participants independently of sex, race, or any other social designation or group affiliation.

5. Reasonably foreseeable risk or discomforts to the subjects (list in detail): We do not foresee any major adverse effects of participation in the study.  No major adverse effects have been reported by investigators in similar studies.  There is very mild risk to participants in this study, as follows.  There may be some personal unease or discomfort experienced by participants during the interview process, as some questions ask about personal and household factors.  These questions have been included as factors that are considered important in the cycle of S. aureus acquisition or transmission.  In order to minimize discomfort, interviews will be conducted by trained study staff, and interviews will be conducted in a private area to ensure confidentiality.  Participants will be told that they may decline to answer any question they prefer not to answer and will be allowed to withdraw from the interview or from participation at any time.  In addition, the participant may experience some slight and very brief discomfort when the sterile swab is briefly inserted into his/her nose during sample collection. The swabbing will be out by trained study staff.  The participant will be instructed to contact study staff and/or their physician if there is any residual physical discomfort from the swabbing procedure.  A 24-hour phone number will be provided to study participants at which they can contact study staff in such a case.

8. Botswana based personnel that will be involved (Name, functions and qualifications):                           Dr Andrew Stenhoff, MD – BUP Associate Country Director, Research Director and Lead Paediatrician, is the Botswana based PI Dr Gerard Morris, PhD – BUP Research Lab Director, will lead all Botswana lab activities, scientific input in design, implementation, co-author, co-investigator. Dr Mike Reid, MD – BUP HIV Physician, will be on the ground lead for this project working with Rebecca and others in the team to make the project happen, specifically for IRB, recruitment, liaising with recruitment sites, also scientific input into design, implementation, co-author, co-investigator. Dr Naledi Mannathoko, PhD – University of Botswana Faculty (Lecturer), PhD work in S.aureus, will work with Gerry and Rebecca on in-country lab aspects of the project, scientific input in design, implementation, co-author, co-investigator

9. Any renumeration given to subjects?   Yes   (  x )   No   ( x ). If yes, specify: A 15Pula ($2USD equivalent) remuneration will be provided to participants following completion of the second sampling visit to defer cost of travel to the clinic.

Section G. Study Details

1. Capacity Building (how will the study build capacity in the country)   The partnership between these academic and research institutions enables sharing of intellectual capacity and building of research and laboratory practice capacity.  In addition, samples retained by the partner institutions may be used for further research into strains of S. aureus and S. pneumonia affecting the population of Gaborone.

Section H. References

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Table 2.i Adult HAART in Botswana (adapted from Botswana National Antiretroviral Therapy Guidelines [2012])

	NRTIs	NNRTIs	PIs
1st Line	Atripla (EFV+TDF+FTC)	EFV
2nd Line*	AZT + 3TC		LPV/r
3rd Line	Resistance assay and consultation with HIV Specialist		DRV/r

Table 2.i Pediatric HAART in Botswana (adapted from Botswana National Antiretroviral Therapy Guidelines [2012])

	NRTIs	NNRTIs		PIs
1st Line	AZT + 3TC OR TDF +FTC*	If < 3 years:	If > 3 years:
		NVP	NVP or EFV
2nd Line*	ABC + 3TC			LPV/r
3rd Line	Resistance assay and consultation with HIV Specialist

*In post pubertal adolescents (Tanner stage IV and V)

Figure 5. Sample report of findings to patient and physician