Clinical trial NCT02589067
Conditions: S. Aureus Oropharyngeal Colonization
Sponsor: Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center
Start date: 2015-10-01; Completion date: 2017-11-01
Enrollment: 67 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The problem of interest is the high rate of recurrent SSTIs in children caused by S. aureus despite the use of systemic antibiotic treatment, due to difficulties in decolonization and the prevalence of antibiotic-resistant strains such as MRSA.

This problem will be studied through a randomized, double-blind, placebo-controlled clinical trial of the use of a 0.12% clorhexidine gluconate (CHG) oral rinse in decolonizing S. aureus in the oropharynx in children 5-17 years old. CHG is an excellent candidate for use in children as its safety has been widely established, and it is readily commercially available. Eligible, consented subjects will be asked to participate in a baseline study visit in which swabs of their oropharynx and nares are obtained, and they are educated on the use of either CHG oral rinse or a placebo oral rinse containing saline. The subjects will be instructed to perform the oral rinse twice daily for seven days. Two follow-up visits will occur at 7 and 28 days post-baseline, where subjects' nares and oropharynx are again swabbed in order to ascertain short- and long-term decolonization of S. aureus. This procedure will serve to 1) determine the efficacy of a CHG oral rinse in decolonization of S. aureus, 2) investigate the safety, tolerability, and compliance of oropharyngeal decolonization among children and their caregivers, and 3) determine the genetic backgrounds of strains of S. aureus associated with continued colonization.

Detailed description:
Each year, children visit doctors offices and emergency rooms over 2 million times due to skin and soft tissue infections (SSTIs). These SSTIs are usually caused by a bacteria called Staphylococcus aureus (S. aureus), and can result in severe negative consequences such as hospitalization, disability, and even death. Despite treatment with antibiotics, 20-70% of children with an SSTI will develop a recurrent infection within the next year. Additionally, this treatment fails to eradicate S. aureus from children nearly 50% of the time. Of the types of S. aureus that cause these infections, antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA), which are associated with higher rates of transmission, have become increasingly more common. Thus, the problem of interest is the high rate of recurrent SSTIs in children caused by S. aureus despite the use of systemic antibiotic treatment, due to difficulties in decolonization and the prevalence of antibiotic-resistant strains such as MRSA.

Arms: arm 1: Randomized to 15mL 0.12% Chlorhexidine Gluconate oral rinse, to be used twice daily for 60 seconds for 7 days. arm 2: Randomized to 15mL saline placebo oral rinse, to be used twice daily for 60 seconds for 7 days.
Interventions: intervention 1: Chlorhexidine gluconate intervention 2: Saline Placebo
Intervention descriptions: intervention 1: Oral rinse of either 0.12% Chlorhexidine Gluconate oral rinse to test the safety/efficacy of 0.12% Chlorhexidine Gluconate oral rinse in decolonizing the oropharynx of children colonized with S. aureus. intervention 2: Placebo saline oral rinse to to test the safety/efficacy of 0.12% Chlorhexidine Gluconate oral rinse in decolonizing the oropharynx of children colonized with S. aureus.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Decolonization of the oropharynx, an important and neglected reservoir of S. 
aureus colonization 
NCT02589067 
September 30, 2015 
 


5 
 
 
 
Skin and soft tissue infections (SSTIs) are an extremely common reason for children to seek care.  
Four in 100 children will experience a medically attended SSTI annually, resulting in over 2 million visits to 
doctors’ offices and emergency departments annually among children in the U.S. Among the general 
population, children are disproportionately affected by CA-S. aureus SSTIs. SSTIs can lead to severe 
disease that can cause hospitalization, bacteremia, disseminated disease, critical illness, and death.  
The most commonly identified cause of SSTIs is Staphylococcus aureus, including methicillin-
resistant S. aureus (MRSA). S. aureus’ success as a bacterial infection is related to its ability to 
asymptomatically colonize patients and later cause disease. Another aspect of its success is its ability to 
cause infection in both immunocompromised and non-immunocompromised hosts. Most SSTIs in children 
occur in otherwise healthy children. Of great concern is that, after an initial S. aureus SSTI, recurrence rates 
of S. aureus skin infections can exceed 50%. 
Because of the large burden of S. aureus SSTIs in the community, there is a large interest in SSTI 
prevention. However, clinical trials of S. aureus SSTI prevention have been very disappointing. Fritz et al. 
found that skin decolonization with chlorhexidine (CHG) of affected children and their household members 
has been found superior to decolonizing just the affected child. However, the magnitude of decrease was 
minimal and 52% of children in the more aggressive decolonization regimen experienced a SSTI during the 
12 month follow up period. Another recent clinical trial by Kaplan et al. randomized children with CA-S. 
aureus infections to bleach baths versus routine hygienic measures. Unfortunately, the skin decolonization 
afforded by bleach baths did not decrease the recurrent SSTI rate. Clearly, more effective means of 
decolonization are needed to meaningfully decrease rates of recurrent infections.  
Traditionally eradication efforts of S. aureus colonization have focused on removing colonization in 
the anterior nares (nose) and skin. A recent investigation by our group found that among persons with an S. 
aureus SSTI and their household members, the most common site of colonization of S. aureus on the 
human body is not the anterior nares, but the oropharynx (26% vs. 24%), with overlap of colonization far 
from universal. In fact, 50% of oropharyngeally colonized persons were not nasally colonized. Other 
investigations show a similar magnitude of S. aureus orpharyngeal colonization. There are mounting data 
that decolonization regimens that ignore the pharynx are not effective at preventing recurrent S. aureus 
SSTIs. However, prospective data on oropharyngeal decolonization of S. aureus in children are lacking. 
We hypothesize that suboptimal S. aureus prevention efforts stem from previous inattention to 
eradication of S. aureus oropharyngeal colonization. To this end, we will conduct a clinical trial on 
eradication of oropharyngeal S. aureus colonization. Data from our “proof of principle” study will form the 
foundation of more comprehensive efforts to prevent recurrent S. aureus infections in children. 
We will perform a prospective, double blind, randomized controlled clinical trial of 0.12% 
chlorhexidine (CHG) oral rinse versus placebo oral rinse for children age 5-17 with S. aureus oropharyngeal 
colonization. We hypothesize that, compared to placebo, 0.12% CHG rinse will reduce oral S. aureus 
colonization. We choose CHG given its wide availability, excellent safety profile, and low cost. We will enroll 
240 children who have a history of S. aureus infection or SSTI in the prior year. Consenting subjects will be 
screened for S. aureus oropharyngeal colonization using a rapid molecular test. Subjects will be eligible if 
they have S. aureus oropharyngeal colonization during a screening visit, are able to gargle, and can be 
followed for 28 days. CHG oral rinse will be used twice daily for 7 days total.  
Our primary outcome is S. aureus eradication at 7 days (test of cure (TOC)). Secondary outcome 
is eradication at 1 month (long term follow up). Study staff will administer surveys to subjects and/or their 
parents about risk factors hypothesized to predict treatment success. Secondary outcomes include safety, 
tolerability, and adherence to oral CHG. We will also examine predictors of eradication, including pathogen 
level factors. Pathogen-level factors will be examined using S. aureus whole genome sequencing, which will 
allow us to quantify the presence or acquisition of the qacA/B gene, which is associated with CHG 
resistance. WGS will also allow us determine if stains associated with persistence after CHG use represent 
pre-existing colonizing strains or acquisition of new strains. 
If CHG oral rinse is efficacious at eradication, our findings will form the foundation for a larger, 
more comprehensive intervention to prevent recurrent SSTIs in children. If CHG oral rinse cannot 
decolonize the oropharynx, then investigations may need to focus on other strategies to prevent S. aureus 
disease. Regardless of results, our trial will forward the field of prevention of this extremely common and 
potentially deadly infection.
Scientific	  Abstract	  (Do	  not	  exceed	  one	  page)	  


6 
 
 
 
 
Structured	  Abstract	  (Do	  not	  exceed	  one	  page)	  
 
Describe the medical problem as it relates to children 
• 
Community-associated (CA) Staphylococcus aureus, including methicillin-resistant S. aureus 
(MRSA), is the most commonly identified cause of skin and soft tissue infections (SSTIs). 
and affects children disproportionately. 
 
State the incidence/prevalence of problem in children 
• 
Four in 100 children in the U.S. will experience a SSTI annually, resulting in over one million 
infections annually, a problem further complicated in that over 50% of children who have a S. 
aureus skin infection will develop a recurrent infection. 
 
Background that will lead to the research gap (may use 2 sentences)  
• 
Our group found that, despite common belief, the most common site of colonization of S. 
aureus on the human body is not the anterior nares (nose), but the oropharynx. There are 
mounting data that decolonization regimens that do not eradicate oropharyngeal colonization 
are ineffective at preventing recurrent S. aureus SSTIs. 
 
State the primary research gap this study will address  
• 
Because trials on prevention of recurrent S. aureus infections have been disappointing, we 
will examine the efficacy of oropharyngeal S. aureus decolonization with 0.12% chlorhexidine 
gluconate (CHG) oral rinse, a widely available commercial product with an excellent safety 
profile.  
 
Hypotheses 
• 
Oral CHG rinse (0.12%) will be more effective than placebo at eradiating S. aureus 
oropharyngeal colonization after treatment completion. 
 
Specific Aims  
• 
To determine if CHG oral rinse can decolonize S. aureus oropharyngeal colonization better 
than placebo in children. 
 
Study design 
• 
Randomized, prospective, double-blind, placebo-controlled clinical trial of 0.12% CHG oral 
rinse vs. placebo oral rinse to decolonize the oropharynx among children. 
 
Describe study population or sample material  
• 
Subjects 5-17 years of age who have confirmed colonization of the oropharynx with S. 
aureus and who are able to gargle. 
 
Sample size/power of primary endpoint 
• 
The primary endpoint is eradication of S. aureus oropharyngeal colonization. To have 85% 
power to detect a 40% absolute difference in eradication between groups (10% for placebo 
and 50% for CHG), we will require 54 subjects total who complete the trial. We will need to 
screen potentially eligible children with rapid molecular tests for S. aureus oropharyngeal 
colonization to find eligible subjects. Estimating 25% of screened children will be eligible, and 
accounting for a 10% attrition rate, we will need to enroll 240 children for screening with the 
expectation that 60 will be eligible and participate and 54 will complete the clinical trial. 
 
Assuming the project is successful, state the next step moving down the pathway to clinical applicability 
• 
A randomized, prospective, multi-center, clinical trial of children of prevention of S. aureus 
skin infection that includes CHG oral rinses (in addition to body and nasal disinfectants) with 
a comparator group that will depend on standard of care at the time of development of the 
clinical trial protocol. 
 


7 
 
 
 
Hypothesis(es)	  and	  Aims	  (Do	  not	  exceed	  one	  page)	  	  
A.	  Hypothesis(es)	  to	  be	  Tested	  
 
Our primary hypothesis is 0.12% CHG oral rinse will be superior to placebo at eradicating S. aureus from 
the oropharynx of children age 5-17 colonized with S. aureus compared to placebo oral rinse.  
 
We have several secondary hypotheses: a) decolonization of oropharyngeal S. aureus colonization with 
0.12% CHG will be sustained at 28 days; b) recolonization of the oropharynx will come from isolates that 
colonize patients nose and not from development of antimicrobial resistance, including the emergence of 
CHG-resistant S. aureus strains that contain the qac A/B genes; and c) predictors of failure to decolonize 
with CHG will include poor treatment adherence, nasal S. aureus colonization, and younger age. 
 
If our primary hypothesis is correct, findings will form the foundation for a larger, randomized, prospective, 
multi-center, placebo-controlled clinical trial comparing skin, nasal, and oropharyngeal decolonization vs. 
standard of care decolonization regimen for the prevention of recurrent S. aureus skin infections. If oral 
CHG rinse is unable to decolonize the oropharynx, then other more aggressive oropharyngeal S. aureus 
colonization eradication strategies in children will need to be tested.  
	  
B.	  Specific	  Aim(s)	  of	  the	  Project	  
	  
1) Determine the efficacy of oropharyngeal decolonization among children with a history of CA-
MRSA, CA-S. aureus or skin and soft tissue infection (SSTI). To determine if oropharyngeal S. aureus 
colonization can be eradicated, we will perform a randomized, double blind placebo-controlled controlled 
clinical trial comparing 0.12% oral chlorhexidine (CHG) oral rinse to placebo among children age 5-17 with 
S. aureus oropharyngeal colonization. We hypothesize that 0.12% CHG rinse will reduce oral S. aureus 
colonization better than placebo treatment. Our findings will provide “proof of principle” data on the ability of 
oral antiseptics to eradicate oropharyngeal S. aureus colonization in children. 
 
2) Assess the safety, tolerability, and compliance of oropharyngeal decolonization among children 
and their caregivers. Using a structured instrument, we will survey trial participants and their 
parents/guardians on side effects, tolerability, and adherence of the 0.12% oral CHG rinse and placebo. 
Findings will serve as a counterpoint to any benefits derived from oral decolonization. 
 
3) Determine the S. aureus genetic backgrounds associated with breakthrough oropharyngeal 
colonization. We will molecularly characterize all baseline S. aureus isolates and “breakthrough” (failure) 
isolates using whole genome sequencing (WGS) for key S. aureus molecular markers, including emergence 
of S. aureus isolates with the qacA/B gene, which is associated with CHG resistance.  
 
 


8 
 
 
 
 
Background	  and	  Significance	  (See	  General	  Instructions	  for	  page	  limitations/recommendations)	  
Skin and soft tissue infections (SSTIs) are an extremely common cause of visits to healthcare 
practitioners. Among U.S. children, there are over 2 million visits to doctors’ office and emergency 
departments annually for SSTIs.1 SSTIs can results in significant morbidity, hospitalization, bacteremia, and 
death. Children are disproportionately affected by S. aureus SSTIs in the U.S., with a relative risk of 1.51 
[1.19-1.92] higher relative risk compared to adults.2 Furthermore, racial and ethnic minorities are 
disproportionally affected by SSTIs,3 and African-Americans have CA-MRSA rates over 2 times higher than 
Caucasians.2 SSTI incidence in the U.S. is increasing and the economic cost of hospitalization for SSTIs in 
children is estimated to be $184 million annually in the U.S.4  
In the first decade of the 21st Century, there has been a dramatic rise methicillin-resistant S. aureus 
(MRSA) incidence. Between 2000 and 2006, SSTI hospitalization incidence in children has risen from 23.2 
cases per 100,000 children to 62.7 cases per 100,000 children.4 When including outpatient visits, 
approximately four in 100 children will experience a SSTI annually.5   
The most common identified cause of SSTIs is Staphylococcus aureus6 and the majority of CA-
MRSA skin infections in the U.S. are caused by S. aureus isolates from the USA300 genetic background. 
This strain is particularly problematic as it is associated with high rates of household transmission and 
recurrent disease.7 Approximately 20-70% of children with a S. aureus SSTI will develop a recurrent 
infection in the following year.8 And approximately 13% of household contacts of persons with CA-MRSA 
will experience a similar skin infection in the subsequent 6 months.9 Because of these high recurrence 
rates, there is tremendous interest in interventions to prevent recurrent S. aureus SSTIs.  
Even after systemic antibiotic therapy, S. aureus colonization persists in nearly one-half of 
patients.10 Understanding persistent colonization may be important for identifying patients at higher risk for 
recurrence. In hospitalized patients, S. aureus colonization is clearly associated with higher risk of 
subsequent S. aureus infection.11 In the ambulatory care setting, there are data suggesting a relationship 
between colonization and invasive disease, although the data are less robust.12 In one military study, 38% 
(9/24) of recruits who were nasally MRSA colonized suffered a subsequent SSTI in the next 2 months 
compared to 3% (8/229) with MSSA colonization (P <0.001).13 In a study of ambulatory children, Fritz et al. 
found that 32% (7/22) of children who were nasally MRSA colonized suffered a subsequent SSTI in the next 
12 months compared to 10% (14/142) with MSSA colonization and 9% (33/370) in those without baseline S. 
aureus colonization (P=0.03).14 Additionally, patients with MSSA colonization commonly suffer from 
recurrent infection.15 These data strongly suggest that MRSA and S. aureus colonization is a risk factor for 
subsequent infection in ambulatory patients.  
Studies on prevention of recurrent infections have been disappointing. Most SSTI prevention 
efforts have focused on body decolonization of S. aureus. Typical decolonization regimens have involved 
combined nasal and skin decontamination. For example, Fritz et al. randomized children with SSTIs to 
either skin decolonization with chlorhexidine (CHG) and nasal decolonization with mupirocin of the affected 
child or CHG skin decolonization + mupirocin given to the whole household.8 While decolonization the 
whole household was superior (54% SSTI recurrence rate vs. 72% during the 12 month follow up period), 
the trial had disappointing results in that even in the more aggressively treated group, recurrence rates of 
SSTIs in the affected children exceeded 52%. The results suggest that even skin and nasal decolonization 
is insufficient to prevent recurrent SSTIs in children. Another recent study by Kaplan et al. randomized 
children with SSTIs, most caused by CA-S. aureus infections to bleach baths versus routine daily hygienic 
measures.16 Unfortunately, the skin decolonization afforded by bleach baths did not result in a decrease 
rate of recurrent SSTIs compared with routine daily hygienic measures (17% vs. 21%, P=0.15).16 These 
findings suggest that skin decolonization is ineffective at prevention of recurrent S. aureus-associated 
SSTIs. Clearly, more effective means of decolonization are needed to further decrease rates of recurrent 
infections.  
Data from our group and others have demonstrated that extra-nasal S. aureus colonization is much 
more common than previously believed.17,18 We found that among household contacts of children and 
adults with S. aureus SSTIs, the most common body site of S. aureus colonization on the human body is 
not the anterior nares (nose) but the oropharynx.18 In this study, conducted in Los Angeles and Chicago, 
30% of household contacts were colonized with S. aureus in the oropharynx, but on 25% in the nares. 


9 
 
 
 
Among this subset of 268 household contacts of persons with CA-MRSA skin infection, 31% were 
oropharyngeally colonized with S. aureus, but on 23% in the nares. Oropharyngeal S. aureus colonization 
has been found in other populations with a prevalence similar or higher than nasal colonization.17-19 
However, to date, virtually all interventions to prevent S. aureus SSTI have ignored oropharyngeal 
decolonization. And there are, to our knowledge, no data on eradication of S. aureus oropharyngeal 
colonization in children. Given the very high prevalence of oropharyngeal S. aureus colonization, we believe 
that the oropharynx may be an important unaddressed sanctuary site for S. aureus that, when not targeted, 
may lead to post-eradication re-colonization and recurrent S. aureus disease. 
 
Chlorhexidine gluconate (CHG) is an ideal treatment for eradication of S. aureus on the skin and the 
oropharynx. CHG has been safely used for bathing, showering and dental hygiene for over 50 years. CHG 
is available over-the-counter product as a 4% topical solution to be directly applied to the skin as an 
antimicrobial skin cleanser. CHG is associated with marked reductions in skin bacteria following serial CHG 
bathing or showering,20-26 and it is widely used as a pre-operative showering agent based upon CDC 
guidelines that recommend its use.27 CHG is also the gold standard in periodontal hygiene, including oral 
care in ventilated patients.28,29 However, CHG is poorly studied as means to eradicate S. aureus 
colonization in patients, especially children, suffering from recurrent S. aureus infections.  
 
Data on CHG oral rinse for eradication of oropharyngeal S. aureus colonization are extremely sparse. 
One recent investigation in adults examined CHG oral rinses in adults, most of who were elderly, with S. 
aureus infection or colonization after hospital discharge for S. aureus. In this investigation, oral CHG rinses 
eradicated over 50% of S. aureus oropharyngeal colonization compared to no oral CHG rinses (no placebo 
was used in this study).19 No data on CHG oral rinses for S. aureus colonization are available in children 
and examination of recolonization after a course of oral CHG rinses are lacking. 
 
One concern with antibiotic use is the emergence of antibiotic resistance. Decreased susceptibility to 
CHG in S. aureus has been recently associated with a plasmid-mediated biocide resistance gene qac A/B, 
which encodes an energy-dependent export system that pumps chlorhexidine from the bacterial cell.30 
Although the clinical significance is not fully elucidated with large studies, qac A/B has been identified in a 
small percent of S. aureus isolates in the US, Europe, and Asia and there is interest in correlating the 
presence of the qac A/B gene with clinical outcomes, such as failure to eradicate colonization.30-34 In a 
recent investigation among adults and children with SSTI, 1% of adults and children with SSTIs harbor the 
qac A/B gene.35 Another study of 281 MRSA isolates from a children’s hospital, 18.5% harbored the qac 
A/B gene,36 suggesting that prevalence of this resistance gene among S. aureus may be high in selected 
populations. There are no data about the emergence qac A/B containing S. aureus after exposure to CHG 
products.  Because of the concern of emerging resistance in trials of antibiotic treatment37 studies using 
CHG products for decolonization purposes should to perform careful monitoring for the development of 
resistance.33,38,39 
 
In summary, S. aureus SSTIs are extremely common, often recur, and efforts to prevent recurrent 
infections have been extremely disappointing. Because decolonization efforts almost always ignore 
oropharyngeal S. aureus colonization, there is a need to quantify a safe, well-tolerated oral rinse that can 
eradicate S. aureus oropharyngeal colonization. This trial will be the first in children to test such an 
approach and form the foundation for further more comprehensive studies to prevent this common and 
potentially deadly infection. 
 
 


10 
 
 
 
 
Supportive	  Preliminary	  Data	  (See	  General	  Instructions	  for	  page	  limitations/recommendations)	  
Oropharyngeal colonization is common but its role as a predictor of recurrent SSTIs is unexplored  
The body site of greatest S. aureus colonization has traditionally been believed to be the anterior 
nares.40,41 S. aureus has been known to colonize the axilla, perineum, rectum, vagina, and the throat 
(oropharynx).42-45 However, only recently have data regarding the importance and scale of non-nasal 
colonization been appreciated.42-45 An investigation conducted by our group in an area of high MRSA 
endemicity found that among persons with S. aureus SSTIs cultured at the anterior nares, oropharynx, and 
inguinal region, S. aureus colonized 40% (137/350) and 50% (405/812) of their household contacts.18 A 
nares-only survey would have missed 48% of S. aureus and 51% of MRSA colonized persons.18 Among the 
subset of patients with CA-MRSA infection, the proportion of oropharyngeal-only colonization was 12%, 
suggesting nasal-only screening for S. aureus colonization misses a sizable proportion of colonized patients 
with community onset SSTI. In fact oropharyngeal colonization was more prevalent than nasal colonization 
(26.0% vs. 24.0%) in our cohort of >1000 persons.18 Investigations of patients with acute skin infections and 
other populations at high risk for S. aureus infection by our group and others have demonstrated the 
prevalence of S. aureus oropharyngeal colonization are similar or exceed that of nasal colonization.17,43,44,46-
49 
The failure of nares plus body decolonization at decolonizing patients and preventing recurrent S. 
aureus infections is disappointing. However, as outlined above, major efforts to prevent infection have 
employed mupirocin plus CHG (or bleach).8,16 Both nasal and body decolonization are well-established 
methods to decrease S. aureus in hospitalized patients.50-53 However, data on the efficacy of oral CHG 
rinses to eliminate oropharyngeal S. aureus colonization are extremely limited. Those few data that exist are 
from adults, largely elderly adults;19 thus the applicability of these limited data to children is unclear. To 
truly perform studies of comprehensive S. aureus decolonization, oropharyngeal S. aureus 
colonization must be addressed therapeutically. However, there are few data on the efficacy of CHG 
at oropharyngeal S. aureus eradication and none in children. If CHG is found to be effective in children, 
strategies that incorporate oropharyngeal decolonization as part of a more global eradication strategy may 
be critical for S. aureus SSTI prevention. 
 
Few data exist on the efficacy of oropharyngeal decolonization of S. aureus 
 
Virtually all decolonization efforts to prevent S. aureus infections have focused on nasal and body 
decolonization.10 However, these efforts ignore S. aureus oropharyngeal colonization. In children with cystic 
fibrosis, a population at high risk for S. aureus infections, it has been noted that S. aureus persistence is 
more common in the oropharynx than the nares.54 A similar observation of higher S. aureus persistence in 
the pharynx compared to the nares was seen in adults and staff in an orthopedic ward.55 These data 
suggest that the oropharynx can be reservoir of S. aureus colonization. 
 
Traditional methods to eradicate S. aureus colonization in the skin and nose do not appear to 
eradicate oropharyngeal colonization. In a Dutch study, two hospital staff found to be MRSA colonized were 
given traditional topical (skin) and nasal decolonization regimens. However, this traditional regimen, similar 
to regimens used for eradication of children with S. aureus infections, did not eradicate pharyngeal S. 
aureus colonization.56 Only after systemic eradication with oral antibiotics was performed, were these 
persons’ throats decolonized. The authors suggested that MRSA throat (oropharyngeal) colonization might 
be a reason why MRSA eradication fails in otherwise healthy persons.56 Of note, systemic antibiotic use for 
S. aureus decolonization is limited by toxicity, tolerability, and collateral damage to healthy normal flora, 
which are disrupted by systemic antimicrobial therapy.57,58 Another study looked at predictors of failure of 
MRSA decolonization. In their multivariate they found that oropharyngeal MRSA carriage was an 
independent risk factor for decolonization failure.59 These findings further reinforce that oropharyngeal 
S. aureus carriage is associated with failure of decolonization if just skin and nares are 
decolonization targets. 
 
Our group is one of the few to conduct studies of oropharyngeal decolonization in S. aureus. The 
trial, called Project CLEAR,60 is an ongoing trial of decolonization of recently hospitalized adults with S. 
aureus infection and/or colonization. In this ongoing clinical trial of over 2,000 patients, we randomized 
patients to either standard of care (education) or decolonization with nasal mupirocin, topical CHG, and 


11 
 
 
 
0.12% oropharyngeal CHG oral rinse. Data from the first 976 subjects demonstrated that oropharyngeal 
colonization decreased over 50% (from a baseline of 21% to 10%) by 1 month with only a 22% to 19% 
decrease in the control group (P<0.05). (Figure 1) This data provides proof of principle that oropharyngeal 
S. aureus can be eradicated with 0.12% chlorhexidine. However data in other populations, especially 
children, are lacking. It is important to investigate S. aureus oropharyngeal eradication in children given 
results of studies in adults cannot simply be generalized or extrapolated to children, and adolescents.61 
 
 
Rapid diagnostic testing can identify 
extra-nasal S. aureus colonization  
 
To identify children and patients 
with oropharyngeal colonization who are to 
be target for decolonization, a simple and 
rapid identification system is required. 
Several rapid molecular diagnostic 
technologies to directly identify S. aureus, 
including MRSA, for the detection of S. 
aureus in oropharyngeal and nares 
specimens. Rapid molecular S. aureus tests 
employ real-time polymerase chain reaction 
(RT-PCR) technology that targets spa, 
mecA, and SCCmec genes found in MRSA. 
They have a turn-around-time of ~ 1 hour,62-
65 as opposed to 48-72 hours for culture-
based techniques commonly used in clinical 
microbiology laboratories. Test characteristics suggest these rapid molecular tests are of high clinical utility. 
Our group has used rapid molecular tests to identify patients with extra-nasal (inguinal) colonization.66 
Others have used rapid molecular tests for identifying S. aureus oropharyngeal colonization.49,64 
 
Whole genome sequencing has high level discriminatory power to distinguish small changes 
among S. aureus isolates  
 
With advances in sequencing technologies and the availability of large computing power and 
bioinformatics, whole genome sequencing (WGS) is becoming a method of choice to characterize emerging 
phenotypes and virulent subtypes of microbial pathogens. A comparative WGS can identify single 
nucleotide polymorphisms (SNPs), indels, small genetic rearrangements, and the acquisition and loss of 
mobile genetic elements in S. aureus strains associated with disease phenotypes.67 WGS can distinguish 
differences in strains that cannot be appreciated by commonly used methods such as pulse field gel 
electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing,68-70 PFGE, MLST, and spa 
typing have been used for decades as the standard for S. aureus molecular epidemiologic investigations.71 
However, these methods suffer from poor discrimination of S. aureus isolates, especially among community 
isolates found in the U.S. 
 
The discriminatory power of WGS is illustrated in studies of community-associated MRSA spread, in 
which the vast majority of isolates come from the USA300 background. These USA300 isolates, which form 
the vast majority of CA-MRSA strains in the U.S.6,40 are indistinguishable via traditional typing methods even 
among isolates that come from very distinct sources and differences in geography of hundreds or 
thousands of miles.6 WGS, however, is an extremely powerful tool that demonstrates inter-person spread 
and distinguishes differences in USA300 strains that cannot be appreciated by traditional methods.72-74 The 
high discriminatory power of WGS is increasingly used to investigate outbreaks of S. aureus and is rapidly 
becoming the method of choice for molecular epidemiologic investigations of S. aureus spread. There are 
no data however, investigating strain relatedness within patients who have recurrence of colonization or 
failures of decolonization regimens. Given the very high discriminatory power of WGS for S. aureus, and the 
observation that colonizing strains within a given individual even with low discriminatory ability are 
frequently different,18 we hypothesize that in patients using CHG oral rinses, isolates that are associated 
with recolonization after initial decolonization success will represent new strains or be related to nasal 
Figure 1. Figure 1: Efficacy of nasal mupirocin, topical 
(skin) chlorhexidine, and orophayngeal chlorhexidine 


12 
 
 
 
isolates from the same patient. No investigation has characterized isolates associated with treatment failure 
of decolonization regimens.  
In summary, oropharyngeal colonization is extremely common among patients with S. aureus 
infections and there are mounting data suggesting that decolonization regimens that fail to decolonize the 
oropharynx leave behind a critical reservoir of S. aureus. This reservoir can then serve as a persistent 
source of S. aureus colonization that can be the nidus for subsequent infections. Because recurrence rates 
of S. aureus skin infection are so high, there is an urgent need to study the efficacy of oropharyngeal 
decolonization regimens for S. aureus in children before they can be used in more comprehensive 
decolonization regimens.  	  
	  
Experimental	  Design	  and	  Methodology	  (See	  General	  Instructions	  for	  page	  
limitations/recommendations)	  
This investigation is a randomized, prospective, multi-center, placebo-controlled clinical trial to 
examine eradication of S. aureus from the oropharynx of children age 5-17 who recently experienced an 
SSTI and have proven oropharyngeal S. aureus colonization. We will compare eradication of oropharyngeal 
S. aureus colonization among those randomized to oral chlorhexidine (CHG) oral rinse compared to those 
randomized to placebo. Our investigative team has experience in S. aureus treatment and prevention, 
Pediatrics, Infectious Diseases, Emergency Medicine, Microbiology, Molecular Biology, Infectious Diseases 
Epidemiology, and Health Services Research. We have ample experience enrolling subjects in the Pediatric 
Emergency Department (PED) setting. Over the last 6 years, Dr. Miller’s group has enrolled over 3,500 
subjects into epidemiologic studies and clinical trials focused on infectious diseases, a third of which were 
children.9,17,18,75-85 Drs. Miller and Young have collaborated on a National Institutes of Health (NIH) multi-
center clinical trial comparing TMP-SMX and clindamycin for the treatment of skin and soft tissue infections 
in children and adults in the ambulatory care setting.86 Ms. Eells has worked with Dr. Miller for over seven 
years on multiple federally-sponsored investigations including clinical trials sponsored by the NIH, Centers 
for Disease Control and 
Prevention (CDC), and the 
Agency for Healthcare 
Research and Quality 
(AHRQ). 18,75,77-82 Starting 
early 2015, Dr. Young will 
lead a team of research 
assistants whose mission is 
to help identify patients who 
are eligible for clinical trials 
and connect them with the 
appropriate recruitment 
team. The study activities 
and milestones are outlined 
in Figure 2. 
 
 
Enrollment of a cohort of pediatric patients with S. aureus skin and soft tissue infection 
 
Enrollment 
We will enroll 240 subjects with a history of SSTI in the Pediatric Emergency Department (PED) at 
Harbor-UCLA Medical Center. Subjects who meet the inclusion and exclusion criteria outlined below will 
undergo rapid screening for oropharyngeal S. aureus colonization. Assuming a 25% oropharyngeal 
colonization prevalence,18 60 subjects will have oropharyngeal colonization with a S. aureus and be eligible 
for the trial’s decolonization regimen (Figure 3).  


13 
 
 
 
All patients who participate in the 
clinical trial will be followed for 28 days for 
the purpose of determining which 
subjects achieve and maintain 
eradication. Participants will be enrolled 
regardless of race, ethnicity, or gender. 
While younger children commonly suffer 
from SSTIs, for this investigation we will 
only enroll children 5 years of age and 
older, as younger children may not be 
able to gargle. In a pilot study of 64 
children from a general dentistry practice 
asked to gargle, among the 12 children 
ages 3-4, only 2 (17%) could complete a 
30 second gargle, whereas 51/52 (98%) 
of those ages 5-8 could gargle 
successfully (Mitra Evans, DDS, personal 
correspondence).  
 To recruit subjects, study 
coordinators will screen the PED three 
times daily. During screening, they will 
query PED physicians, and nursing staff about potentially interested patients (e.g., those with a history of 
SSTIs) whose parents/guardians are willing to discuss possible participation in a study. PED staff will also 
be in-serviced about the study and encouraged by our PED physician Co-Investigator to call study 
coordinators as needed. To complement these efforts, flyers will be placed in the PED advertising to 
medical staff to call or page study coordinators about potentially eligible patients. These strategies have 
been highly successfully at each of the sites for enrolling subjects with SSTIs in the PED for our previous 
investigations. Once an interested patient is identified, the study coordinator will complete the informed 
consent process in a private setting in the patient’s parent/guardian preferred language (English or 
Spanish). 
 
Inclusion and Exclusion criteria 
For this investigation, we will use strict inclusion and exclusion criteria. Inclusion criteria will include: 
a) History of skin or soft tissue infection, as per patient report 
b) Age > 5 years and < 18 years. 
c) Able to gargle (if needed, ability to gargle can be assessed at screening using water). 
d) Willing and able to undergo nares and oropharyngeal swabbing. 
e) Able to come to the research clinic for study follow-up visits for the study period. 
 
Exclusion criteria for subjects will include: 
a) Suspected or confirmed infection requiring systemic antibiotics. 
b) Receipt of systemic antibiotics in the prior 28 days. 
c) Plans for administration or likely receipt of systemic antibiotics in the next 28 days (e.g., if the patient 
suffers from recurrent infections such as otitis media or has a planned surgery that requires 
prophylactic antibiotics). 
d) Plans for hospitalization or likely hospitalization in the next 28 days (e.g., if the patient suffers from 
recurrent infections). 
e) Any of the following in the prior 6 months: hemodialysis, peritoneal dialysis, central venous catheter 
placement, and systemic chemotherapy for cancer, any immunecompromising condition. These 
criteria ensure that subjects enrolled with have CA-S. aureus infections and are likely colonized with 
USA300 S. aureus, the most common cause of SSTIs in the U.S.  
f) Previous participation in the study. 
 
 


14 
 
 
 
Baseline visit 
 
Once a patient consents to study participation, study coordinators will obtain a specimen from the 
subject’s nares, and oropharynx for both rapid testing and standard culture based testing. A research 
assistant will then process the swab for rapid molecular testing (GeneXpert MRSA/SA platform) to 
determine if the purulent fluid has MRSA, MSSA, or neither. The sensitivity and specificity of this FDA 
approved assay in the nares are 97 and 96%, respectively;65 sensitivity for throat specimens is 75%-81% 
with a very high sensitivity of 98%.87,88 The specimen will be processed in the Miller Laboratory on the 
Harbor-UCLA Medical Center campus, which is a <5 minute walk from the PED. Rapid molecular testing 
results will be available within one hour and conveyed to the subject. Participants who are negative for S. 
aureus oropharyngeal colonization will be told they are not eligible for the intervention and doscontinued 
from the study.  
Participants who are positive for S. aureus oropharyngeal colonization will be allowed to participate 
in the trial and data collection will commence. Of note, participants who are rapid test positive for S. aureus 
and later using clinical microbiology lab testing with culture-based methods are found to be negative for S. 
aureus will not be included in the analysis, although given the very high sensitivity of the GeneXpert assay 
in throat specimens (98%), we think this is an unlikely event87 and these events are built into our expected 
attrition rate (see below). For ethical purposes, all subjects, regardless of rapid testing results, will receive 
educational materials regarding optimal hygienic practices to reduce risk of transmission, basic education 
about S. aureus, SSTI, and recognition of warning signs of serious skin infections. These materials were 
developed for previous investigations and available in English and Spanish.  
 Based on previous investigations of patients with a history of skin infection from our group,18 we 
estimate 33% of children with a history of SSTIs will be positive for S. aureus (n=80, Figure 2). Assuming a 
75% sensitivity of the GeneXpert assay, and 75% of those participants (n=60) will be positive at the 
oropharynx (see section on Power and Sample size, below). Study coordinators will complete the baseline 
visit for participants who are S. aureus positive on rapid molecular testing. 
Subjects will also be swabbed for S. aureus and MRSA colonization at the anterior nares and 
oropharynx. Testing will be performed using two methods: direct microbiology cultures and rapid molecular 
testing. Microbiologic cultures will be performed by culturing swabs from sites taken to identify S. aureus 
and MRSA carriage will be transported to the research microbiology laboratory at LA BioMed at Harbor-
UCLA and plated within 24 hours of collection. Swabs will be processed for S. aureus using blood agar and 
CHROMagar plates for rapid identification of S. aureus and MRSA. All S. aureus and MRSA isolates will be 
banked for pulse-field typing, determination of SCCmec type, and presence of the PVL locus (lukF-PV and 
lukS-PV). Rapid molecular testing will be performed using the GeneXpert MRSA/SA platform, which is 
currently available at our site.  
Upon confirmation of eligibility, the study coordinators will call the pharmacy to randomize the 
participant and obtain the oral rinse. The randomization assignment list will be generated in advance by 
pharmacy. Randomization procedures by an assignment list will be prepared by a computer method. To 
ensure adequate balance of subjects between the CHG and placebo groups, treatment allocation will be 
assigned using a random number generation with randomization of blocks of four subjects each. The 
randomization list will be kept by the pharmacy and treatment allocation randomization will not be shared 
with either the study staff of patients. The research pharmacists will dispense the appropriate oral rinse to 
the participant and their parent/guardian. Placebo will be created using the inert vehicles components of 
CHG, as has been done previously.89 Participants and their parent/guardian will be given detailed 
instructions on the use of the oral rinse both verbally and in a handout in either English or Spanish as 
preferred by the participant and their parent/guardian. 
Subjects randomized to oral CHG will be provided verbal and written instruction on use of CHG oral 
rinse. Subjects will be asked to use CHG oral rinse twice daily for 7 days. Subjects will be educated to rinse 
their mouth and gargle with CHG oral rinse for 60 seconds twice a day. Subjects will be instructed to use a 
watch or other timing device (e.g., smartphone) to ensure that at least 60 seconds have elapses. Research 
staff will demonstrate the technique for all subjects using drinking water. All subjects randomized to the 
CHG arm will be provided a new soft bristle toothbrush to prevent recontamination of the oropharynx with S. 
aureus from a contaminated toothbrush. Subjects randomized to usual care will not be provided with any of 
the above. Regardless of treatment allocation, study staff will educate all subjects’ parent/guardian, and 
subject (if of sufficient age) with the clinical significance of the results of their positive test for oropharyngeal 


15 
 
 
 
S. aureus colonization. More specifically, they will be educated that currently testing for S. aureus 
oropharyngeal colonization is considered experimental and that if the test is positive, the clinical significance 
is unclear. They will also be educated that tens of millions, perhaps over 100 million Americans are 
colonized in the throat with S. aureus, and the vast majority do not suffer consequences of having this 
bacteria reside in the throat. Upon request, subjects will be provided with a written copy of their results as 
well as a written description of the clinical significance of oropharyngeal colonization that they can share 
with their provider, if they wish. All subjects, regardless of treatment allocation, will be provided written and 
verbal information about S. aureus and skin infection prevention and recognition of early disease. These 
materials were developed for previous studies done by our group.18 
Of note, we debated whether the control group not receiving CHG should receive no therapy or a 
placebo oral rinse. Obviously, placebo controlled trials are of stronger study design compared to non-
placebo controlled studies. More specifically, in non-placebo studies, both patients and investigators will 
know the treatment allocation, which can lead to bias by both subjects and the investigative team. We did 
have concern that oral rinses/gargling with placebo or an inert substance may affect S. aureus colonization. 
However, a 1970 investigation examined this issue using quantitative techniques. Investigators asked 
subjects to gargle with saline and took serial quantitative cultures. They found that gargling specimens 
taken at 5 minute intervals for 25 minutes showed no significant decrease in organisms concentration, with 
mean organism concentrations at baseline of 7.11, 6.85 at 5 minutes, and 6.92 at 25 minutes.90 This 
suggests that placebo will have no ability to decolonize oropharyngeal S. aureus from study participants.  
At the baseline visit, study coordinators will also administer a pediatric S. aureus risk factor survey 
similar to surveys used for previous S. aureus and MRSA epidemiologic investigations by our 
group.9,17,18,76,80,84,85 Items from this survey have been based on careful reviews of the literature on S. aureus 
risk factors and have incorporated items used previously by the CDC, Health Departments, and other 
investigations examining MRSA risk.80,84 The baseline survey will collect information about demographics, 
medical history, antibiotic use, and behavioral risk factors for S. aureus infection and colonization. 
Participants and their parent/guardian will be given phone numbers of the study coordinator for questions. 
Participants and their parent/guardian will be compensated for their time for this and all study visits. 
 
Follow-up visits 
 
Participants will be asked to come to the research clinic 7 days after initiation of oral CHG, i.e., at the 
end of their treatment (Visit 2). A subsequent visit will occur at 28 days (Visit 3). The purpose of these visits 
is to ascertain oropharyngeal eradication of S. aureus both short term (primary study outcome) and longer 
term. Based on previous investigations conducted at our center(s), we estimate a 10% attrition 
rate.75,77,78,81,82 At Visit 2, participants will be swabbed the nares and oropharynx for the presence of S. 
aureus and to complete a follow-up survey about any recurrent infections, new medical history, or receipt of 
antibiotics. At Visit 3, participants will return to the research clinic to complete a follow-up survey. Study staff 
will also call all subjects remind them of their upcoming study appointments and at days 14 and 21 to 
address any questions the participant or their parent/guardian may have about the study. Adverse events 
will be measured at each scheduled and unscheduled (see below) follow-up visits. Adverse events will be 
measured using the Medical Dictionary for Regulatory Activities Terminology (MedDRA) version 14.0 or 
later terms.  
 
Unscheduled visits 
 
If participants or their parent/guardian experience potential adverse drug events that occur during 
the follow up period, subjects or their parent/guardian will be encouraged to contact study staff. If the event 
may be of clinical concern, the event will be referred to a study physician and the oral rinse discontinued, if 
applicable. If deemed necessary by the study physician or the subjects’ primary physician, participants will 
be asked to return to the research clinic for evaluation by a study investigator. 
 
Aim 1: Determine the efficacy of oropharyngeal decolonization among children suffering from CA-
MRSA and other S. aureus skin infections 
To determine if oropharyngeal S. aureus colonization can be eradicated, we will perform a prospective, 
double blind randomized controlled clinical trial of 0.12% oral chlorhexidine (CHG) oral rinse versus placebo 
for children age 5-17 with S. aureus oropharyngeal colonization. We hypothesize that 0.12% CHG rinse will 


16 
 
 
 
reduce oral S. aureus colonization better than placebo treatment. The findings from this Aim will provide 
“proof of principle” data on the ability of oral antiseptics to eradicate oropharyngeal S. aureus colonization in 
children. 
 
Power and Sample Size 
Using an alpha of 0.05 and expected cure rates of 50% and 10% in the CHG and placebo arms 
respectively, 54 subjects (27 per group) would be needed for 85% power to detect a 40% absolute 
difference in cure (p<0.05) between groups.  Correcting for a 10% attrition rate, 60 enrolled subjects would 
be needed. To identify 60 subjects with oropharyngeal colonization, and assuming 33% of patients who are 
high risk for S. aureus colonization will have oropharyngeal colonization, 240 subjects will need to be 
screened to obtain 80 patients that are oropharyngeally colonized with S. aureus. Of these, 75% (n=60) will 
have S. aureus detected by the GeneXpert rapid test and will undergo randomization. 
 
Data Analyses and Data and Safety Monitoring 
The decolonization rate will be calculated as the number of subjects who remain decolonized divided 
by the number of subjects, and treatment group differences as differences in these rates. The precision of 
estimated rates and rate differences will be expressed with two-sided confidence intervals calculated as 
asymptotically normal approximations to binomial or differences in binomial random variables. The level of 
confidence for these intervals will be 95%. Statistical significance for differences between treatment groups 
in cure rates will be determined from Fisher’s exact tests. The level of significance for these tests is 0.05. 
Factors associated with failure of decolonization in the CHG treatment group will also be examined. Based 
on prior investigations of S. aureus colonization from our group and others, these will include demographics 
(age, gender, race/ethnicity), co-morbidities, measures of hygiene, number of prior skin infections in the 
prior year, antibiotic use in the prior year, household history of skin infection, adverse effects and tolerability 
(see Aim 2, below), and treatment adherence. Additionally, we will examine pathogen level predictors of 
decolonization failure, based on molecular characterization of baseline isolates (see Aim 3). These factors 
will include baseline colonization with USA300 strain, presence of qac A/B gene, presence of nasal 
colonization, presence of nasal colonization with a strain identical to the oropharyngeal colonizing strain, 
and presence of nasal colonization with a strain differing from the oropharyngeal colonizing strain. 
Associations will be examined using Chi-squared, Fisher Exact test, Wilcoxon rank sum test, or T-test, as 
appropriate. Small sample size will preclude multivariable analyses of factors independently associated with 
failure to decolonize. This analysis will be key to identifying factors associated with decolonization failures. 
Identification of mutable factors associated with decolonization failure will be key towards improving the 
success of future efforts at oropharyngeal eradication of S. aureus. Identification of non-mutable factors will 
be key towards identifying groups that may need more intense intervention and or education.  
 
Given this investigation is minimal risk, Data and Safety Monitoring (DSM) will be performed by the 
Investigative Team. DSM will be performed according to standards of The Eunice Kennedy Shriver National 
Institute of Child Health and Human Development (NICHD) Policy for Data & Safety Monitoring.91 These will 
include reviewing monthly summaries of the number of patients enrolled, age, ethnic and gender makeup, 
Number of patients retained in the study, number of dropout and lost to follow up, adverse events (AEs), 
and serious adverse events (SAEs) (See Aim 2, below for further details). Subjects that report treatment 
related adverse events will discontinue the oral rinse and come to the research clinic for an unscheduled 
visit. 
 
Aim 2: Assess the safety, tolerability, and compliance of oropharyngeal decolonization among 
children and their caregivers. We will administer a detailed and systematic survey of side effects, 
tolerability and compliance of the oropharyngeal decolonization regimen to determine the effects, concerns, 
and usability of this regimen in children for both the child participants and their parents/caregivers. Findings 
will identify adverse effects and barriers to use to be considered when possibly administering this regimen in 
the future. 
Data Analyses 
Adverse events will be coded to a Medical Dictionary for Regulatory Activities Terminology 
(MedDRA) version 14.0 or later terms.  Verbatim description and the MedDRA System Organ Class and 
Preferred Term for all adverse events will be contained in the subject data listings. A separate listing sorted 


17 
 
 
 
by MedDRA System Organ Class and Preferred Term will include all verbatim descriptions associated with 
the Preferred Terms. 
Data for adverse events will be analyzed using the treatment-emergent signs and symptoms philosophy.   
 
Treatment emergent signs and symptoms are defined as adverse events where: 
• 
onset occurs during exposure to study medication or within 7 days after the last dose of study 
medication, having been absent prior to receiving study medication, or  
• 
onset reoccurs during exposure to study medication or within 7 days after the last dose of study 
medication, having been present but stopping prior to receiving study medication, or 
• 
worsening in severity occurs during exposure to study medication relative to the pre-treatment state, 
when the adverse event is continuous. 
 
All reported adverse events (regardless of treatment-emergent or not) will be included in a by-subject 
adverse event listing. Only treatment-emergent adverse events will be included in summary tables. The 
number of subjects (incidence) of treatment-emergent adverse events will be presented as well as the 
frequency of all adverse events reported. 
 
Summary tables of adverse events will include the following: 
• 
Summary of adverse events by severity and relationship 
• 
Summary of related adverse events 
• 
Summary of Serious adverse events 
• 
A listing of any on-study pregnancies 
 
Percentages of subjects who reported adverse events will each be compared separately among each group 
using Chi-Square or Fisher Exact tests, as appropriate. 
 
Aim 3: Determine the S. aureus genetic backgrounds associated with breakthrough oropharyngeal 
colonization.  
Advances in sequencing technologies and the availability of large computing power and bioinformatics have 
allowed WGS to become the method of choice to characterize emerging phenotypes.72 WGS also allows 
examination of the relationship between presence of the qac A/B gene in S. aureus at baseline and 
treatment response. WGS allows detection of this plasmid-based resistance gene for comparative analysis 
among a subset of S. aureus isolates from our population, as described below. 
We will molecularly characterize all baseline S. aureus isolates and “breakthrough” (failure) isolates 
using whole genome sequencing (WGS) for identification of breakthrough S. aureus isolates. We will 
compare breakthrough isolates with baseline nasal and orophangyeal isolates to determine if breakthrough 
isolates represent either: a) recolonization with the original oropharyngeal isolate, b) recolonization with the 
baseline nasal isolate, or c) acquisition of a new S. aureus strain. Additionally, we will investigate whether S. 
aureus associated with treatment failures have acquired or possess the qac A/B gene.  
 
Sample Size 
Data Analyses 
WGS will be done in the laboratory of Barry Kreiswirth, PhD (see Letter of Support), using 
established techniques.92,93 Dr. Kresiworh’s lab has ample experience performing WGS on S. aureus and 
other bacterial pathogens.92-94 Dr. Kreiswirth’s lab will also supply analytic support to assist with 
interpretation of findings.  
WGS sequencing will addresses several hypothesis in those subjects randomized to CHG oral 
rinses, specifically: a) strains that are found in the oropharynx at end of treatment (Day 7) will be identical 
genetically to those at baseline and not represent acquisition of new S. aureus strain; b) recolonizing S. 
aureus strains at Day 28 among subjects successfully eradicated at Day 7 will represent (re)colonization 
with the original isolate that colonized the nasopharynx from the same individual (if present at baseline) or a 
new S. aureus strain not present at baseline and; c) nasal isolates found at baseline and follow up will be 
identical to synchronously identified oropharyngeal S. aureus isolates.  


18 
 
 
 
Based on data in older adults,19 we estimate that of the 30 subjects randomized to the CHG arm, 
50% will be decolonized, leaving 15 subjects who will be S. aureus positive at the oropharynx at the end of 
treatment. Based on data of recolonization after decolonization in which recolonization can occur after the 
decolonization regimen is completed,10 we estimate that a 10% increase in colonization will be seen at Day 
28, i.e., 18 subjects will be oropharyngeally colonized with S. aureus at Day 28. Thus, we anticipate we will 
have results in 33 pairs of oropharynx isolates (15 between baseline and Day 7, and 18 between baseline 
and Day 28, for a total of 66 isolates total). Additionally we will conduct whole genome sequencing on 
baseline nasal S. aureus isolates associated with the subsequent oropharynx isolates identified at Day 7 
and/or Day 28. Based on previous data suggesting that ~45% of those colonized at the oropharynx will be 
colonized at the anterior nares,18,46,95 we estimate an additional 30 (45% x 66) nasal S. aureus isolates from 
baseline and follow-up will be present synchronously with oropharyngeal isolates.  
In total 96 isolates will undergo whole genome analysis to address the hypotheses outlined above. 
Specifically, we will examine if decolonization failure of the oropharynx is due to any of the following: 1) a 
new and distinct S. aureus strain; 2) an isolate found at the nares at baseline; 3) an isolate found in the 
oropharynx at baseline; or 4) an identical strain from baseline but that has acquired with new resistance 
genes to CHG, specifically qac A/B. WGS typing of nasopharyngeal S. aureus isolates also allows us to 
determine if “breakthrough” oropharyngeal strains at Day 7 and/or 28 arise from endogenous or exogenous 
sources. Given that oropharyngeal and nasal isolates taken from the same person at the same time, are not 
always genetically identical,18 one cannot assume that baseline strains will be concordant. Finally testing of 
nasal oropharyngeal and nasal S. aureus at baseline and the Day 7 and 28 follow up visits, can assist in 
determination if resistance genes to CHG, specifically qac A/B, emerged after treatment in non-
oropharyngeal, specifically nares sites, as it is known that antimicrobial therapy is associated with collateral 
damage among strains not specifically targeted by that treatment.37 I.e., oral CHG may result in the 
emergence of qac A/B containing S. aureus strain in the nares. Analytically, the above analyses will be 
descriptive thus there are no power calculations for this aim.   
 
Limitations 
 
This investigation is limited as it is a single center study. However, our center serves a ethnically and 
socioeconomically diverse population and findings from our investigations have been comparable to similar 
investigations in other populations.18,96,97 Our intervention is also singular in decolonization efforts 
(oropharynx only). However, no investigation has looked at the effect of S. aureus eradication on the 
oropharynx in children, so the addition of other decolonizing agents would prevent us from determining the 
efficacy of CHG oral rinse. Additionally, our trial is a “proof of principle” study that is required prior to a more 
comprehensive trial on S. aureus decolonization as a means for SSTI prevention. Finally, we are not 
performing WGS typing on all colonizing S. aureus isolates at all study time points, as it is known that S. 
aureus can colonize not only the nares and oropharynx, but the axilla, inguinal fold, perirectal area, vagina, 
and areas of skin that have lost integrity.42,47,98,99 However, the extra cost of testing these sites for possible 
sources of S. aureus that can later recolonize the oropharynx would be extremely expensive. Thus we 
chose just to perform testing in sites in which S. aureus is most commonly found (nares, oropharynx). 
 
Future Directions 
Investigation on prevention of recurrent S. aureus SSTI have been extremely disappointing, 
suggesting more comprehensive decolonization regimens need to be developed, especially those that focus 
on sites of colonization not previously targeted in clinical trials. Our investigation will be the first study to 
examine the efficacy of CHG to eradicate S. aureus from the oropharynx of children. Dr. Miller and his team 
provide ample experience in the area of MRSA infection and colonization, infectious disease expertise, 
clinical trials, and pediatrics. Besides the aims addressed for this investigation, our team is poised to 
address future aims related to S. aureus decolonization efforts and SSTI recurrences by generating 
information on the efficacy of CHG. In summary, the results of this investigation will provide the critical 
foundation for the development of future clinical trials. Data generated from our investigation will further 
investigative work on prevention of recurrent S. aureus SSTIs, which remain an extremely common and 
vexing clinical problem for clinicians, and more importantly, patients. 
 
 


19 
 
 
 
Human	  Subjects/Investigational	  New	  Drug	  (See	  General	  Instructions	  for	  page	  
limitations/recommendations)	  
For this investigation, Harbor-UCLA Medical Center pediatric emergency department providers (MD, 
NPs) will refer patients agreeable to being approached by research coordinators about the study. If patient 
and their guardian are agreeable, research staff will approach patients and initiate the informed consent 
process. If the patient and/or their parent or guardian consent and the patient is eligible, the patient will 
undergo the first part of the study (oropharyngeal screening for S. aureus, which will be evaluated using the 
GeneXpert platform). Study staff will explain the study to the potential subject’s parent or guardian. The 
informed consent process will be conducted by a study team member in their preferred language (English or 
Spanish). Written informed consent will be sought, obtained, and documented by a research coordinator 
and the site’s PI using a form approved by the institution’s IRB. Subject’s who are capable will participate in 
the informed consent process and those 7-12 will be asked to sign an assent form. Subject’s older than 12 
will co-sign the consent form with their parent/legal guardian. The informed consent process will explicitly 
state that if the oropharyngeal screening for S. aureus is positive, they agree to participate in the clinical trial 
and that if the oropharyngeal screening for S. aureus is negative, they are not eligible for the clinical trial. 
All subjects who have consented will have the rapid S. aureus test performed with results shared 
with the subject’s parent/guardian, the subject, and the clinician, if approved by the parent/guardian. Study 
staff will educate and the subject’s parent/guardian, and subject (if of sufficient age), the clinical significance 
of the results, either positive or negative. More specifically, they will be educated that currently testing for S. 
aureus oropharyngeal colonization is considered experimental and that if the test is positive, the clinical 
significance is unclear. They will also be educated that tens of millions, perhaps over 100 million Americans 
are colonized in the throat with S. aureus, and the vast majority do not suffer consequences of having this 
bacteria live in the throat. Upon request, subjects will be provided with a written copy of their results as well 
as a written description of the clinical significance of oropharyngeal colonization that they can share with 
their provider, if they wish.  
Subjects with a positive screening test will undergo 3 study visits total: baseline/enrollment visit (Visit 
1), Day 7 (Visit 2), and Day 28 (Visit 3). Consenting subjects will have their anterior nares and oropharynx 
swabbed at Visits 1, 2, and 3. A survey will be administered at the initial visit and a follow-up survey will be 
administered at Visits 2, and 3. The survey administered at Visit 1 will collect information about 
demographics, medical history, and behavioral risk factors for skin and soft tissue infection and colonization. 
Subjects will be randomized to 0.12% CHG oral rinse or placebo use for 7 days. All subjects will receive 
educational materials regarding S. aureus and skin infections and their prevention. Those randomized to the 
intervention will receive additional verbal and written information on use of CHG oral rinse. The survey 
administered at Visit 2 will collect information about the adherence, tolerability, and adverse events during 
the intervention period. The survey administered at Visit 3 will collect information about (late) adverse 
events, changes in household dynamics and individual behavior (e.g., hygiene) that may affect results of 
Visit 3 nasal and oropharyngeal colonization studies.  
Patients at Harbor-UCLA Medical Center come from a diverse racial and ethnic background, with 
approximately 51% Hispanic, 26% Caucasian, 18% African-American, and 6% “other” (largely Asian and to 
a lesser extent, mixed race). Two hundred and forty patients are anticipated to be enrolled over 18 months 
to randomize 60 subjects. Subjects constitute a cross-section of pediatric patients either presenting to our 
health care facilities that had a history of a skin and soft tissue infection, and will thus consist of pediatric 
patients age 5-17 and both genders. 
There are risks to subject confidentiality, which will be mitigated by all PHI information being kept at 
the individual site with secure locked filing cabinets and/or on a password-protected computer file. This 
likelihood of subject confidentiality being broken is minimal. Risks to confidentiality will be protected by 
keeping all study data in a locked office with access restricted to study personnel. All identifiers will be 
removed and destroyed when data is logged into an Access database. We will protect against psychological 
risk to the patient by including an educational discussion with the patient including the nature of bacterial 
colonization, the risks, current treatments, the lack of acceptance of routine treatments, and the ability to 
attempt treatment in the case of recurrent infections. If a subject suffers psychological harm, they may 
contact the Principal Investigator and they will discuss further options. Subjects will be told that their 
information will not be shared with any persons except for study personnel unless the subject provides 


20 
 
 
 
permission. Finally, subjects will be told they can choose not to participate in the study or end their 
participation at any time without any effect on the care they will receive from their treating physicians or at 
their treating institutions. 
 
Risks associated with study medication is believed to be minimal. Chlorhexidine-gluconate (CHG) 
oral rinse is an FDA-approved medication for the treatment of gingivitis. The known side effects of oral CHG 
oral rinse include staining of teeth and other oral surfaces, an increase in calculus formation; and an 
alteration in taste perception.100 All of these potential side effects are transient, reversible, or preventable 
with education and standard oral hygienic practices (e.g., tooth brushing).  All subjects will be educated on 
the proper use of the oral rinse. Subjects will be asked to report any adverse events to study personnel and 
to stop the use of the oral rinse if any severe side effects develop (swelling, pain, etc.). Staff will also be 
able to counsel subjects about the risks and benefits of the oral rinse to decolonize the oropharynx. Study 
personnel will also have cell phones, which subjects can call to ask questions about the study, use of the 
intervention oral rinse, and report adverse events. Subjects that report serious adverse events or other 
medical needs will be referred to the Harbor-UCLA Medical Centers’ Pediatric Emergency Department.   
 
An Investigational New Drug (IND) application will not be required, as a clinical investigation of a 
marketed drug is exempt from IND requirements if all of the criteria for an exemption in § 312.2(b) are met: 
 
• 
The drug product is lawfully marketed in the United States. 
• 
The investigation is not intended to be reported to FDA as a well-controlled study in support of a new 
indication and there is no intent to use it to support any other significant change in the labeling of the 
drug. 
• 
In the case of a prescription drug, the investigation is not intended to support a significant change in the 
advertising for the drug. 
• 
The investigation does not involve a route of administration, dose, patient population, or other factor that 
significantly increases the risk (or decreases the acceptability of the risk) associated with the use of the 
drug product (21 CFR 312.2(b)(1)(iii)).  
• 
The investigation is conducted in compliance with the requirements for review by an IRB (21 CFR part 
56) and with the requirements for informed consent (21 CFR part 50). 
• 
The investigation is conducted in compliance with the requirements of § 312.7 (i.e., the investigation is 
not intended to promote or commercialize the drug product).  
 
Because the use of 0.12% CHG oral rinse in this investigation meets all of the above criteria for required 
for an IND exemption.  
 
Data and safety monitoring will be conducted as described in Aim 2. 
 


21 
 
 
 
 
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