the clinical presentation of fusobacterium-positive and streptococcal-positive pharyngitis in a...

The Clinical Presentation of Fusobacterium-Positive andStreptococcal-Positive Pharyngitis in a University Health ClinicACross-sectional StudyRobert M. Centor, MD; T. Prescott Atkinson, MD, PhD; Amy E. Ratliff, MLS; Li Xiao, PhD; Donna M. Crabb, MT (ASCP);Carlos A. Estrada, MD, MS; Michael B. Faircloth, MD; Lisa Oestreich, DO; Jeremy Hatchett, MD; Walid Khalife, PhD; andKen B. Waites, MD

Background: Pharyngitis guidelines focus solely on group A-hemolytic streptococcal infection. European data suggest thatin patients aged 15 to 30 years, Fusobacterium necrophorumcauses at least 10% of cases of pharyngitis; however, few U.S.data exist.

Objective: To estimate the prevalence of F. necrophorum; My-coplasma pneumoniae; and group A and C/G -hemolytic strep-tococcal pharyngitis and to determine whether F. necrophorumpharyngitis clinically resembles group A -hemolytic streptococ-cal pharyngitis.

Design: Cross-sectional.

Setting: University student health clinic.

Patients: 312 students aged 15 to 30 years presenting to a stu-dent health clinic with an acute sore throat and 180 asymptom-atic students.

Measurements: Polymerase chain reaction testing from throatswabs to detect 4 species of bacteria and signs and symptomsused to calculate the Centor score.

Results: Fusobacterium necrophorum was detected in 20.5%of patients and 9.4% of asymptomatic students. Group A

-hemolytic streptococcus was detected in 10.3% of patientsand 1.1% of asymptomatic students. Group C/G -hemolyticstreptococcus was detected in 9.0% of patients and 3.9% ofasymptomatic students.Mycoplasma pneumoniae was detectedin 1.9% of patients and 0 asymptomatic students. Infection rateswith F. necrophorum, group A streptococcus, and group C/Gstreptococcus increased with higher Centor scores (P < 0.001).

Limitations: The study focused on a limited age group and tookplace at a single institution. Asymptomatic studentsrather thanseasonal control participantsand a convenience sample wereused.

Conclusion: Fusobacterium necrophorumpositive pharyngitisoccurs more frequently than group A -hemolytic streptococcalpositivepharyngitis in a student population, andF. necrophorumpositive pharyngitis clinically resembles streptococcalpharyngitis.

Primary Funding Source: University of Alabama at Birming-ham and the Justin E. Rodgers Foundation.

Ann Intern Med. 2015;162:241-247. doi:10.7326/M14-1305 www.annals.orgFor author afliations, see end of text.

Recent European studies have estimated that Fuso-bacterium necrophorum, an obligate anaerobicgram-negative bacillus, causes approximately 10% ofendemic pharyngitis in adolescents and young adults(13). Fusobacterium necrophorum is also the mostcommon cause of peritonsillar abscess in this agegroup and the primary cause of the Lemierre syndrome(4, 5), which starts a few days after the onset of a sorethroat. The syndrome includes suppurative internal jug-ular thrombophlebitis with subsequent metastatic in-fections (most commonly lung, brain, or joints). A re-view estimated that F. necrophorum causes at least80% of the cases of this syndrome (6). A 2009 analysisshowed that F. necrophorum pharyngitis caused moremorbidity and mortality than streptococcal pharyngitiscaused by Streptococcus pyogenes (group A) for thisage group (7).

Current pharyngitis management guidelines focusonly on the diagnosis of group A streptococcal pharyn-gitis. A recent Infectious Disease Society of Americaguideline, titled Diagnosis and Management of GroupA Streptococcal Pharyngitis (8), presents the rationalefor treating group A streptococcal pharyngitis (that is,decrease probability of acute rheumatic fever, de-crease suppurative complications [primarily peritonsil-

lar abscess], decrease the duration of symptoms,and decrease contagion). However, Streptococcusdysgalactiae subspecies equisimilis, which includes-hemolytic organisms expressing Lanceeld groupC/G polysaccharide antigens, also causes pharyngitis inthis age group (911). Antibiotics can decrease symp-tom duration in patients with pharyngitis caused bythese organisms (7). Group C streptococci also causeperitonsillar abscess (11).

Mycoplasma pneumoniae is also a cause of bacte-rial pharyngitis, mainly affecting children but also oc-curring in adults (12). As is the case for F. necrophorum,most recent information about the role of M. pneu-moniae in pharyngitis comes from pediatric studiesdone in Europe with little or no prevalence data fromthe United States. In constructing this study, we focusedon the adolescent or young adult age group (15 to 30years) because pharyngitis differs greatly in these pa-tients versus preadolescents (13). They have the high-est rates of both peritonsillar abscess and the Lemierre

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Editorial comment . . . . . . . . . . . . . . . . . . . . . . . . . 311

Annals of Internal Medicine ORIGINAL RESEARCH

2015 American College of Physicians 241

syndrome (5, 14), and it is known that group A strepto-coccus occurs less frequently than in younger children(13). Therefore, we designed a clinical study to address2 questions: How common is F. necrophorum com-pared with other agents of bacterial pharyngitis in per-sons aged 15 to 30 years? Is the clinical presentation ofF. necrophorum pharyngitis similar to streptococcalpharyngitis?

METHODSStudy Design

We developed and validated a real-time poly-merase chain reaction (PCR) assay for detecting F.necrophorum from throat swabs to address these majorquestions about the occurrence and presentation of F.necrophorum pharyngitis. We then used the PCR assayto sample university students seeking medical care forpharyngitis in an ambulatory care setting and a com-parison group of healthy students. In addition, wesought other potential agents of bacterial pharyngitis,including M. pneumoniae and groups A and C/G-hemolytic streptococci, using modications of previ-ously established real-time PCR assays for theseorganisms (15, 16). We also obtained clinical datato characterize the presentations of these bacterialpharyngitides.

Patient and Comparison PopulationsWe enrolled students aged 15 to 30 years seeking

medical care at a university student health clinic with achief symptom of sore throat between March 2013and March 2014. Potential study patients received aninformation sheet that clearly informed them that they

had a choice about whether to participate. On somebusy days, the physician and nursing staff did not enrollall patients. We estimate that approximately 10% to15% of patients either were not offered or declinedparticipation. We excluded patients currently receivingantibiotics or who had been prescribed antibioticswithin the previous 4 weeks. For comparison purposes,research physicians and staff collected throat swabsfrom a population of healthy students (aged 15 to 30years) who did not report any clinical manifestations ofpharyngitis; had no evidence of swollen tonsils, exu-date, or lymphadenopathy; and were not receiving an-tibiotics. Research staff recruited these students outsidethe student health site. Most students (91%) were re-cruited during 4 days (the rst week of April 2013) atthe medical school lecture halls.

Clinical DataClinical information that was obtained included his-

tory of fever; presence of cough; swollen, tender cervi-cal lymphadenopathy; and tonsillar exudates. We usedthese clinical indicators to calculate the Centor scoreand assessed whether that score, originally developedto predict the probability of streptococcal pharyngitis(17), would similarly stratify patients with pharyngitistesting positive for F. necrophorum or group C/G-hemolytic streptococcus. This study was performedwith approval from the University of Alabama at Bir-mingham Institutional Review Board for Human Use(Birmingham, Alabama). Informed consent was not re-quired because of the minimal risk involved.

Microbiological MethodsSpecimen Collection and Initial Processing

Plastic shaft polyester swabs were used for speci-men collection. The tongue was depressed with awooden tongue depressor, and the swab was rubbedover the tonsillar area and posterior pharynx. The swabwas then placed in a vial of phosphate-buffered saline(pH, 7.4) PCR transport buffer and swirled, and excessuid was extracted by pressing the swab against theinside of the vial. The swab was then removed from thespecimen and discarded. The solutions were stored at4 C for up to 24 hours before delivery to the researchlaboratory. We extracted DNA using the proteinase Kmethod, as previously described (18). In brief, approx-imately 400 L of specimen was centrifuged at 14 000xg for 20 minutes at 4 C, and pellet was digested with200 L of proteinase K lysis buffer (10 mM of Tris-HCl[pH, 8.0]; 100 mM of KCl; 2.5 mM of MgCl2; 0.5% ofTriton X-100; 0.5% of Tween 20; and 0.5 mg/mL of pro-teinase K) for 1 hour at 60 C. Proteinase K was theninactivated by incubation at 95 C for 5 minutes. In caseof inhibition of the PCR reaction, proteinase Kdigestedsamples were puried using QIAamp DNA Blood MiniKit (Qiagen). The processed DNA samples were storedat 80 C until analyzed by real-time PCR for F.necrophorum and groups A and C/G streptococci.

EDITORS' NOTES

Context

Fusobacterium necrophorummay cause peritonsillarabscess and life-threatening internal jugular thrombo-phlebitis. However, it is not known how commonly thisbacillus is seen in patients presenting with symptoms ofpharyngitis.

Contribution

This study of patients presenting with pharyngitis to astudent health clinic found that F. necrophorum waspresent more often than was group A -hemolytic strep-tococcus. Infection with either bacterium increased withincreasing Centor scores.

Caution

The study included a convenience sample and did nothave a matched, asymptomatic control group forcomparison.

Implication

Further study is warranted to evaluate testing and treat-ing F. necrophorum pharyngitis.

ORIGINAL RESEARCH Presentation of Bacterial Pharyngitis in a University Health Clinic

242 Annals of Internal Medicine Vol. 162 No. 4 17 February 2015 www.annals.org

Real-Time PCR Assay for F. necrophorumThe PCR method targeting a 254 base-pair frag-

ment of the F. necrophorum rpoB gene was adoptedfrom Aliyu and colleagues (19) with modications usingthe LightCycler 2.0 (Roche). The PCR reaction was car-ried out in a 20-L mixture containing 500 nM of eachprimer, 200 nM of each probe, 0.4 U of uracil-DNA gly-cosylase, and 4.00 L of LightCycler FastStart DNAMasterPLUS HybProbe buffer. Polymerase chain reactionconditions were as follows: uracil-DNA glycosylase di-gestion at 40 C for 10 minutes followed by FastStartDNA polymerase activation and DNA denaturation at95 C for 10 minutes; 50 cycles of amplication andquantication (denaturing at 95 C for 0 sec, annealingat 60 C for 5 seconds, and extension at 72 C for 15seconds); 1 melting curve cycle held at 95 C for 0 sec-onds, 45 C for 30 seconds, and 85 C for 0 secondswith continuous uorescence acquisition at a ramp rateof 0.1 C per second; and 1 nal cooling cycle of 40 Cfor 30 seconds. Polymerase chain reaction results wereanalyzed by LCS4 software (Roche). To calculate theanalytic sensitivity for the real-time PCR assay, serial1:10 dilutions of F. necrophorum subsp. necrophorum(ATCC 25286) were made from a saline mixture (Mc-Farland standard 1.0) of colonies grown anaerobicallyon trypticase soy agar with 5% sheep blood. The num-ber of colony forming units (CFUs) of the original mix-ture was measured by incubating 100 L of each dilu-tion on blood agar plates under anaerobic conditionsand counting the discrete colonies. The calculated bac-terial concentration in the original culture mixture was6.75 107 CFU/mL. The DNA was prepared using 200L of each dilution by the proteinase K digestionmethod, as mentioned previously. Polymerase chain re-action was then performed on each dilution in triplicateand a standard curve was obtained. A total of 120 dif-ferent F. necrophorum reference strains and clinical iso-lates, as well as other bacteria that may be related ge-netically or known to coexist with Fusobacterium in theupper respiratory tract, was tested to determine the an-alytic specicity of the assay (Appendix Table 1, avail-able at www.annals.org).

Real-Time PCR Assay forM. pneumoniaeDetection of M. pneumoniae in extracted DNA was

performed by real-time PCR using an assay that wasdeveloped and validated in our laboratory with theLightCycler 2.0 on the basis of a method originally de-scribed by Dumke and colleagues (15), targeting an89-bp fragment of the repMp1 noncoding DNA se-quence present in several copies in the genome. Thisassay has been shown previously in our laboratory todetect 0.45 CFU/mL of specimen and is known to bespecic for detection of M. pneumoniae.

Real-Time PCR Assay for -Hemolytic StreptococciExtracted DNA from patients and asymptomatic

students were shipped frozen to the Department ofMolecular Diagnostics at Sparrow Health System (Lan-sing, Michigan) for testing by real-time PCR using the

LightCycler 2.0 for the presence of -hemolytic strepto-coccus, according to a method described previously byUhl and colleagues (16). The gene target of this assay isa 198-bp fragment of the ptsI gene of group A strepto-coccus. This PCR assay can distinguish this organismfrom group C/G streptococcus, for which there is a 1base-pair difference from the gene sequence of ptsI.However, the assay does not distinguish between thelatter 2 organisms.

Statistical AnalysisWe calculated descriptive statistics for the preva-

lence of each bacterial pathogen detected in patientsand asymptomatic students using real-time PCR. Wedened nonbacterial as patients with negative PCRtests for F. necrophorum; M. pneumoniae; and groupsA and C/G streptococci. To begin to estimate the car-riage rate of F. necrophorum within this community, westandardized the prevalence in a convenience sampleof students without symptoms of pharyngitis (describedpreviously) to the age and sex distribution of the pa-tients and found condence bounds for this estimate(dstdize command in Stata, version 11.2 [StataCorp]).For each patient, we calculated the Centor score byassigning 1 point each to a fever history; the lack of acough; swollen, tender anterior cervical adenopathy;and tonsillar exudates (17). We measured the strengthof the association between the Centor score and bac-terial species using the chi-square test for trend. Wethen dichotomized the Centor score as 0 to 1 and 2 to4, because Infectious Disease Society of Americaguidelines use the cutoff of 2 for clinical decisions (8).We also compared patients with positive PCR resultsfor F. necrophorum and those with negative PCR resultsfor F. necrophorum using the chi-square test. Stata wasused for all statistical analyses and a P value lessthan 0.05 was used to designate statistical signicance.No formal sample size or power calculations wereperformed.

Role of the Funding SourceThis study was funded by the University of Alabama

at Birmingham and the Justin E. Rodgers Foundation.The funding source did not participate in the study de-sign, data collection, analysis and interpretation of thedata, or the decision to submit the manuscript forpublication.

RESULTSPerformance Characteristics of Real-Time PCRAssay for F. necrophorum

The analytic sensitivity of the rpoB LightCycler 2.0assay using an F. necrophorum reference strain was de-termined to be 0.675 CFU/mL, meaning that this tech-nique can theoretically detect a single organism in athroat swab. Specicity of the assay was conrmed bythe lack of cross-reactivity with other Fusobacteriumspecies or other commensal and pathogenic oropha-ryngeal microorganisms that were examined (Appen-dix Table 1). Amplication occurred only in type spe-cies and clinical isolates of F. necrophorum, including

Presentation of Bacterial Pharyngitis in a University Health Clinic ORIGINALRESEARCH

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both subspecies (F. necrophorum subsp. funduliformeand F. necrophorum subsp. necrophorum).

Demographic Characteristics of Patients andAsymptomatic Students

The demographic characteristics of the patientsand asymptomatic students are provided in Table 1.There were substantially more women and studentsaged 15 years to younger than 20 years in the patientgroup.

Prevalence of Bacterial Species in Patients WithPharyngitis Versus Asymptomatic Students

The prevalence of each bacterial species in pa-tients with pharyngitis and asymptomatic students isshown in Table 2. Fusobacterium necrophorum was themost common organism detected in patients with phar-yngitis, occurring in 64 (20.5%), versus only 17 (9.4%) ofasymptomatic students. The adjusted estimated car-riage rate of F. necrophorum was 12.2% (95% CI, 3.3%to 21.1%). However, small numbers among the groupsof asymptomatic students in whom other bacterialspecies were identied precluded meaningful calcula-tions of standardized carriage rates. Fusobacteriumnecrophorum was present alone in 47 of these 64 pa-tients. In the remaining 17 patients, there were 9 simul-taneous infections with group A streptococcus; 7 withgroup C/G streptococcus; and 1 with group A and C/Gstreptococci and M. pneumoniae. Group A streptococ-cus was the second most common organism detected,occurring in 32 (10.3%) of patients versus only 2 (1.1%)asymptomatic students. This organism was the solepathogen detected in 21 patients and was found in as-sociation with other organisms in 11 patients. GroupC/G streptococcus was almost as common as group Astreptococcus, being detected in 28 (9.0%) patientscompared with 7 (3.9%) asymptomatic students. Theseorganisms were present alone in 19 patients and incombination with other bacteria in 9 patients. Myco-plasma pneumoniae was an uncommon agent of phar-yngitis in this age group, being detected in only 6 pa-

tients (1.9%), 2 of whom also had F. necrophorum orstreptococcus. Mycoplasma pneumoniae was not de-tected in any of the asymptomatic students. Thesesmall numbers preclude meaningful assessment of theclinical characteristics of M. pneumoniae pharyngitis inthis patient population. Overall, 110 of the 312 patientswith pharyngitis (35.3%) had positive results on PCR for1 or more of these bacterial species.

Application of the Centor Score for PharyngitisAccording to Bacterial Species

Table 3 shows Centor scores by bacterial species;higher scores were associated with bacterial species(P < 0.001). Appendix Table 2 (available at www.annals.org) shows Centor scores dichotomized at a score of 2by bacterial species. Patients with scores 2 to 4 werenearly twice as likely to have a bacterial pathogen (76of 169 [45.0%]) than those with scores 0 to 1 (34 of 143[23.8%]). Table 4 shows the individual components ofthe Centor score by bacterial species.

DISCUSSIONThese data document clearly that F. necrophorum

causes pharyngitis in persons aged 15 to 30 years. Wefound a carrier rate of 9.4% for F. necrophorum inasymptomatic students. We also found a similar per-centage of patients with positive results on PCR for F.necrophorum who had a Centor score of 0. A total of20.5% of our patients with pharyngitis had positive re-sults on PCR for F. necrophorum, meaning that approx-imately 11% of cases of pharyngitis in patients comingto this university health clinic were caused by F.necrophorum. Our nding that F. necrophorum was themost common bacterial agent of pharyngitis in this

Table 1. Demographic Characteristics of Patients andAsymptomatic Students*

Characteristic Patients(n 312)

AsymptomaticStudents(n 180)

Mean age (SD), y 22.3 (3.1) 24.1 (1.8)Age, n (%)1520 y 111 (35.6) 7 (3.9)2125 y 141 (45.2) 135 (75.0)2630 y 60 (19.2) 38 (21.1)

Women, n (%) 232 (74) 82 (46)Race, n (%)White 231 (74) 132 (73)Black 43 (14) 21 (12)Asian 21 (7) 19 (11)Hispanic 5 (2) 4 (2)Other or unknown 12 (4) 4 (2)

* Percentages may not sum to 100 due to rounding. Patients were students aged 15 to 30 y recruited between March2013 and March 2014. Asymptomatic students were recruited during 4 d in April 2013.

Table 2. Summary of Bacterial Species Identied inPatients and Asymptomatic Students*

Species Patients(n 312)

AsymptomaticStudents(n 180)

Fusobacterium necrophorum 64 (20.5) 17 (9.4)F. necrophorum alone 47 (15.1) 16 (8.9)F. necrophorum and GAS 9 (2.9) F. necrophorum and GCS/GGS 7 (2.2) 1 (0.6)F. necrophorum, GAS, andMycoplasma pneumoniae

1 (0.3)

GAS 32 (10.3) 2 (1.1)GAS alone 21 (6.7) 2 (1.1)GAS and GCS/GGS 1 (0.3)

GCS/GGS 28 (9.0) 7 (3.9)GCS/GGS alone 19 (6.1) 6 (3.3)GCS/GGS andM. pneumoniae 1 (0.3)

M. pneumoniae 6 (1.9) 0 (0)M. pneumoniae alone 4 (1.3)

None 202 (64.7) 155 (86.1)

GAS = group A -hemolytic streptococcus; GCS = group C -hemolytic streptococcus; GGS = group G -hemolytic streptococcus.* Values are numbers (percentages). Percentages may not sum to thetotals due to rounding. Data were missing for 1 patient.



population of young adults is consistent with previousreports from Europe (1, 2). We also saw that group C/Gstreptococcus was almost as common as group A strep-tococcus in young adults, consistent with a recent U.S.study (20). The data on M. pneumoniae from our pa-tient population do not provide evidence that this or-ganism is a common cause of adolescent or youngadult pharyngitis. This nding argues against diagnos-tic testing or including coverage for M. pneumoniae inempirical treatment strategies. Using the Centor scoreprediction model previously validated for group Astreptococcal pharyngitis (17), we found similar resultsfor F. necrophorum and groups A and C/G streptococ-cal pharyngitis. As the Centor score increased, wefound a trend for increased positive PCR testing for all3 bacteria. These results support the Centor score as apredictor of bacterial pharyngitis caused by F.necrophorum as well as nongroup A streptococcusrather than just group A streptococcal pharyngitis. Sim-ilar conclusions about the application of the Centorscore to nongroup A streptococcal pharyngitis werealso reported by Tiemstra and Miranda (20), but theydid not evaluate the occurrence of F. necrophorum.

How should we interpret these ndings and usethem for clinical decision making? United States guide-lines support prescribing antibiotics for known group Astreptococcal pharyngitis. However, no current guide-lines from any professional or governmental organiza-tion directly address management of nongroup Astreptococcal or F. necrophorum pharyngitis.

Throat cultures with specic Lanceeld typing canidentify group C/G pharyngitis. However, although thatstrategy is likely able to decrease suppurative compli-cations if antibiotic treatment is given based on a pos-itive culture, it may not lead to decreased symptom du-ration. This approach would require at least 1 to 2 daysto obtain results because no point-of-care tests cur-rently exist.

Routine throat cultures do not identify F. necropho-rum because it is an anaerobe requiring special mediaand incubation conditions. Although we developed aresearch PCR assay to detect and identify F. necropho-rum, we are not aware of any commercially availablerapid testing for this organism. We do not believe thatone could ethically do a randomized, controlled trial ofantibiotic therapy for F. necrophorum pharyngitis giventhe potential severity of the Lemierre syndrome. There-fore, we suspect that many physicians would prescribeantibiotics for patients with F. necrophorum pharyngitisif there were a point-of-care diagnostic test proving itspresence. Fortunately, F. necrophorum and all of the-hemolytic streptococci are usually susceptible topenicillin and rst-generation cephalosporin, althoughF. necrophorum is intrinsically resistant to gentamicin,and uoroquinolone and tetracycline have relativelypoor activity. Signicant erythromycin resistance inboth F. necrophorum and group A -hemolytic strepto-coccus has been reported during the last several years(2123). Despite in vitro susceptibility, there have beenreports of clinical failures of treating invasive F.

Table 3. Centor Scores, by Bacterial Species*

CentorScore

Bacterial Species, n (%) Total, n

Fusobacteriumnecrophorum

GAS GCS/GGS >1 Species None

0 4 (10) 2 (5) 0 (0) 0 (0) 33 (85) 391 12 (12) 5 (5) 6 (6) 5 (5) 76 (73) 1042 18 (17) 6 (6) 4 (4) 15 (14) 63 (59) 1063 6 (14) 6 (14) 6 (14) 1 (2) 25 (57) 444 7 (37) 2 (11) 3 (16) 2 (11) 5 (26) 19Total 47 (15) 21 (7) 19 (6) 23 (7) 202 (65) 312

GAS = group A -hemolytic streptococcus; GCS = group C -hemolytic streptococcus; GGS = group G -hemolytic streptococcus.* Percentages may not sum to 100 due to rounding. Data were missing for 1 patient. Includes Mycoplasma species.

Table 4. Centor Score Components, by Bacterial Species

Centor ScoreComponent

Bacterial Species, n (%) None(n 202)

Fusobacteriumnecrophorum(n 47)

GAS*(n 21)

GCS/GGS*(n 19)

>1 Species(n 23)

Fever 18 (38) 6 (29) 9 (47) 9 (39) 55 (27)Lack of cough 29 (62) 14 (67) 12 (63) 18 (78) 100 (50)Swollen, tender anterior cervicallymphadenopathy

31 (66) 15 (71) 16 (84) 16 (70) 115 (57)

Tonsillar exudates 16 (34) 8 (38) 7 (37) 3 (13) 27 (13)

GAS = group A -hemolytic streptococcus; GCS = group C -hemolytic streptococcus; GGS = group G -hemolytic streptococcus.* Data were missing for 1 patient. Includes Mycoplasma species.



necrophorum infections with penicillin, leading to somerecommendations that metronidazole may be a betteralternative for invasive, suppurative conditions, such asthe Lemierre syndrome (21).

Our data show that in patients with Centor scoresof 2 or higher, the probability of having group A or C/Gstreptococcal or F. necrophorum pharyngitis achievedthe 40% range; in those with a score of 4, more than70% had positive PCR results for at least 1 of thesebacteria. These data support avoiding macrolides forempirical treatment of adolescent or young adult phar-yngitis because most F. necrophorum are not sensitiveto macrolides. These data should stimulate furtherstudy and consideration of both testing and treatmentin patients aged 15 to 30 years presenting with acutesore throat.

Although the Lemierre syndrome is uncommon, itoccurs much more frequently in adolescents or youngadults than acute rheumatic fever in the United States(7). The Lemierre syndrome often causes long, complexhospitalizations (often including intensive care), sub-stantial subsequent morbidity, and an estimated mor-tality rate of 5% (7). No data on the efcacy of treatingF. necrophorum pharyngitis with a goal of markedly de-creasing the suppurative complications of peritonsillarabscess and the Lemierre syndrome currently exist. Wenote that 17 of the patients who were PCR-positive forF. necrophorum had more than 1 bacterial speciespresent, including group A and C/G streptococcus,with M. pneumoniae also present in 1 patient. It hasbeen postulated that simultaneous infection with otherbacteria or viruses represents a risk factor for F.necrophorum suppurative complications (2, 21). Thesendings deserve further investigation.

Limitations of this study include the following. ThePCR assay we used does not detect group F strepto-coccus, which may cause some cases of pharyngitis.Although we were able to demonstrate a likely bacte-rial cause of acute pharyngitis in many of our universitystudent population, we did not attempt to detect viralcauses of pharyngitis. Many patients who were PCR-negative for F. necrophorum or group A or C/G strep-tococcus likely had viral infections. Our data supportfurther study of adolescent or young adult pharyngitisdiagnosis and treatment, but our ndings and conclu-sions do not apply to preadolescents because theyrarely have either group C/G streptococcus or F.necrophorum (13). The design of this study to deter-mine the bacterial cause of pharyngitis using molecularmethods was limited to a single health care center ofcollege-aged students and did not include bacterialcultures, any type of follow-up component, or a deni-tive treatment regimen. These topics require furtherstudy.

These data show that the occurrence of F.necrophorum pharyngitis in university students aged15 to 30 years exceeds group A streptococcal pharyn-gitis. The clinical presentation for this bacterial pharyn-gitis resembles the clinical presentation of group Astreptococcal pharyngitis.

From Huntsville Regional Medical Campus, University of Ala-bama at Birmingham, and Birmingham Veterans Affairs Med-ical Center, Birmingham, Alabama, and Sparrow Hospital,Lansing, Michigan.

Disclaimer: The contents of this article are solely the respon-sibility of the authors and do not necessarily represent theofcial views of the U.S. Department of Veterans Affairs.

Acknowledgment: The authors thank the medical and nursingstaff of the University of Alabama at Birmingham StudentHealth Clinic for their assistance in participant enrollment andspecimen collection. They also thank the entire staff of theUniversity of Alabama at Birmingham Student Health andWellness Center for their hard work in contributing to datacollection.

Financial Support: By the University of Alabama at Birming-ham and the Justin E. Rodgers Foundation.

Disclosures: None. Forms can be viewed at www.acponline.org/authors/icmje/ConictOfInterestForms.do?msNum=M14-1305.

Reproducible Research Statement: Study protocol: Availablefrom Dr. Centor (e-mail, [email protected]). Statistical codeand data set: Available from Dr. Estrada (e-mail, [email protected]).

Requests for Single Reprints: Robert M. Centor, MD, Profes-sor, General Internal Medicine, Faculty Ofce Tower 720, Uni-versity of Alabama at Birmingham, 1530 3rd Avenue South,Birmingham, AL 35294-3407; e-mail, [email protected].

Current author addresses and author contributions are avail-able at www.annals.org.

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8. Shulman ST, Bisno AL, Clegg HW, Gerber MA, Kaplan EL, Lee G,et al; Infectious Diseases Society of America. Clinical practice guide-line for the diagnosis and management of group A streptococcalpharyngitis: 2012 update by the Infectious Diseases Society of Amer-ica. Clin Infect Dis. 2012;55:e86-102. [PMID: 22965026] doi:10.1093/cid/cis6299. Turner JC, Hayden FG, Lobo MC, Ramirez CE, Murren D. Epide-miologic evidence for Lanceeld group C beta-hemolytic strepto-cocci as a cause of exudative pharyngitis in college students. J ClinMicrobiol. 1997;35:1-4. [PMID: 8968872]10. Meier FA, Centor RM, Graham L Jr, Dalton HP. Clinical and mi-crobiological evidence for endemic pharyngitis among adults due togroup C streptococci. Arch Intern Med. 1990;150:825-9. [PMID:2327842]11. Brandt CM, Spellerberg B. Human infections due to Streptococ-cus dysgalactiae subspecies equisimilis. Clin Infect Dis. 2009;49:766-72. [PMID: 19635028] doi:10.1086/60508512. Waites KB, Atkinson TP. The role of Mycoplasma in upper respi-ratory infections. Curr Infect Dis Rep. 2009;11:198-206. [PMID:19366562]13.Mitchell MS, Sorrentino A, Centor RM. Adolescent pharyngitis: areview of bacterial causes. Clin Pediatr (Phila). 2011;50:1091-5.[PMID: 21646249] doi:10.1177/000992281140957114. Klug TE. Incidence and microbiology of peritonsillar abscess: theinuence of season, age, and gender. Eur J Clin Microbiol InfectDis. 2014;33:1163-7. [PMID: 24474247] doi:10.1007/s10096-014-2052-815. Dumke R, Schurwanz N, Lenz M, Schuppler M, Luck C, Jacobs E.Sensitive detection of Mycoplasma pneumoniae in human respira-tory tract samples by optimized real-time PCR approach. J Clin Mi-crobiol. 2007;45:2726-30. [PMID: 17537933]

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Current Author Addresses: Dr. Centor: Professor, General In-ternal Medicine, Faculty Ofce Tower 720, University of Ala-bama at Birmingham, 1530 3rd Avenue South, Birmingham,AL 35294-3407.Dr. Atkinson: Birmingham Children's Hospital, Children's ParkPlace M220, 1601 4th Avenue South, Birmingham, AL 35233.Ms. Ratliff, Ms. Crabb, and Dr. Waites: Diagnostic MycoplasmaLaboratory, 1720 2nd Avenue South, SHEL 407, Birmingham,AL 35294.Dr. Xiao: Diagnostic Mycoplasma Laboratory, 1720 2nd Ave-nue South, SHEL 476, Birmingham, AL 35294.Dr. Estrada: Division of General Internal Medicine, Universityof Alabama at Birmingham, 732 Faculty Ofce Tower, 51020th Street South, Birmingham, AL 35294-3407.Drs. Faircloth: University of Alabama at Birmingham StudentHealth and Wellness Center, 1714 9th Avenue South, Birming-ham, AL 35294.Dr. Oestreich: Assistant Professor, University of Alabama atBirmingham Student Health and Wellness Center, 1714 9thAvenue South, Birmingham, AL 35294.Dr. Hatchett: 159 Moss Stone Lane, Calera, AL 35040.Dr. Khalife: Department of Laboratories, Sparrow Hospital,1215 East Michigan Avenue, Lansing, MI 48909.

Author Contributions: Conception and design: R.M. Centor,T.P. Atkinson, J. Hatchett, K.B. Waites.Analysis and interpretation of the data: R.M. Centor, T.P.Atkinson, A.E. Ratliff, L. Xiao, D.M. Crabb, C.A. Estrada, W.Khalife, K.B. Waites.Drafting of the article: R.M. Centor, T.P. Atkinson, C.A.Estrada, W. Khalife, K.B. Waites.Critical revision of the article for important intellectual con-tent: R.M. Centor, T.P. Atkinson, A.E. Ratliff, L. Xiao, D.M.Crabb, C.A. Estrada, M.B. Faircloth, L. Oestreich, K.B. Waites.Final approval of the article: R.M. Centor, T.P. Atkinson, A.E.Ratliff, D.M. Crabb, C.A. Estrada, K.B. Waites.Statistical expertise: C.A. Estrada.Obtaining of funding: R.M. Centor.Administrative, technical, or logistic support: R.M. Centor, T.P.Atkinson, A.E. Ratliff, L. Xiao, D.M. Crabb, K.B. Waites.Collection and assembly of data: T.P. Atkinson, A.E. Ratliff, L.Xiao, D.M. Crabb, M.B. Faircloth, L. Oestreich, J. Hatchett, K.B.Waites.

Annals of Internal Medicine

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Appendix Table 1. Microorganisms Used to Evaluate theSpecicity of Fusobacterium necrophorum Using aReal-Time Polymerase Chain Reaction Assay

Acholeplasma laidlawii PG8Acholeplasma oculiAcinetobacter baumanniiAcinetobacter lwofiAcinetobacter speciesActinomyces odontolyticusAdenovirusArcanobacterium haemolyticumBacteroides fragilisBordetella pertussisBrevibacillus brevisBuffalopox virus BP4Burkholderia cepaciaCandida albicansCandida tropicalisChlamydophila (formerly Chlamydia) pneumoniaeCitrobacter braakiiCorynebacterium diphtheriaeCorynebacterium jeikeiumCorynebacterium pseudodiphtheriticumEikenella corrodensEnterobacter cloacae subsp. cloacaeEnterococcus faecalisEnterococcus faeciumEscherichia coliFusobacterium gonidiaformansFusobacterium mortiferumFusobacterium naviformeFusobacterium necrogenesFusobacterium necrophorumFusobacterium necrophorum subsp. funduliformeFusobacterium perfoetensFusobacterium polymorphumFusobacterium periodontiumFusobacterium russiiFusobacterium simiaeFusobacterium variumHaemophilus inuenzaeHaemophilus parainuenzaeHelicobacter pyloriHerpes simplex virus type 1Herpes simplex virus type 2Human genomic DNAKlebsiella pneumoniae subsp. pneumoniaeLactobacillus speciesLegionella pneumophilaListeria monocytogenesMicrococcus luteusMoraxella catarrhalisMorganella morganiiMycoplasma amphoriforme A39Mycoplasma arthritidis PG27Mycoplasma buccaleMycoplasma collisMycoplasma fauciumMycoplasma fermentans PG18Mycoplasma genitalium PG37Mycoplasma hominisMycoplasma hyorhinisMycoplasma lipophilumMycoplasma murisMycoplasma neurolyticumMycoplasma oraleMycoplasma penetransMycoplasma pirum BER p9Mycoplasma pneumoniaeM129Mycoplasma primatumMycoplasma pulmonis ASH3 PG34

Continued

Appendix Table 1Continued

Mycoplasma salivarium PG20Mycoplasma spermatophilumNeisseria gonorrhoeaeNeisseria meningitidisPaenibacillus polymyxaPasteurella speciesPeptostreptococcus anaerobiusPeptostreptococcus speciesPeptostreptococcus type APeptostreptococcus type BPorphyromonas gingivalisPrevotella melaninogenicaProteus mirabilisProteus vulgarisPseudomonas aeruginosaShewanella putrefaciensStaphylococcus aureusStaphylococcus aureus subsp. aureusStaphylococcus aureus subsp. aureus strain 328Staphylococcus capitisStaphylococcus epidermidisStaphylococcus haemolyticusStaphylococcus lugdunensisStaphylococcus pseudintermediusStaphylococcus saprophyticusStaphylococcus sciuriStaphylococcus simulansStaphylococcus warneriStaphylococcus xylosusStenotrophomonas maltophiliaStreptococcus agalactiaeStreptococcus anginosusStreptococcus canisStreptococcus dysgalactiae subsp. dysgalactiaeStreptococcus dysgalactiae subsp. equisimilisStreptococcus equi subsp. equiStreptococcus mutansStreptococcus oralisStreptococcus pneumoniaeStreptococcus pyogenesStreptococcus salivariusStreptococcus sanguisStreptococcus sobrinus (mutans group)Treponema denticolaUreaplasma parvum serovar 3U. urealyticum serovar 10Veillonella parvulaVeillonella species

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Appendix Table 2. Centor Scores Dichotomized at a Score of 2, by Bacterial Species

CentorScore

Bacterial Species, n (%) Total, n

Fusobacterium necrophorum GAS GCS/GGS >1 Species* None

01 16 (11) 7 (5) 6 (4) 5 (3) 109 (76) 14324 31 (18) 14 (8) 13 (8) 18 (11) 93 (55) 169Total 47 (15) 21 (7) 19 (6) 23 (7) 202 (65) 312

GAS = group A -hemolytic streptococcus; GCS = group C -hemolytic streptococcus; GGS = group G -hemolytic streptococcus.* Includes Mycoplasma species.

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