JOURNAL OF CLINICAL MICROBIOLOGY, June 1992, p. 1529-15340095-1137/92/061529-06$02.00/0Copyright © 1992, American Society for Microbiology

National Hospital Survey of Anaerobic Culture and SusceptibilityTesting Methods: Results and Recommendations for Improvement

ELLIE J. C. GOLDSTEIN,l12* DIANE M. CITRON,' AND RONALD J. GOLDMAN" 3

R. M. Alden Research Laboratory, Santa Monica Hospital Medical Center, Santa Monica, California 904041;UCLA School of Medicine, Los Angeles, California 900732; and Department of Political Science,

University of Southern California, Los Angeles, California 900893

Received 3 February 1992/Accepted 23 March 1992

The methods for performing anaerobic bacterial isolation and identification continue to change and improve.Anaerobic susceptibility testing has become controversial, and method-dependent variability has been noted.To assess the status of clinical anaerobic bacteriology in the United States, we surveyed, by means of aquestionnaire, 120 hospitals, selected at random, with bed capacities of 200 to 1,000, and we received responsesfrom 88 (73%). All hospitals performed cultures for anaerobes. The media and methods used for transport,initial processing, incubation, and identification varied between the different regions in the United States.Thirty percent of laboratories did not perform susceptibility studies, 16% used a reference laboratory, and54% performed them in house. For half the laboratories, susceptibility testing was performed on isolatesdepending on the source; in this case, blood cultures were tested by 97% of the laboratories, serious infectionswere tested by 60%, sterile body sites were tested by 73%, pure cultures were tested by 47%, and tests weredone by physician request by 39%o. For laboratories doing testing, the broth disk method, no longer sanctionedby the National Committee for Clinical Laboratory Standards, was used most often (56%), followed bymicrodilution (33%), P-lactamase testing (25%), macrotube dilution (2%), and agar dilution (2%). Theantimicrobial agents tested were as follows: penicillin-ampicillin, 94%; clindamycin, 94%o, metronidazole,90%o; chloramphenicol, 80%o; cefoxitin, 76%; tetracyclines, 51%; and erythromycin, 45%. All other agentswere tested by <25% of laboratories; the methods used could be improved to make the results more timely andconsequently more clinically relevant.

Anaerobic bacteria are important human pathogens andcause a wide variety of human infections (12). Technologicaladvances have resulted in improved methods for transport,identification, and susceptibility testing of anaerobes (8,10-12, 24, 30, 31). However, diminishing reimbursement andlimited financial resources of hospitals have led to theimplementation of cost-cutting strategies, including changesin the procedures and activities of the clinical microbiologylaboratory. Anaerobic bacteriology has been consideredlabor intensive and requires a higher level of expertise thanother laboratory activities. These practical considerations,coupled with scientific problems and controversies in anaer-obic bacteriology and susceptibility testing, have resulted ina microbiologist's dilemma and the feeling that "it is notrealistic or rational for a microbiologist in a nonteachinghospital to do detailed anaerobic bacteriologic studies androutine anaerobic susceptibility testing" (11). This statementhas been misinterpreted and has led some laboratories toabandon anaerobic bacteriology and susceptibility testingentirely. When performed, the results of anaerobic identifi-cation and susceptibility studies are often returned to thechart long after there is any potential for clinical relevance.This results in a situation in which clinicians are faced withthe need to choose antibiotics for antianaerobic therapy onthe basis of literature surveys. Several studies (17, 34) havenoted that the susceptibility results, especially with resistantisolates or "infections involving strains with MICs near thebreakpoint" (34), appear to predict therapeutic failure.Bourgault et al. (5) noted that the prognosis and outcome ofpatients' anaerobic infections were improved when physi-

* Corresponding author.

cians utilized specific anaerobic susceptibility data that wereprovided within a clinically relevant time frame. Conse-quently, there has been a clinical need, even among non-teaching hospital physicians, to have an individual patient'ssusceptibility data available for the specific anaerobic bac-teria isolated despite all the related problems and controver-sies (1, 3, 7, 11, 14, 32, 33).

Clinical microbiology laboratories vary in their capacitiesto isolate and identify anaerobes. To assess these differ-ences, we performed a national survey to determine whatmethods are in use for the isolation, identification, andsusceptibility testing of anaerobic bacteria in relatively largenonteaching hospitals across the United States. As thesurvey was performed in the spring and summer of 1990, thethen-in-force Methods forAntimicrobial Susceptibility Test-ing ofAnaerobic Bacteria standard (24) was used as a basisfor some of the questions.

MATERIALS AND METHODS

In the spring of 1990, a sample of 120 hospital microbiol-ogy laboratories was randomly selected from the AmericanHospital Association's Guide to the Health Care Field, 1989edition (2). The sample was limited to general medicine andsurgical hospitals with capacities between 200 and 1,000beds. To test for regional differences, a stratified samplingprocedure was utilized, with 20 hospitals selected from eachof six geographical areas (Northeast, Mid-Atlantic, South,Great Lakes, Central, and Western regions). The person incharge of the microbiology laboratory (or reference labora-tory used by the hospital) was identified by telephone. Athree-page questionnaire and self-addressed stamped enve-lope were sent to the identified person. The questionnaire

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was made up by the authors and consisted of 14 questionsregarding elements of anaerobic bacteriology practice in thelaboratory (see Results for specific questions). If a supervi-sor declined participation in the study, a random selection ofanother hospital in that region was made and the sameprocedure was followed. Follow-up phone calls to the super-visors were made 1 month after sending the questionnaire torequest cooperation for completion of the mailing. Statisticalanalysis was performed by one of us (R.J.G.). The chi-square test was used to determine the significance of regionaland other differences.

RESULTS

Of the 120 hospital laboratorians contacted, 6 declinedparticipation in the study because of workload consider-ations or hospital policy and 1 was eliminated because thelaboratorian used the same reference laboratory as anotherhospital in the sample. These seven were replaced throughthe random selection of another hospital in that region. Ofthe 120 participants, 88 (73%) returned completed question-naires. There was equal compliance in all geographic re-gions.

Questions and answers. Questions and answers in thesurvey were as follows.

(i) "Do you process specimens for anaerobes in your micro-biology laboratory?" All respondents in the survey proc-essed anaerobic specimens. Eighty (91%) processed thecultures in-house, and eight (9%) utilized outside referencelaboratories.

(ii) "How many anaerobic specimens are processed eachmonth (excluding blood cultures)?" Twenty-eight percent oflaboratories processed <50 specimens per month; 25% proc-essed 50 to 99 specimens per month; 24% processed 100 to199 specimens per month; 14% processed 200 to 299 speci-mens per month; and 9% processed >300 specimens permonth.

(iii) "What specimen transport system(s) is/are used?"Ninety-six percent of laboratories used a swab system, and27% used a vial system. All but two of the vial system usersalso used a swab technique, and one laboratory usedchopped meat glucose broth. Seven percent of the respond-ents used a bifunctional system consisting of a tube contain-ing prereduced anaerobically sterilized Cary and Blair me-dium with a Hungate screw cap which accommodates bothaspirates and swabs; these laboratories were counted as bothvial and swab users.

Regional differences were found in the use of both tech-niques. Swab use was 100% in all the regions except theNortheastern region (77%), where 3 of 13 laboratories didnot report usage (X = 16.99, P < 0.01). Almost all bifunc-tional system users (five of six) were from the Westernregion. Vial use was highest in the West (32%) and Northeast(27%), compared to the South (14%), Great Lakes (14%),Mid-Atlantic (9%), and Central (5%) regions (X = 11.85, P <

0.05).(iv) "What blood agar base do you use to isolate anaer-

obes?" The most commonly used agar base was CDC (51%),followed by brucella agar (27%), Columbia agar (13%), brainheart infusion agar (6%), Trypticase soy agar (6%), andSchaedler agar (5%). CDC agar base use was highest in theMid-Atlantic (77%), Southern (67%), Northeastern (57%),and Great Lakes (56%) regions, compared to the Central(23%) and Western (21%) regions (X = 14.28, P < 0.02).Brucella agar was used more often in the Western region(71%) compared to <30% use in the other regions (X = 18.16,

TABLE 1. Use of selective media for primary isolation ofanaerobic bacteria by geographic region

Regions (no. of % of laboratories using medium'laboratories)' LKV PEA BBE CNA

Northeast (13) 85 46 0 31Mid-Atlantic (13) 85 54 0 8South (14) 79 43 7 21Great Lakes (14) 50 43 29 29Central (11) 100 45 27 9West (14) 86 71 57 17

an = 79; one questionnaire did not identify location.* LKV, laked blood, kanamycin vancomycin agar; PEA, phenylethylalco-

hol agar; BBE, bacteroides bile esculin agar; CNA, colistin nalidixic acidagar.

P < 0.01). Three institutions used more than one agar baseroutinely.

(v) "Do you use selective media for primary isolation? If so,which ones?" Ninety-one percent (80 of 88) of laboratoriesused selective media for primary isolation. Of those labora-tories, kanamycin vancomycin laked blood agar (LKV) wasused by 80%, phenylethylalcohol agar (PEA) was used by57%, bacteroides bile esculin agar (BBE) was used by 20%,and colistin nalidixic acid agar (CNA) was used by 19%. Twolaboratories used Neomycin agar, and one used paromomy-cin-vancomycin agar.

Forty-one percent of laboratories used both LKV andPEA, and an additional 11% used LKV, PEA, and BBE.LKV was frequently used across all regions (79 to 100%),except in the Great Lakes, where usage was 50% (X = 11.17,P < 0.05). BBE was used most often in the Western region(X = 20.82, P < 0.001) (Table 1).

(vi) "Which of the following do you use and how long do youincubate the primary plates before initial examination?"Anaerobic jars with gas generators were the most popularsystem for incubation (58%) among this sample. Anaerobicpouches were used by 47% of respondents, an anaerobicchamber was used by 31%, and gas evacuation-replacementjars were used by 6%. Most laboratories (68%) used only onesystem, but 27% used two systems and 4% used three ormore systems. Holding jars or tanks were used by 14% oflaboratories.Anaerobic chambers were used more often in the Central

region (54%) and Great Lakes Region (50%) than in the West(36%), South (20%), Northeast (14%), and Mid-Atlantic (8%)regions (X = 12.33, P < 0.05). Many laboratories (56%) usinganaerobic chambers examined their primary plates after 24 hof incubation, whereas laboratories using other systemsexamined them after 48 h. Specific results are presented inTable 2.

(vii) "If the primary plates appear negative, how long arethey incubated before discarding?" One-third of the labora-tories discarded the primary plates after 48 h. An additional24% of the laboratories discarded them after 72 h, 39%discarded them after 4 to 6 days, and 3% discarded themafter 7 or more days.

(viii) "What liquid back-up medium is used?" Thioglyco-late liquid medium was used by 74% of laboratories andchopped meat glucose was used by 26%, with 5% using bothmedia. Four laboratories reported use of other media: brainheart infusion agar with 1% agar (2), B-D peptone broth (1),and Schaedler agar supplemented with vitamin K1 and hemin(1).

(ix) "To what extent do you identify anaerobes?" Sixty-four

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TABLE 2. Length of incubation of anaerobic culture primaryisolation plates prior to examination in various systems

No. of incubation % of laboratories using systemdays Chambera Jars/Gb Jars/ER' Pouchesd

1 56 40 22 37 84 60 803 7 8 104 8 7

a Anaerobic chambers.b Anaerobic jars with gas generators.Anaerobic jars with the evacuation replacement method.

d Anaerobic pouches.

percent of the laboratories usually identified anaerobes tospecies level, 32% identified them to genus level, and 5%identified general groupings. A trend of negative correlation(not significant) was found between the more-exact identifi-cation and laboratory workload, as defined by the specimensprocessed and number of hospital beds (i.e., the lower theworkload, the more exact the identification).

(x) "Which anaerobes do you usually speciate and fromwhich sources?" Ninety-five percent of laboratories usuallyidentified all blood culture isolates to species level. Of the80% which usually identified any nonblood source isolates,97% would identify anaerobes from sterile body sites, 86%would identify from serious infections, and 81% wouldidentify pure culture isolates. As a write-in response, 11%noted they would identify isolates only if there were three orfewer organisms isolated from the culture.

Sixty-percent of laboratories identified anaerobes to spe-cies level for specific isolates. Of those laboratories, 91%made this kind of identification for Bacteroides fragilis, 72%did it for other B. fragilis group species, 68% did it forBacteroides or Prevotella species, 66% did it for Fusobac-terium species, 79% did it for Clostndium species, 45% did itfor gram-positive rods, 42% did it for gram-positive cocci,and 17% did it for other isolates.

(xi) "What method(s) do you use for identification?" Iden-tification methods were requested by brand names andclassified as carbohydrate fermentation kits, preformed en-zyme kits, and classical methods (e.g., gas-liquid chroma-tography, prereduced anaerobically sterilized biochemicals)(18). Most laboratories (75%) used preformed enzyme kitsfor identification, while 33% used carbohydrate fermentationkits and 7% used classical methods. In all, 78% used onlyone method within their laboratories, with 18% using twoand 5% using all three methods. Carbohydrate fermentationkits were used most often in the South (60%) and least oftenin the West (8%). There were no regional differences in therate of use of the preformed enzyme kits.

(xii) "Do you perform anaerobic susceptibility studies? Ifso, which isolates are tested?" Seventy percent of laborato-ries performed anaerobic susceptibility testing. Of these70%, 77% performed them in-house and 23% sent isolates toa reference laboratory.For 51% of the laboratories, testing of isolates depended

upon the specimen source. Of these laboratories, bloodculture isolates were tested by 97%, normally sterile bodysite isolates were tested by 73%, serious infection isolateswere tested by 60%, and pure culture isolates were tested by47%. Thirty-nine percent of laboratories volunteered theresponse that they conducted susceptibility testing onlyupon physician request.

Isolates routinely selected for susceptibility studies werethe B. fiagilis group (44%) and other Bacteroides species(24%). All other species were tested less often.

(xiii) "What method do you use for susceptibility testing?"Of laboratories doing susceptibility testing, most (56%) usedthe broth disk elution method. Other methods used includedbroth microdilution (33%), P-lactamase testing (25%), mac-rotube dilution (2%), and agar dilution (2%). Six percentstated they relied on published national surveys for suscep-tibility data.

(xiv) "Which antibiotics do you test against anaerobes?"The frequencies of testing of specific antibiotics were asfollows: penicillin or ampicillin, 94%; clindamycin, 94%;metronidazole, 90%; chloramphenicol, 80%; cefoxitin, 76%;tetracycline, 51%; erythromycin, 45%; carbenicillin, 25%;imipenem, 20%; ampicillin-sulbactam, 20%; piperacillin,18%; cefotetan, 16%; cefotaxime, 14%; ceftizoxime, 10%;mezlocillin, 8%; cephalothin, 8%; ticarcillin-clavulanic acid,4%; amoxicillin-clavulanic acid, 4%; ticarcillin, 4%; vanco-mycin, 2%; doxycycline, 2%; and trospectinomycin, 2%.

DISCUSSION

There has been a worldwide trend to standardize the fieldof clinical microbiology. Clinical anaerobic bacteriology,originally propelled by the increasing recognition of anaer-obes as important pathogens, has been less standardized.Inability to isolate anaerobes is due, in part, to inadequatemethods of collection and transport, medium variability (20),cumbersome and sometimes ill-defined identificationschema, and the lack of agreement about susceptibilitystudies (1, 7, 14, 31-33). Final reports frequently reach thepatient's chart and the clinician well after the informationhas ceased to be clinically relevant. It is no wonder, then,that in the face of cost containment measures imposed by thenew economic realities, interest in clinical anaerobic bacte-riology has declined. Even the utility of obtaining routineanaerobic blood cultures from febrile patients has beenquestioned (21, 27). One possible remedy to potential regres-sion is to assess the methods currently in use in order toconsider methods to improve clinical utility of anaerobiccultures.Our survey found a broad spectrum of methods employed

in every phase of clinical anaerobic bacteriology and notedsome regional variations. It appears, however, that mosthospitals have some capacity at various levels for isolation,identification, and susceptibility testing of anaerobes. Allrespondents processed anaerobic cultures, with -50% re-ceiving > 100 anaerobic cultures per month, excluding bloodcultures.

Studies from the 1970s (15, 22, 26, 29) compared a varietyof agar media for the recovery of anaerobic bacteria fromeither clinical specimens or stock clinical isolates. Sheepblood, MacConkey, and PEA plates are recommended as theminimum set to be used for aerobic culture of the primaryspecimen (30). For primary isolation of obligate anaerobes,use of the following media has been suggested: brucella agar,supplemented with 5% sheep blood, vitamin K1, and hemin,for general isolation; BBE for selection and presumptiveidentification of the B. fragilis group isolates; LKV for theselection of pigmented and other Bacteroides and Prevotellaspecies; and PEA for inhibition of facultative gram-negativebacilli (30). The wide variety of media used to recover andgrow anaerobes in our study was surprising. CDC blood agarwas generally the most commonly used medium, especiallyin the Mid-Atlantic, Southern, Northeastern, and Central

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regions, despite its reported lesser ability to grow Prevotellaspecies (28). Brucella agar was commonly used in theWestern region, probably because of the influence of theWadsworth Anaerobic Bacteriology Manual (30). Brucellaagar was chosen by the Wadsworth group because of overallsuperiority in supporting growth of a variety of anaerobicbacteria, including peptostreptococci and fusobacteria.Studies of the variability of growth of clinical anaerobicbacteria on a variety of media noted that agar freshnessappeared to be more critical than the agar base used (15, 22).Schaedler agar with colistin and nalidixic acid (CNA) failedto support growth of more than half of the anaerobes tested(29), yet 19% of laboratories we surveyed used CNA. Evenwhen a single agar base is used, there may be markedvariation because of freshness of the agar or even bymanufacturer. Mangels and Douglas (20) studied four differ-ent commercially prepared brucella blood agar plates anddemonstrated a vast variation between the products. Thecurrent National Committee for Clinical Laboratory Stan-dards guidelines for quality assurance of commercially pre-pared media for anaerobes (25) requires that blood agarplates support the growth of B. fragilis, Clostridium perfrin-gens, Peptostreptococcus anaerobius, Bacteroides levii, andFusobacterium nucleatum at the time of preparation, al-though no recommendation is made for shelf life assurancefor each lot of media prepared.Most laboratories used selective media for primary isola-

tions, especially LKV and PEA. However, despite thepossibility that use of BBE would allow more rapid presump-tive identification and therefore more rapid reporting of B.fragilis group isolates, which constitute one-third of allclinical isolates (13), only a minority (20%) used BBE.Again, regional differences were noted and BBE use wasgreatest in the Western region, perhaps reflecting the influ-ence of the Wadsworth Anaerobe Laboratory Manual (30).Anaerobic jars (56%) and pouches (47%) were used fre-quently by laboratories. The high use of anaerobic pouchesis surprising in this era of cost containment. The cost perplate for incubation is considerably higher for the pouchesthan for the other systems. An additional 31% of laboratoriesused anaerobic chambers and were more likely to examinethe plates earlier (24 h). Laboratories which used jars orpouches were more likely to examine plates after 48 h ofincubation. Consequently, the use of selective media, suchas BBE, and an anaerobic chamber should lead to theavailability of clinically relevant results of the presence ofthe B. fragilis group within 24 h. The marked regionalvariation showed more chamber use in the Central and GreatLakes areas. One-third of the laboratories discarded primaryisolation plates after 48 h of incubation if negative and mighttherefore miss more slowly growing isolates such as Porphy-romonas, Actinomyces, and some Prevotella species.The use of a liquid backup medium is standard procedure.

However, the efficacy of the medium is important not onlyfor the primary isolation of isolates, but also for storage ofisolates that may require future susceptibility studies. Thio-glycolate was the usual (74%) backup medium used. Only26% of laboratories used chopped meat glucose. Use ofthioglycolate for storage of isolates may present some prob-lems. Citron et al. (9) noted that 25% of pure cultures ofvarious anaerobes died after 2 weeks of storage in thiogly-colate broth. Chopped meat glucose maintained viability ofall strains tested for a minimum of 5 weeks. This wouldimply that laboratories that do not do routine anaerobicsusceptibility testing on clinical isolates but await a physi-cian request or the development of a clinical therapeutic

failure may not have the opportunity to recover the isolate ifit is stored in thioglycolate (9). This would place furtherreliance upon literature surveys rather than specific therapyeven when susceptibility studies are indicated (11, 30, 32,33).Brook (6) reported that anaerobes were isolated in 12% of

bacteremic patients; approximately half of the isolates wereclinically significant. Several studies (6, 12, 13) noted that B.fragilis was the most frequent anaerobic blood culture iso-late, followed by B. thetaiotaomicron. Almost all laborato-ries (95%) will attempt to identify blood culture isolates tospecies level. Fewer (80%) will attempt to identify nonbloodculture isolates to species level and will usually attempt to doso for normally sterile sites, pure-culture and serious-infec-tion isolates, and B. fragilis group species. Most laboratories(75%) use preformed enzyme kits to identify the isolates.Eleven percent of laboratories identify isolates only if threeor fewer anaerobes grow from a culture. Some will identifythe specimen results as normal flora or contaminated. Thiscan cause a clinical dilemma. Anaerobes are opportunisticpathogens of the normal flora and are typically part ofpolymicrobial infections. Finding three anaerobes in a spec-imen is to be expected and does not necessarily representcontamination unless a specimen was improperly collected.A more desirable approach would be to report the quantityand types of organisms present, identified to a general grouplevel by using simple tests, such as Gram stain and growthon selective media, etc.Most recent attention has been focused on anaerobic

susceptibility testing, and numerous articles have addressedthe different methodologies, results, and controversies sur-rounding this procedure (1, 7, 14, 31-33). In an era of limitedand diminishing resources, it has been suggested that theclinician can often "use a logical empiric approach totreatment of a patient" (11) and that "routine susceptibilitytesting of anaerobes is generally not required" (33) despite adiminishing ability to predict susceptibility patterns. Resis-tance of isolates to all agents, including clindamycin, met-ronidazole, imipenem, and 1-lactamase-inhibitor combina-tions, has been reported (2, 4, 19, 23, 33). If susceptibilityresults could be obtained in a clinically relevant time frame,the rate of therapeutic failures might be decreased (5).

Thirty percent of laboratories surveyed do not performanaerobic susceptibility studies, and of those that do, 77%perform these studies in-house. Isolates tested usually con-formed to those recommended by Swenson (31) and others(11, 24, 32), such as isolates from blood cultures andnormally sterile body sites. Surprisingly, only 47% of labo-ratories that perform susceptibility studies would performthem on pure-culture isolates and 39% wrote in that they willperform testing only after a physician request. The brothdisk method was the most commonly employed susceptibil-ity method (56%) despite reports of its unreliability forseveral drug-organism combinations (3). The current Na-tional Committee for Clinical Laboratory Standards docu-ment specifically recommends not using the broth diskmethod, so laboratories will have to seek alternatives. Wenoted a slight increase, from 26% in the Amsterdam andJones study (3) to 33% in our study, in the use of amicrodilution system. Additionally, 25% of laboratoriestested ,-lactamase production in isolates.The variety of antimicrobial agents tested was quite sur-

prising, with almost 50% of laboratories testing the activityof tetracycline and erythromycin, two agents that are rarelyused to treat anaerobic infections. The use of commerciallyprepared and therefore predetermined panels may be re-

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flected in these data, especially for agents tested by <30% ofthe laboratories. It would seem that newer methods that are

rapid, reliable, able to be used on an individual basis, limitedin selection of clinically relevant agents, and relativelyinexpensive are needed. One possible new method whichmeets these criteria is the E test, which can yield resultswithin 24 h for rapidly growing anaerobes, 1 day earlier thanbroth microdilution methods. Our laboratory evaluated theE test against 105 clinical anaerobic isolates and comparedMICs with those obtained by using the reference agar

dilution method with Wilkins-Chalgren and supplementedbrucella agars, and we found the E test to be reliable, as wellas easy to perform and read (10).

Despite the current economic environment, almost alllaboratories are still processing cultures for anaerobic bac-teria and a majority are performing susceptibility studies.However, it appears that the methods employed could beimproved considerably and allow presumptive identificationof many important pathogens at 24 h and susceptibility data24 h later. This would certainly make anaerobic bacteriologymore clinically relevant and should help improve patientcare.

ACKNOWLEDGMENTS

We thank Judee H. Knight and Alice E. Goldstein for variousforms of assistance.

This study was funded, in part, by grants from Merck Sharp &Dohme and The Maurice Goldstein Public Health Research Fund.

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