1045

Principles

and Pitfalls of Clinical Trials

Design* M.K.

Jeffcoat

Periodontitis and gingivitis trials can involve many complex experimental designs. The selection of a specific design and the details of the protocol can influence the magnitude of any effect observed, the generalizability of the results, and the clinical significance of the findings. The purpose of this paper is to review selected aspects of clinical trials including the overall clinical experimental design, controls, outcomes, sample size, and patient selection. Particular emphasis will be placed on Periodontitis trials. /Periodontol 1992; 63:1045-1051.

Key Words: Clinical trials; research design; experimental design.

THE HYPOTHESIS There is no single experimental design and protocol that is ideally suited to testing all potential agents or modes of therapy. Rather, the investigator must first be guided by the overall hypothesis underlying the rationale for the trial, taking into account the proposed mechanism of action of the mode of therapy, the planned target population, and the anticipated useful clinical effect. A frequently encountered pitfall is to fail to clearly define the aims of the study. For example, most efficacy studies for Periodontitis agents seek, at least in part, to determine whether or not a significant decrease in probing attachment loss or bone loss is associated with the test therapy. Such trials do not automatically lend themselves to answering all questions relating to periodontal diseases. The reason this may present difficulty is that a study to determine efficacy of a proposed agent, the specific bacterial pathogens responsible for periodontal disease, and the potential role of several gingival crevicular fluid cytokines as markers for periodontal disease activity is most difficult to design. Issues such as sampling strategy, frequency of sampling, and order of samples taken are key to the success of the second two aims, but may be more peripheral to the determination of the efficacy of the agent. Such a trial is not impossible to design and carry out, and certainly is important, but will necessarily be far more labor-intensive and expensive than a simpler efficacy trial. On the other hand, if the parties involved decide to plan the more complex trial, the additional aims must be considered in determining the experimental design and specific outcomes to be used for the trial. Clinical trials (i.e., controlled studies involving patients) may be performed for reasons other than determining the efficacy of an experimental mode of treatment. For exam-

'Department of Periodontics, University of Alabama School Birmingham, AL.

of Dentistry,

pie, administration of test agents is sometimes used to test hypotheses concerning the pathophysiology of Periodontitis. An example of this type of trial would be to use an antibiotic with known efficacy against bacteria x, measure

bacteria

before and after treatment with the antibiotic or and determine the effect on progressive Periodontitis. This type of trial is often designed to shed light on the role of bacteria in the pathogenesis of periodontal disease. This type of trial must be specifically designed to simultaneously provide evidence as to the pathogenic role of the test bacteria as well as to the efficacy of the antibiotic in slowing progressive Periodontitis.

placebo,

THE ISSUES OF PLACEBO AND THE HAWTHORNE EFFECT The double-blind placebo-controlled clinical trial is frequently performed in support of a New Drug Application (or a new indication for an existing drug) to be submitted to the Food and Drug Administration. The double-blind clinical trial requires that neither the patient nor the investigators have knowledge of the treatment regimen administered to each group of subjects. Furthermore, the investigator is not even aware which subjects comprise a single group. The double-blind clinical trial is to be distinguished from an open label trial in which the investigators and patients are administered a known agent. Such open label experiments are essentially large case report studies that lend early credence to the hypothesis under test. Nonetheless, such studies must be followed by double-blind placebo-controlled trials in order to assure that the observed effects are due to the active therapy and not due to participation in the trial itself. Patients frequently appear to improve merely from the effects of being placed in a clinical trial. This often occurs because patients may improve oral hygiene or compliance

J Periodontol 1046

December 1992

CLINICAL TRIALS DESIGN rx

1:

patient numbers 13 4 7 9

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(Supplement)

on

the

assigned regimen throughout the course of the study. with the treatment regimen as a result of the special attention or frequent examinations that often result from study participation. This phenomenon has been termed the Hawthorne effect. A double-blind trial is critical to controlling for the Hawthorne effect. Furthermore, patients who know they have been placed on an inactive regimen may not comply with the entire protocol as well as patients who know they are taking active agent. The double-blind design also serves to reduce investigator bias. For example, it is not unlikely that the many subjective indices used in Periodontitis and gingivitis trials could be affected by the natural and honest desire for successful treatment on the part of the investigators. The need for a placebo may seem obvious, but the formulation of an adequate placebo is not always a simple task. Agents that have a distinctive taste or smell or that stain the teeth may be difficult to duplicate in an inactive form. Side effects can also unblind the investigator as to the treatment regimen of a particular patient. Furthermore, the placebo itself must not affect the course of the disease. Fortunately, the double-blind nature of the controlled clinical trial often works in the investigator's favor. Often patients on the placebo regimen will report mild side effects detailed in the consent form so that the investigator cannot be sure of treatment assignments from patient complaints. ISSUES OF OVERALL EXPERIMENTAL DESIGN The experimental designs in the periodontal literature primarily fall into 4 main categories: 1) the randomized parallel design; 2) the cross-over design; 3) the split mouth design; and 4) the pretreatment period design. The randomized parallel design is illustrated in Figure 1. In this type of experiment all patients are entered into the study based on fixed inclusion and exclusion criteria and randomly assigned to either test or placebo groups. The difference in outcomes across groups determines any significant efficacy of the test mode of therapy. This design has been used in many clinical trials.1"6 It has the advantage of being simple and requiring minimal prior knowledge of the history of the course of the disease in each individual patient. This advantage is also a disadvantage. It is possible

stratification level 3: patient number rx 1 : rx 2: 2 4 5 1 3 6

Figure 2. The randomized parallel design with stratification. Patients are assigned to strata based on criteria defined by investigator. In this example stratification levels 1, 2, and 3 could be comprised ofpatients with probing attachment loss < 5 mm, 5 to 7 mm, and > 7 mm, respectively. Each stratum has its own randomized code. This design is intended to balance treatment groups on the basis of the stratification factor. that the randomization may result in many more patients with active sites in either the test group or placebo group. In an attempt to minimize this potentially damaging problem some investigators have utilized surrogate variables which are believed to be related to disease activity rather than disease activity itself. Examples of such variables, which can be assessed in a single examination and may put patients or sites at increased risk for disease activity, include 8 a history of bone loss;7 age or smoking;9"10 elevated crevicular fluid enzymes such as AST,11"12 beta glucuronidase,1314 collagenase1516 or elastase;17 bone scans;1819 ;22 presence of Prostaglandins;20 immunoglobulins;21 clinical putative periodontopathic bacteria;23"27 and routine measurements such as bleeding on probing.28 33 If the patients are randomized within blocks, the results of these tests may be used in an attempt to balance the test and control groups with respect to disease activity (Fig. 2). The cross-over design is illustrated in Figure 3. In this case all subjects receive both the test and control therapy at some time during the test period.34 38 The advantages of this design include the ability to use the patient as his own control thereby reducing some of the biologic variation inherent to clinical trials. In addition, the use of each patient as his own control is economical both in terms of financial savings and in terms of the power of the design allowing the study of fewer patients. The cross-over design is not appropriate to test all hypotheses. For example, in Periodontitis trials involving antibiotics, which are hypothesized to eliminate periodontopathic bacteria, it does not make sense to use a placebo after the active drug. Other Periodontitis agents, including antibiotics and non-steroidal anti-inflammatory drugs, have been shown to have either loss of effect over time or carry-over effect. In either case, the results of a cross-over design are nearly impossible to interpret. Cross-over designs have been most effectively utilized in gingivitis trials when the test therapy has not

Volume 63 Number 12

JEFFCOAT

Group 1 rx

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The cross-over design. Patients are randomly assigned to first receive either treatment 1 or 2 based on a random code. After a fixed period, the treatments are crossed and the groups now receive treatment 2 or 1, respectively.

treatment

period

Figure 5. The pre-treatment period design. Patients are examined repeatedly for a pre-treatment period and disease activity calculated. This calculated disease activity serves either as an enrollment criterion or is used to stratify patients by disease activity.

Figure 3.

patient

1

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Figure 4. The split mouth design. In this design sites, teeth, quadrants, or half mouths are randomly assigned to receive placebo or test treatments. been hypothesized to eliminate a specific periodontal pathogen and does not appear to have carry-over or loss of effect over time. Nonetheless, the possibility of cross-over effects must always be considered and wash-out periods may be employed to reduce the likelihood of such effects, especially when treatments with high substantivity are under

study.

split mouth design also takes advantage of using the patient as his own control.34'39"41 This design is illustrated in Figure 4. In this case, treatment or control regimens are randomly assigned to half mouths, quadrants, sextants, or when appropriate to the treatment, individually affected teeth. Like the cross-over design the split mouth design attempts The

to account for individual variation and to increase the power

of the experiment using the paired design. Recent literature, however, indicates that there may be some concerns with the use of split mouth designs.34'42 Clearly, such designs

appropriate for certain forms of therapy, such as systemic drugs. Of greater consequence is the more recent literature, both in human subjects and some older literature are not

in animal models, that indicates that if certain local treatments, such as surgery, may have distant effects through the entire mouth of the patient. Further research will continue to shed light on this important matter. For the present, however, split mouth designs have the most potential for utility in the assessment of therapy that has been shown to exert its effect solely on a local level. The data will need to be carefully analyzed to assure a lack of cross-mouth contamination. The pre-treatment period design attempts to address several of the concerns raised by previous designs. The pretreatment period design is illustrated in Figure 5. In this design each patient is studied for a fixed pre-treatment period and the outcome variables for the main efficacy portion of the study are measured on a repeated basis.43 44 Thus, the pre-treatment portion of the study will allow treatment and control groups to be balanced based on the prevalence and severity of progressive disease as determined by comparison of sequential examinations in addition to the usual indices measured in a single examination. Strengths of the pre-treatment period design include the ability to use the treated patient as his own control as well as in comparison to the matched control group. The main weakness of the pre-treatment period design lies in our lack of knowledge of the natural history of periodontal disease progression. Since the average duration of periods of disease activity is uncertain, it is not known if matching test and control groups on the basis of pre-treatment disease activity justifies the additional expense and effort required to do so. THE PATIENT POPULATION Few decisions are as important to the conduct and results of a clinical trial as the patient population. Careful attention to the inclusion and exclusion criteria is needed not only to assure the safety of the patients involved in the study but to make every effort to ensure a population that reflects the

J Periodontol 1048

CLINICAL TRIALS DESIGN

hypothesis being tested. The Proceedings of the World Workshop in Clinical Periodontics45 has defined the clinical appearance of several forms of Periodontitis including: 1) prepubertal Periodontitis; 2) localized or generalized forms of juvenile Periodontitis, 3) rapidly progressive Periodontitis; 4) adult Periodontitis; 5) Periodontitis associated with systemic disease; 6) necrotizing ulcerative Periodontitis; and 7) refractory Periodontitis. Special populations, such as patients with rapidly progressive Periodontitis or juvenile Periodontitis, can make especially interesting populations for study. This is particularly true with respect to the pathophysiology of the disease process and may make it easier

to detect treatment effects because the disease may be more rapidly progressive in these patients in comparison to adult Periodontitis patients. Nonetheless, care must be exercised if attempting to generalize the results of special populations to a target population composed of adult Periodontitis pa-

tients. Little is presently known about the validity of the use of early onset diseases as models for adult Periodontitis and it is therefore difficult to anticipate results in the adult population on the sole basis of studies performed in early onset or

rapidly progressive subjects.

OUTCOMES TO BE MEASURED There is little agreement in the periodontal community as to exactly what outcomes must be measured in a Periodontitis trial. According to a consensus report from the World Workshop in Clinical Periodontics, "the gold standard for determining the presence of active periodontal disease is represented by histologie evidence of periodontal tissue destruction. Unfortunately there is no satisfactory gold standard for the clinical diagnosis of periodontal disease."45 In the absence of this standard for progressive disease comparison, the results of sequential examinations are used to detect sites and patients with active periodontal breakdown.46"48 In general, physical manifestations of soft and hard tissue breakdown are used as the clinical evidence of progressive Periodontitis. Probing attachment levels made with a manual or electronic probe are used to track a measure of the soft tissue attachment.49"52 Radiographic measures including the Schei Ruler technique,53 direct measurement of bone loss in millimeters on bite-wing films,54 or measurements from computer-corrected films,55'56 and subtraction radiography19,57 59 are used to track the bone support. None of these methods is perfect and may result in either false-positive or false-negative tests. For example, an increase in gingival inflammation may increase probe penetration independent of a "true" change in the soft tissue attachment.60 This phenomenon would result in a falsepositive result. On the other hand, the use of interpretive radiography is a relatively crude tool that often does not register bone loss until 30% or more of the bone mineral has been lost. In this case false-negative results may be likely. The choice of both probing and radiographie methods is important because the choice may favor either false-

positive or negative results.

December 1992

(Supplement)

utilizing electronic probes and indicates that the use of these instrudigital radiography ments may improve the sensitivity of measurement, reduce the error associated with repeated measurement, and in some cases improve the accuracy of the measurement itself.61 Furthermore, the use of probes and radiographie techniques that measure in 0.1 to 0.2 mm can provide a nearly continuous measure of disease progression which facilitates analysis of the study. Furthermore, if the new measurement is accurate, the use of the more precise tool does not negate the ability to apply the more crude, but often utilized, clinical criteria for disease activity. For example, if 3.5 mm of attachment loss is observed using the Florida probe,1' it is a simple matter to categorize that site as active using a traditional 2 to 3 mm cut-off for disease activity frequently used with a manual probe. Likewise, the use of the more sensitive instrument may make the effect of truly active agents detectable in a shorter study period. This is because the placebo sites would not have to lose large amounts of bone or soft tissue attachment to be detectable, and smaller gains in attachment level or bone will be readily detectable. For better or for worse, however, the new techniques that appear to offer the best sensitivity and specificity come at a cost in terms of actual expense for the instrumentation required, time of examination, and expertise needed to produce the results. For example, a bite-wing radiograph may be interpreted in a minute or less, measurements of bone height from the same film may take 5 to 10 minutes, and state-of-the-art quantitative digital subtraction radiography could take 20 minutes. In the absence of excellent clinical standards for disease activity for Periodontitis, it is especially important that the outcomes measured match the hypothesis being tested and the proposed mechanisms of action of the mode of therapy under study. For example, an antimicrobial therapy may be postulated to have a major effect on the soft tissue attachment so that probing attachment levels would be used as the prime indicator of efficacy in the slowing of progression of Periodontitis. Measures of the bacterial flora in conjunction with the probing attachment level measurements will give information as to the mode of action of the antibacterial therapy. On the other hand, a mode of therapy that is postulated to have its primary effect on bone should use radiographic measures as the primary measure of efficacy since these outcomes match the hypothesis under study. There are many secondary variables that are also of importance in the full description of the action of a mode of therapy in Periodontitis trials. For example, it is generally considered to be important to track the course of gingival inflammation although all gingivitis is not indicative of active Periodontitis. Other secondary efficacy variables for Periodontitis include bleeding on probing, pocket depth, mobility, and plaque scores. The use of surrogate variables to substitute for actual The

more

recent literature

fThe Florida Probe

Co., Gainesville,

FL.

Volume 63 Number 12

JEFFCOAT

longitudinal measures of disease activity is appealing. Surrogate measures do not directly measure destruction but measure a

struction

parameter that is believed to be related to deputs the patient at risk for destruction. Many

or

measures that may be utilized in a single examination and may put patients or sites at increased risk for disease activity have been referenced above, including a history of probing attachment or bone loss, elevated crevicular fluid enzymes, persistent bleeding on probing, and presence of putative periodontal pathogens. The major reason to use surrogate variables would be that they are more easily measured, or can be measured with greater assurance than the direct measures of progressive disease. It is dangerous to substitute surrogate variables for direct measures such as sequential probing attachment level measurements or radiographie measurements before the validity of the surrogate has been completely tested in the type of patient population under study. Given the present state of the art, since all patients will be studied longitudinally in any of the experimental designs described above, it is safer for the conduct of the experiment to define the surrogates as secondary outcomes and be sure to track probing attachment level and/or bone loss over the course of the study. This assertion does not belittle the need for quick, accurate, and easily-performed measurements related to progressive disease. Rather, it simply states that the actual goal of the clinical trial must always be kept in sight. If the goal is to determine if a mode of therapy reduces the progression of Periodontitis, for example, measures generally believed to be related directly to the hypothesized effect are clearly needed.

surrogate

ISSUES OF SAMPLE SIZE The determination of how many patients are required to attain a given experimental power is a function of the difference expected in outcome between test and control agents as well as the variance within each group. The separate issues of sample size for superiority and equivalence trials have been recently discussed by Fleiss.62 The number of patients needed to demonstrate a statistically significant superiority between a placebo and a test mode of therapy is dependent on both the observed mean difference in the primary outcome between placebo and test groups as well as the variance for the outcome in each group. Protocols that serve to either increase the mean difference between groups or decrease the variance for the group will decrease the needed sample size.63 For example, the use of special pop-

ulations such as rapidly progressive Periodontitis patients may increase the mean observed difference between test and placebo groups since the mean rate of progression is relatively high in this special population in comparison to adult Periodontitis patients. Another method frequently used to increase the observed mean difference in outcomes between test and control groups is to increase the duration of the study. Although the actual model for disease progression in human Periodontitis remains controversial, most

1049

predicated on the overall assumption that, least some of the untreated patients will experience disease progression. Lengthening the study, therefore, will often make it easier to demonstrate differences between groups. On the other hand, some study parameters may increase the needed sample size. For example, the utilization of conventional therapy as part of the protocol, which is usually successful in reducing disease progression in the absence of adjunctive therapy, may tend to decrease the observed mean difference between groups and thereby increase the needed sample size. The measurement tool will also have an effect on the sample size calculation. Measurement methods that do not have precision and a low level of repeatability result in a high variance independent of the actual biological component of the variance.61 This higher variance means that for any given mean difference in outcomes between test and control groups a larger sample size is required to demonstrate a statistically significant difference. Thus, the apparently high expense involved in state-of-the-art measurement tools may not be as high as it initially appears. The possibility of studying smaller populations for a shorter period of time may reduce the overall cost of a trial. clinical trials

given time,

are

at

THE ISSUE OF CONTROLS, INCLUDING CUSTOMARY CARE

As outlined in the section on the determination of sample size, the utilization of usual and customary care, such as scaling and root planing, in the protocol will make it more difficult to detect differences between the test and control group necessitating either larger sample sizes or a longer study period. Difficulty and expense do not in and of themselves, however, dictate the experimental design. Of primary concern is the hypothesis under test. If the hypothesis is that the mode of therapy is an adjunct to conventional therapy, it would appear that a conventional therapy control is needed. Alternatively, if the hypothesis is that the therapy by itself has an effect on disease progression, conventional therapy may not be needed. In such experimental protocols the investigator must always consider the protection of the human subjects. Since the test therapy may not work and since the placebo group will not receive active therapy at all in such designs, safeguards must be in place to assure that a low level of progressive disease is detected, the patient informed, and necessary therapy offered even though the therapy may preclude further study of the patient.

CLINICAL SIGNIFICANCE The determination of statistical significance is a science in and of itself and the reader is referred to the proceedings for the conference on General Design Issues in Efficacy, Equivalency and Superiority Trials.62 Nonetheless, the determination of statistical significance is a science; the determination of clinical significance is to some extent an art with no clear-cut boundaries between a clinically significant effect and an insignificant effect. Clearly some crude out-

J Periodontol 1050

CLINICAL TRIALS DESIGN

counting lost teeth, are easily measured and agreed upon as clinically significant outcomes. Yet for most of the measures determined during a clinical trial, such as progressive probing attachment or bone loss, there is no comes, such

as

consensus on how much counts. These determinations should best be made prior to the start of the study taking into account patient population and intended use for the mode of therapy.

THE TYPE OF TRIAL Given all the variables outlined above, clinical trials may be classified in one of 3 categories as follows: 1) the proofof-principle trial; 2) the efficacy trial; and 3) the pathophysiological mechanism trial. The proof-of-principle trial is intended to determine whether or not a test mode of therapy is at all effective. Usually, the goal is to test the hypothesis in a limited clinical trial. Therefore, the proof-of-principle trial may involve study of special populations for relatively short periods using state-of-the-art measurement methods. In these trials a statistically significant result may be observed in a relatively short study period in a small sample size. The results of a proof-of-principle trial do not represent the definitive answer concerning the efficacy of a test mode of therapy. Rather, the results of this type of trial may be used to carefully design the (often multicenter) efficacy trial. The efficacy trial should utilize a clinical design and study period that follows from the intended use of the mode of therapy. In this regard, the patient population should also reflect the target population for the mode of therapy. Measurement tools may run from the routine clinical instruments, such as manual probing attachment level measurements and clinical indices, to state-of-the-art methods.

REFERENCES 1. Söder P-O, Frithiof L, Wikner S, et al. The effect of systemic metronidazole after non-surgical treatment in moderate and advanced Periodontitis in young adults. J Periodontol 1990; 61:281-288. 2. Al-Joburi W, Quee TC, Lautar C, et al. Effects of adjunctive treatment of Periodontitis with tetracycline and spiramycin. J Periodontol 1989; 60:533-539. 3. Quee TC, Chan EC, Clark C, et al. The role of adjunctive Rodogul

in the treatment of advanced periodontal disease. A longitudinal clinical and microbiological study. / Periodontol 1987; 58:594601. Watts T, Palmer R, Floyd P. Metronidazole: A double-blind trial in untreated human periodontal disease. / Clin Periodontol 1986; 13:939943.. Loesche WJ, Schmidt E, Smith BA, Morrison EC, Caffesse R, Hujoel PP. Effects of metronidazole on periodontal treatment needs. / Periodontol 1991; 62:247-257. Kulkarni GV, Lee WK, Aitken S, Birk , McCulloch CA. A randomized, placebo-controlled trial of doxycycline: Effect on the microflora of recurrent Periodontitis lesions in high risk patients. / Periodontol 1991; 62:197-202. Albandar JM, Rise J, Gjermo P, et al. Radiographic quantification of alveolar bone changes. J Clin Periodontol 1986; 13:195-200. Capilouto ML, Jeffcoat MK, Douglass CW, et al. Radiographic study of the progression of periodontal disease. J Dent Res 1989; 68:(Spec.

therapy

4.

5.

6.

7.

8.

Issue):236 (Abstr. 441).

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9. Beck JD, Lainson PA, Field HM, Hawkins BF. Risk factors for various levels of periodontal disease and treatment needs in Iowa. Community Dent Oral Epidemiol 1984; 12:17-22. 10. Rissin J, House JE, Conway C, et al. Effect of age and removable partial dentures on gingivitis and periodontal disease. / Prosthet Dent 1979; 49:217-223. 11. Chambers DA, Imrey PB, Cohen RL, Crawford JM, Alves MEAF, McSwiggin TA. A longitudinal study of aspartate aminotransferase in human gingival crevicular fluid. J Periodont Res 1991; 26:65-74. 12. Persson GR, DeRouen TA, Page RC. Relationship between gingival crevicular fluid levels of aspartate aminotransferase and active tissue destruction in treated chronic Periodontitis patients. / Periodont Res 1990; 25:81-87. 13. Lamster IB, Vogel RI, Hartley LJ, DeGeorge CA, Gordon JM. Lactic dehydrogenase, ß-glucuronidase and arylsulfatase activity in gingival crevicular fluid associated with experimental gingivitis in man. J Periodontol 1985; 56:139-148. 14. Lamster IB, Oshrain RL, Celenti R, Levine , Fine JB. Correlation analysis for clinical and gingival crevicular fluid parameters at anatomically related gingival sites. / Clin Periodontol 1991; 18:272277. 15. Birkedal-Hansen H, Pierson M, Heaven , Jeffcoat M, Cogen RB. Bone loss, GCF collagenase and GCF flow in human Periodontitis. J Dent Res 1989; 68:(Spec. Issue):324 (Abstr 1221). 16. Gangbar S, Overall CM, McCulloch CAG, Sodek J. Identification of polymorphonuclear leukocyte collagenase and gelatinase activities in mouthrinse samples: Correlation with periodontal disease activity in adult and juvenile Periodontitis. J Periodont Res 1990; 25:257-267. 17. Palcanis K, Larjava I, Wells B, Suggs K, Jeffcoat M. Elastase as an indicator of periodontal disease activity: Two month results. J Dent Res 1990: 69(Spec. Issue):344 (Abstr. 1883). 18. Jeffcoat MK, Williams RC, Reddy MS, English R, Goldhaber P. Flurbiprofen treatment of human Periodontitis: Effect on alveolar bone height and metabolism. J Periodont Res 1988; 23:381-385. 19. Jeffcoat MK, Page R, Reddy MS, et al. Use of digital radiography to demonstrate the potential of naproxen as an adjunct in the treatment of rapidly progressive Periodontitis. J Periodont Res 1991; 26:415421. 20. Offenbacher S, Odie BM, Van Dyke TE. The use of crevicular fluid Prostaglandin E2 levels as a predictor of periodontal attachment loss. J Periodont Res 1986; 21:101-112. 21. Mealey BL, Ebersole JL. Development of a rapid qualitative assay for determining elevated antibody levels to periodontopathic organisms. / Periodontol 1991; 62:300-307. 22. Loesche WJ, Giordano J, Hujoel PP. The utility of the test for monitoring anaerobic infections due to spirochetes. J Dent Res 1990; 69:1696-1702. 23. Savitt ED, Keville MW, Peros WJ. DNA probes in the diagnosis of periodontal microorganisms. Arch Oral Biol 1990; 35(suppl.):153159. 24. DiRienzo JM, Cornell S, Kazoroski L, Slots J. Probe-specific DNA fingerprinting applied to the epidemiology of localized juvenile Periodontitis. Oral Microbiol Immunol 1990; 5:49-56. 25. Zambón JJ, Bochacki V, Genco RJ. Immunological assays for putative periodontal pathogens. Oral Microbiol Immunol 1986; 1:3944. 26. Slots J. Bacterial specificity in adult Periodontitis. J Clin Periodontol 1986; 13:912-917. 27. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. J Clin Periodontol 1988; 15:85-93. 28. Badersten A, Nilvéus R, Egelberg J. Scores of plaque, bleeding suppuration and probing depth to predict probing attachment loss. 5 years of observation following nonsurgical periodontal therapy. J Clin Periodontol 1990; 17:102-107. 29. Claffey N, Nylund K, Kiger R, Garren S, Egelberg J. Diagnostic predictability of scores of plaque, bleeding, suppuration and probing

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35.

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37.

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39.

40.

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42. 43.

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depth for probing attachment loss. VA years of observation following initial periodontal therapy. J Clin Periodontal 1990; 17:108-114. Kaidahl WB, Kalkwarf KL, Patii KD, Molvar MP. Relationship of gingival bleeding, gingival suppuration, and supragingival plaque to

attachment loss. J Periodontal 1990; 61:347-351. Lang NP, Joss A, Orsanic T, Gusberti FA, Siegrist BE. Bleeding on probing. A predictor for the regression of periodontal disease? / Clin Periodontal 1986; 13:590-603. Lang NP, Adler R, Joss A, Nyman S. Absence of bleeding on probing. An indicator of periodontal stability. / Clin Periodontal 1990; 17:714-721. Haffajee AD, Socransky SS, Lindhe J, Kent RL, Okamoto H, Yoneyama T. Clinical risk indicators for periodontal attachment loss. / Clin Periodontal 1991; 18:117-125. Antczak-Bouckoms AA, Tulloch JFC, Berkey CS. Split-mouth and cross-over designs in dental research. J Clin Periodontal 1990; 17:446453. Moran J, Addy M, Newcombe R. A clinical trial to assess the efficacy of sanguinarine-zinc mouthrinse (Viadent) compared with Chlorhexidine mouthrinse (Corsodyl). / Clin Periodontal 1988; 15: 612-616. Jenkins S, Addy M, Newcombe R. Triclosan and sodium lauryl sulphate mouthwashes. II. Effects of 4-day plaque regrowth. / Clin Periodontal 1991; 18:145-148. Jenkins S, Addy M, Newcombe R. Comparison of two commerciallyavailable Chlorhexidine mouthrinses. II. Effects of plaque reformation, gingivitis and tooth staining. Clin Prevent Dent 1989; 11:1216. Kalaga A, Addy M, Hunter B. The use of 0.2% Chlorhexidine spray as an adjunct to oral hygiene and gingival health in physically and mentally handicapped adults. / Periodontal 1989; 60:381-385. Kaidahl WB, Kalkwarf KL, Patii KD, Molvar MP. Responses of four tooth and site groupings to periodontal therapy. / Periodontal 1990; 61:173-179. Anderegg CR, Martin SJ, Gray JL, Mellonig JT, Gher ME. Clinical evaluation of the use of decalcified freeze-dried bone allograft with guided tissue regeneration in the treatment of molar furcation invasions. / Periodontal 1991; 62:264-268. Wennstrom JL, Heijl L, Dahlen G, Grondahl . Periodontic subgingival antimicrobial irrigation of periodontal pockets. I. Clinical observations. J Clin Periodontal 1987; 14:451-550. Hujoel PP, Loesche WJ. Efficiency of split-mouth designs. J Clin Periodontal 1990; 17:722-728. McCuIloch CA, Birek P, Overall C, Aitken S, Lee Kulkarni G. Randomized controlled trial of doxycycline in the prevention of recurrent Periodontitis in high-risk patients; antimicrobial activity and collagenase inhibition. J Clin Periodontal 1990; 17:616. Williams RC, Jeffcoat MK, Howell , Rolla A, Stubbs D, Teoh KW, et al. Altering the progression of human alveolar bone loss with the nonsteroidal anti-inflammatory drug flurbiprofen. J Periodontal 1989; 60:485^190. The American Academy of Periodontology. Proceedings of the World Workshop in Clinical Periodontics. Consensus Report, Discussion Section I. Periodontal Diagnosis and Diagnostic Aids. Chicago: The American Academy of Periodontology; 1989:123-131.

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Haffajee AD, Socransky SS, Goodson JM. Comparison of different data analyses for detecting changes in attachment level. / Clin Per-

iodontal 1983; 10:298-310. 47. Jeffcoat MK, Reddy MS. Progression of probing attachment loss in adult Periodontitis. J Periodontal 1991; 62:185-189. 48. Jeffcoat MK, Capilouto ML. Problems in risk assessment in periodontal disease—use of clinical indicators. In: Bader JD, ed. Risk Assessment in Dentistry. Chapel Hill: University of North Carolina Dental Ecology; 1990:109-113. 49. Aeppli DM, Pihlstrom BL. Detection of longitudinal change in Periodontitis. / Periodont Res 1989; 24:329-334. 50. Birek , McCuIloch CAG, Hardy V. Gingival attachment level measurements with an automated periodontal probe. / Clin Periodontal 1987; 14:472-477. 51. Gibbs CH, Hirschfeld JW, Lee JG, et al. Description and clinical evaluation of a new computerized periodontal probe—the Florida Probe. J Clin Periodontal 1988; 15:137-144. 52. Jeffcoat MK, Jeffcoat R, Captain K. A periodontal probe with automated cemento-enamel junction detection-design and clinical trials. IEEE Trans Biomed Eng 1991; 38:330-333. 53. Schei O, Waerhaug J, Lovdal . Alveolar bone loss as related to oral hygiene and age. J Periodontal 1959; 30:7-16. 54. Hausmann E, Allen , Christersson L, Genco RJ. Effect of x-ray beam vertical angulation on radiographie alveolar crest level measurement. / Periodont Res 1989; 24:8-19. 55. Jeffcoat MK, Jeffcoat R, Williams RC. A new method of the comparison of bone loss measurements on non-standardized radiographs. J Periodont Res 1984; 19:434-440. 56. Webber RL, Ruttimann UE, Groehuis RAJ. Computer correction of projective distortions in dental radiography. / Dent Res 1984; 63:1032-

1036. 57. Webber RL, Ruttiman UE, Grondahl HG. X-ray image subtraction as a basis for assessment of periodontal changes. / Periodont Res 1982; 17:509-511. 58. Hausmann E, Christersson L, Dunford R, et al. Usefulness of subtraction radiography in the evaluation of periodontal therapy. J Periodontal 1985; 56:4-7. 59. Hausmann E, Dunford R, Wikesjö U, Christersson L, McHenry K. Progression of untreated Periodontitis as assessed by subtraction radiography. / Periodont Res 1986; 21:716-721. 60. Listgarten MA. Periodontal probing: What does it mean? / Clin Periodontal 1980; 7:165-175. 61. Jeffcoat MK, Reddy MS. A comparison of probing and radiographie methods for detection of periodontal disease progression. Current Opin Dent 1991; 1:45-51. 62. Fleiss JL. General design issues in efficacy, equivalency and superiority trials. / Periodont Res 1992; 27:306-313. 63. Jeffcoat MK. The effect of experimental design parameters on the determination of sample size. J Periodont Res 1992; 27:320-322. Send reprint requests to: Dr. M.K. Jeffcoat, Department of Periodontics, University of Alabama School of Dentistry, UAB Station, Birmingham, AL 35294-0007.