Journal of Neuro-Ophthalmology, June 1982, Volume 2, Issue 2

Is penicillin therapy always infallible in syphilis?

Penicillin is undoubtedly the antibiotic most effective on Treponema pallidum, but is it possible to prescribe it according to a standard regimen? Experimentation proves that such a uniform therapeutic plan cannot be determined for the following reasons: 1) Treponema pallidum may divide every 30 to 33 hours, but this concept is only established during the period of exponential growth in the initial lesion. Moreover, if Treponema pallidum are very quickly disseminated throughout the organism, then they do not divide at the same rate. In addition, we take into account a whole series of factors which can interfere with their rate of multiplication. 2) A penicillinemia of 0.03 U/ml may kill all the Treponema pallidum when they divide, but Dr. Eagle's data, although this would be a fairly active serum level, showed that the effective maximal serum concentration should be far higher, about 0.820 U/ml. The experimental data prove it is not possible to point out an accurate correlation between blood and tissue levels; thus, the penicillin levels in the cerebrospinal fluid are about 1/100 lower than those obtained in the serum. Among other factors it is necessary to take into account not only the age of the patient but also the penicillin complex chosen. Although the total of injected penicillin can be the same, the kinetics of serum levels are essentially variable, both regarding the increase of levels and duration according to the drug used. 3) As noted it follows that penicillinotherapy prescribed early with high and prolonged doses may allow a bacteriologic sterilization of primary syphilis.(ABSTRACT TRUNCATED AT 250 WORDS)

/. CJin. Neuro-ophth.llnllli. 2: 77-83, 1<)82. Is Penicillin Therapy Always Infallible in Syphilis? P. COLLART M. POITEVIN Abstract Penicillin is undoubtedly the antibiotic most effective on Treponema pallidum, but is it possible to prescribe it according to a standard regimen? Experimentation proves that such a uniform therapeutic plan cannot be determined for the following reasons: I) Treponema pallidum may divide every 30 to 33 hours, but this concept is only established during the period of exponential growth in the initial lesion. Moreover, if Treponema pallidum are very quickly disseminated throughout the organism, then they do not divide at the same rate. In addition, we take into account a whole series of factors which can interfere with their rate of multiplication. 2) A penicillinemia of 0.03 U/ml may kill all the Treponema pallidum when they divide, but Dr. Eagle's data, although this would be a fairly active serum level, showed that the effective maximal serum concentration should be far higher, about 0.820 U/ml. The experimental data prove it is not possible to point out an accurate correlation between blood and tissue levels; thus, the penicillin levels in the cerebrospinal fluid are about 1/100 lower than those obtained in the serum. Among other factors it is necessary to take into account not only the age of the patient but also the penicillin complex chosen. Although the total of injected penicillin can be the same, the kinetics of serum levels are essentially variable, both regarding the increase of levels and duration according to the drug used. 3) As noted it follows that penicillinotherapy prescribed early with high and prolonged doses may allow a bacteriologic sterilization of primary syphilis. At the secondary stage, however, it usually is not possible to kill all the Treponema pallidum which have invaded an organism for a long time, even with very high and repeated doses. This latent state is shown by an irreducible positive serology and may look like "a clinically apparent healing," but is not at all a bacteriologic sterilization. No one can deny the remarkable efficiency of penicillin on Treponema pallidum. However, be- From the Laboratoires de Recherches I'll Syphili' [xp"rillH'IlI,'il'. Institut A. Fournier. Paris, Fr,mee. June 1982 cause of inability to grow this spirochete, there are many gaps in our biological knowledge about it. For example, we have no criterion at all of bacte­riological sterilization with which to assess the post-therapeutic results. As a matter of fact, clinical signs (chancre, roseola) are nothing but warning signals which, generally speaking, heal sponta­neously. Their disappearance, however, cannot al­low one to assert at all that infection will not proceed in a latent phase. III The serological results alone may bring us to this conviction, but they are so numerous that they have been described as "a serological Babel Tower." We do need to know the biologic signifi­cance of the various serologic tests in order to be able to interpret accurately the different results obtained. 1M Of course, repeated totallv negative results (V.D.R.L., Kolmer, F.T.A.-Abs, TP.L. and T.P.H.A. tests) would suggest a recovery. But. it is quite different when these serological results are either doubtful or dissociated. Then, we are quite often obliged to interpret them following personal empiric conceptions, which are not alwavs in har­mony with well-proven biological facts. It is surprising that syphilis is one of the rarest infectious diseases for which a standard ther.1p" has been suggested. This olle is based c~n the following three arguments I": I) all trepl)nemes .He said to divide every 30 to 33 hl)urs. 21 penicillin acts on these germs only when the" di,'ide .md when this antibiotic rises to.1 levell)f 0.03 LJ/ml in serum, and 3) such a titer of penidllinemi.1 fl)r .1 duration of three times 30 to 33 hours C.1I1 destrl)" all the treponemes which h.l\'e in\'.lded .1n l)r~.l­nism. By ,lpplying Sll(h ,1 tl'er,lpeutic pl.m. s,)mc syphilogr,lphers h,lVe thought it pl)ssibk tl) cr.1di­cate syphilis, but st,ltistics h.l\'l' prl1\'l'n the llPP')­site result. (From the st,ltistics publi,lwd bv the United St.1tes Dep,Htment l)f Hl',llth ,llld Hum.1n Services: 10 57, b,251 C,lSl'S l)f primary ,llld sE'cL)nd­, uy syphilis ,md 10 74-28.874 c,lses.~I) Clinical observations ,md epidemiolngical d,lt,l have sup­ported those who, from their experiments, have concluded that such a concept was based on a two biologic,ll mista kes 1:1: I) a bacteriologic error, and 2) ,1 ther,lpeutic error. 77 Penicillin ThC'r.lpy Bacteriological Argument As desirable ,IS it might be to find out the multiplication rate of Treponema p,llIidum, it is nllt possible to cultivate this organism on which such ,10 experiment,ll dat,) hypothesis can be based, wnsidering th.lt it is not likely all of the germs would divide at the same time. 'M In his book The Biology of Treponematoses,"7 Professor T. B. Turner has written: "Under suitable conditions treponemes divide at the rate of about once every 30-33 hours and increase roughly ten­fold e.lch 4 days. Clinically recognizdble lesions begin to dppedr when the treponeme count in a local area reaches the order of 10 million trepo­nemes ... There is dmple experimental evidence thdt during the incubdtion period the inoculum is increasing logarithmically." From only the duration of the incubation period it might be possible to deduce the medn generation time; however, exper­imental datd now prove thdt it is not possible to make such a generdl stdtement. Indeed, numerous factors are capdble of interfering with the rate of the growth of these germs. In his thesis, J. Monod:l'l proved thdt all micro­bidn cultures in vitro would develop in five periods following Gause's graph. In need of being to able to cultivate treponemes, when these are injected in the testicle of a rabbit, one can admit to a sort of culture in vivo. 10 An evolution simildr to that de­scribed by J. Monod in microbian cultures can be observed. After a stage of incubation during which the germ adapts to its medium (corresponding to a phdse of disappearance), there follows a period of exponential growth, characterized by an accel­erated multiplication of spirochetes dnd resulting in the formation of the acute orchitis. Then the number of treponema pallidum in the lesion does not dppear to increase: this is the stationary phase. An indurdtion appears at this point in the testicles where the Treponema pdllidum become rarer: this is the stdge of regression. Finally, there is the hedling of these local signs in which the trepo­nemes are very difficult to pick out, despite their invasion of the whole orgdnism when they Me going to persist, without appearing to multiply: this is the phase of latency. . It must be emphasized that the multiplic.ltil)n rate of 30 to 33 hours has been est'lblished llnly during the incubation period, i.e., the exponenti.~1 growth period, with the Nichols strain. Thus, bced with such obviously varying sequenti,ll ph,lses of the experimental infection, it is hardly possible tl) admit th.lt the Trepllnema p.lllidulll const.lI1tly di­vide .It the s.lnH' r.lte .15 during the initi.11 peri~ld. In the ,14ueous humor of recently infected r,lb­bit<" we found very few TrepIHwm.; p.lllidulll, but th,'ir vit.llity W.lo., evidently well preserved since the I).I'..,.I'~'· "f l11i·,,·d .1lIU(·puo., humpr W.IS .Ible til ~..~, provide a positive passage. However, a year and a half later, one of these animals developed an inter­stitial keratitis, yet the number of treponemes dis­covered in the aqueous humor was more or less the same, thus proving that during the year and a hdlf the richness of these germs had barely in­creased. This certainly does not represent a graph of continual growth on a logarithmic scale. On 49 cerebrospinal fluid smears from infected rabbits, we have been able to show Treponema pallidum in all but two cases. Since the quantity of cerebrospinal fluid extracted by suboccipital punc­ture is extremely small (approximately 0.5 mm), we constituted "pools" of fluids on four occasions. Each of these pools had been infected for similar periods: 24 hours, 9 days, 12 days, and 65 days respectively.l~The result of the bacteriological ex­amination was positive in all four cases; however, after intratesticular injection of these four pools into four fresh rabbits, we obtained a positive result only twice with the mixture of cerebrospinal fluid from four rabbits that had been infected with syphilis 9 and 12 days previously, after incubation periods of 21 and 26 days respectively. The other two remained negative. Previous experience has proved, however, that the duration of the incuba­tion period is rather variable and depends on a series of factors as reported by Professor J. Monod. The Medium upon which the Inoculum Develops T. B. Tumer~7 pointed out that "the treponemes in the testis and in the skin multiplied at about the same rate, but a larger inoculum was required for the same incubation period in the testis by a factor of approximately 100." It would seem in such condition, therefore, that the Treponema paIlidum must divide at a much slower rate in the skin than in the testicle. Age of the Inoculum With a material of similar richness, a passage nude With ground down syphiloma (signs on an average 3 weeks after initial infection) induces a lesion much later than with a suspension of Tre­ponema pallidum extracted from an orchitis (which usually occurs 6-8 days after the intratesticular injection of Treponema pallidum). We have ascer­t. lined~l. 10. 1:1 thJt node transfers carried out using rabbit infected with 50 million of Treponema pal­IIdum of the Nichols strain for 4 months-but not treJted-responded on an average in a positive manner after 18 days. However, after 2 years in­tection (still untreated), the incubation period fol­lowing such passages was 41 days, and after 3 years, this can extend to 4 months.4 • ~ Journal of Clinical Neuro-ophthalmology Strain We have proved experimentally and statistically" that if 100,000 Treponema pallidum of thl? Nichols strain would produce an orchitis in 14 d.lyS, with an equal number of treponemes from thl? Grand strain (not adapted to the production of an acute orchitis). the time span would extend to 38 days. This example clearly proves that the multiplication of germs of different strains does not take place at the same rate. The Effects of Earlier Treatments Professor Turner points out that"Any incuba­tion period much in excess of these values must indicate some interfering influence producing less than optimum conditions. We have observed an incubation period of 72 days in an animal which was maintained in a warm environment; and in other animals receiving intermittent doses of pen­icillin we have deliberately produced prolonged incubation periods of up to 167 days.,,~t; We were able to statistically establish" that if 100,000 Treponema pallidum of the Nichols strain induced an orchitis in 14 days, the incubation period for the same material of a similar richness but having undergone several passages through animals treated with cortisone was no longer than 5 days. Thus, this multiplication rate was trebled. As a result, for an equal richness in treponemes, infectious materials can present longer or lesser variations in the duration of their phase of expo­nential growth, caused by a whole series of factors. So, it must be accepted that the elements compos­ing these strains certainly did not present a rate in multiplication analogous to that of the Nichols strain. This outline refers to phenomena observed during experiments on rabbits, but it must be noted that on mice, P. D. Rosahn44 • 4 '. calculated that Treponema pallidum would divide every 24 hours, far from the 30-33 hours. Such data are unknown for monkeys, and in man can not be undertaken. It is obvious that the prelesional stage characterized by the phase of exponential growth is missed in the majority of human cases because the clinician sees the patient once the initial lesion has appeared. In man, A. Sezary and P. Boutteau,'I'i. ·1; as well as ourselves,cl have proved that in the adenitis caused by the primary accident, Treponema palli­dum can be easily found in nearly 80% of cases; at the secondary stage this is possible in only about 20%. Yet, no clinician would question the pl?rsist­ence of the infection in this lymphatic tissue. If the treponemes continued dividing every 30 to 33 hours it should be infinitely easier to pick them out during the secondary stage. It Finally, it is generally accepted in bacteriology that all microbian strains are made up of heterog­enous elements, and nothing prevents us from June 1982 Colldrl, Poitevin affirming that this is also the case with a trl?poneme strain. Also, there is no reason to conclude that in an infl?cting material, there should not be spiro­chetl? s which do not divide at the same acceleratl?d rate as the others, and thus l?scape a rather brief action of penicillin.1:l These few examples clearly proVl? that one cannot gl?neralize the rate of division of all treponemes as every 30 to 33 hours, but, as T. B. Turner pointed out, "under optimum condi­tions," during the period of exponential growth. Thus, if it is admitted that Trl?ponema pallidum will divide at a logarithmic scale before the for­mation of the initial lesion, it is not possible to think that this multiplication takes place at such a rate some months later. The spirochetes are then in a commensal state in different tissues, invaded from the beginning of the infection, and having kept their vitality. Therefore, it seems obvious that at that quiescent period (latency). they cannot have the same receptivity towards penicillin. J",. I" Action of Penicillin on Treponema Pallidum Argument According to research carried out on Gram pos­itive germs,r,r,· r,t; the action of penicillin by blocking the synthetic functions of the transpeptidase of the cell wall of treponemes would contribute to the formation of swollen cytoplasmic structures; as a result of the hydrostatic pressure, this cell wall would finally break, thus bringing about the death of the germ.:l1 With Pechere lJ we proved that Treponema pal­lidum (Nichols strain) incubated during 48 hours in the Mayer medium (treponemes do not divide in this case; it is only a medium of preservation). to which were added 0.005 to 500 U/ml of benzvl­penicillin. The treponemes retained their vitality since they were still able to infect the rabbit after an intratesticular inoculation, supporting the fact that the period of quiescence penicillin does not effect these germs.):l On the other hand, if we refer to the lVork l)f Eagle et a!./" the average .1Ctive concentr.ltil1l1 l)f penicillin on rabbits, whatever the strength of in­oculum and the age of orchitis, would be between 0.0083 U/ml and 0.0167 U/ml; but the effective concentration would be fifty times superil~r. i.e.. 0.6 U/ml, an amount which is rarelv .ltt.lined dur­ing a standard treatment of syphilis in m.m.•md .1 long way from the 0.03 U/ml CL)nsidered bv nu­merous syphilographl?rs .15 being ,1Iw.lVS leth.l!' But, the saml? authors .ldd "of .111 b.lctl?ri.l, Trepo­nema pallidum is at the s.lme time one of the nwst sl?nsitive and most resist.mt gl?rms to the action of penicillin;" an opinion confirmed by A. Kern"l who wrote, "Although Treponem.l pallidum is highly sensitive to pl?nicillinemia it is capabll? of persisting not only after the administration of sub­liminal doses, but also aftl?r a maximal one which 79 Penicillin Ther.Jpy produces the bacteriostdtic effect. However the phenomenon is not identicdl to a true genetic re­sistance." This would dppedf to dfgue in favor of treatments giving a rate of penicillinemia; certdinly high, but above dll, prolonged. Research on the Kinetics of Penicillinemia As edrly.Js 1952, Heite~!1 showed in rabbits, with sulph ur:l7. :IX Idbeled penicillin, thdt the dctivity of that antibiotic depended less on the blood level than on the concentrdtion of the dntibiotic dt the lesion sites. More recently, in 1961, an editoridl in the ]ourndI of the Americdn MedicdI Associdtion ~4 concluded that it was not possible to stdte d precise correldtion between serum levels dnd c1inicdl dctiv­ity of the dntibiotic. As d mdtter of fact, the result of penicillin therapy depends mdinly on the phdr­mdcokinetics of the drug obtdined during infection, not only in the serum but more especidlly in tis­sues. However, since the Idtter is impossible to get in the clinic, the serum levels can be used as d reference; dnd, although they constitute noth­ing but a relative ddtum, the concentrations in tissue and in sera are very different following orgdns.l~. 22. :1I. 40 In 1964, having done comparative determina­tions of penicillin in serum and cerebrospinal fluid of 12 patients, it was established that penicillin levels in the cerebrospindl fluid were about 1/100 lower than those obtained in serum.G • 3 :J Not long ago, Niwat Polnikorn et al. 41 showed that "There were no detectable concentrations of penicillin in the cerebrospinal fluid after administration of ben­zathine penicillin 2.4 mega units, benzathine pen­icillin 7.2 mega units, procaine penicillin in dlu­minum monostearate (PAM) 12 megd units, or aqueous procaine penicillin G 2.4 mega units. Only after high doses of aqueous penicillin G 24 mega units daily or aqueous penicillin G 2 mega units daily together with oral probenecid 2 g daily was penicillin detectable in the cerebrospinal fluid. The concentrations after the latter regimen were the highest and much higher than the minimum inhi­bitoryconcentration for Treponema pallidum."" Moreover, from the research carried out by Pro­fessor N. M. Ovcinnikov et al./6.:l9 Azar et dl., I dnd Lauderdale et al.,:12 Treponema pallidum may pen­etrate early in all the cellular elements, where they can vegetate in a commensal state. Is penicillin, however, able to penetrate in close relationship with cells? On the other hand, we have shown in rabbits 19 that after a single injection of two penicillin com­plexes (benzathine-penicillin complex* and bene- • Benzathine benzyl penicillin, 600,000 I.U.; penicillin G prl)­caine, 300,000 I.U.; .lnd penicillin G sodium, 300,000 I.U. I) [xlencilline Bipenicilline" Pendural .lll purposes," Cep.lcillina.'· Cenlocillin;" and 2) Bicillin all purposes 6:3:3, Panbiolic.'1 Pen­ddur 6:3:3," Benzaleclil Reforcado," and Longdcillin." thamine-penicillin complex **), although the total number of units of penicillin might seem to be similar in quantity, the kinetics of the seru.m co,:,­centrations are shown to be extremely vanable In concentration as well as in time, following the chosen drug. Thus, two groupS of rabbits received two similar regimens. One was 40,000 i.U./kg of the benzathine-bipenicillin complex and the other 43,000 I.U./kg of the benethamine-penicillin com­plex in a single injection done at the same time as the inoculation of virulent Treponema pallidum. The serum concentrations were significantly dif­ferent at the confidence interval of 95% as soon as the second day, the latter complex being eliminated within 3 days. IX The results of inoculations were 100% bacteriological sterilization in inoculated rab­bits treated with the former complex, and 0% for those which were given the latter one. If we compare serum concentrations, the blood levels obtained after injection of 86,000 I.U./kg of the benethamine complex and those obtained with 40,000 I.U./kg of the benzathine-bipenicillin com­plex do not present any significant statistical dif­ference as soon as the second day.18. 19 However, therapeutical results were 12% of bacteriological sterilization with 86,000 I.U./kg of the benetham­ine- penicillin complex, when 40,000 i.U. of the benzathine-bipenicillin complex gave 100% of bac­teriological sterilization. It is necessary to inject 172,000 I.U./kg of the benethamine-penicillin complex to animals, that is, a dose 4.3 times higher than the dose injected with the benzathine com­plex, to obtain 100% of bacteriological sterilization with an effective penicillinemia above 0.03 U/ml for 168 hours. This proves that with equal dosages of penicillin, the lethal activity of the drug on Treponema pallidum is far from being the same, considering the complex chosen. Thus, it is not possible to think of a uniform standard treatment based only on the sole dosage of that antibiotic. Finally, do the kinetics of different penicillin complexes vary according to other factors? Not long ago we proceeded to do a comparative study between serum concentrations and curves of elim­ination in serum of two long-acting penicillin com­plexes after a single injection given in two groups of patients, the younger group 20 years old and the older 75 years old. The following results were established and confirmed by a statistical study.17, 18 Age was a major factor in the variations in serum penicillin concentrations and in their persistence in the serum. The penicillin was absorbed faster in young than in elderly subjects, even when a long­acting complex was used. Serum concentrations below the level regarded as lethal for treponemes appeared much earlier and more frequently in .. Benethamine penicillin, 600,000 I.U.; penicillin G porassium, 300,000 I.U.; and Biclinocilline.R Journal of Clinical Neuro-ophthalmology young than in old people. Finally, the bioequival­ence between penicillin preparations could not be estimated solely from the number of units of the agent used but from the bioavailability of the formulation chosen. Thus, a uniform and standard penicillin dosage allowing no safety margin may help in the superficial healing of a syphilitic chan­cre or the resolution of a roseola, but it will cer­tainly be insufficient to kill Treponem,l pallidum, even if they have just invaded the organism of the patient. Conclusions From these data, it is deduced that it is impos­sible to set up an effective treatment only on the basis given by the three previously cited reasons. If Treponema pallidum are able to multiply at a logarithmic scale only during the experimental growth period (that is, before the warning signals become visible), it cannot be argued that Trepo­nema pallidum would divide at the same rate dur­ing the latent period. At present, all the authors who studied this problem notice that the presence of Treponema pallidum in a commensal state, although keeping their vitality, disseminated in different tissues even during the early period.~' 4.7. 14-16. ~O. ~5. ~6-~H. :15. :17-:lI<. 4~. 4,1. 4H-',Il. ',H-ol If it cannot be denied that penicillin is one of the best therapies against Treponema pallidum, we do not know very well the kinetics of the different types of penicillins or individual factors which may in­terfere with the activity of the penicillin chosen. The penicillin levels found in the cerebrospinal fluid are lower than those found in serum or other tissues. What is the lethal concentration of this antibiotic for Treponema pallidum? It is of great interest to read again the remarkable work of Professor H. Eagle (and not to refer to superficial abstracts which have been written about them) to under­stand that it is wrong to assert that a serum level of 0.03 U/ml of penicillin kills all Treponema pallidum. Setting up a standard treatment gener­alized on such reasoning appears to be dangerous when we know that the arguments are quite wrong and that we do not have any criterion of bacterio­logical sterilization. Thus, a standard uniform dos­age of penicillin without any explanation about the drug chosen and without any safety margin may help to heal superficially a chancre or to remove a roseola, but certainly does not give a bacteriologic sterilization in 100% of cases of those who have been invaded by Treponema pallidum in a com­mensal state. It is therefore absolutely essential to undertake treatment at high doses and for a long time to avoid a latent infection which is likely to be the cause of future serious relapses. June 1982 Collart, Poitevin References 1. Azar, H.A., Than Duc, P., and Kurban, AX.: An electron microscopic study of a syphilitic chancre, Arch. Patho/. 90: ]43-/50, 1970. 2. Boncinelli, U., and Lucellotti, M.: La prognosi della infezione sifilitica. C. ltal. Derm. 107: 463, /966, 3. Collart, P.: Etudes de Syphilis Experimenta/e, Ar­nette These, Ed. /936. 4. Collart, P., Borel, L.J., and Durel, P.: Recherches sur I'efficdcite de 1.1 penicillinotherapie ddns Id syphilis tardive experimentdle et humdine. I. Ld syphilis ex­perimentdle. Ann. Inst. Pasteur 102: 596-615. ] 962; II. La syphilis tdrdive humaine. Ann, Tmt, Pasteur 102: 693-704, 1962; lII. Etude de I'action de Id pen­icilline ddns Id syphilis tdrdive, Persistance du tre­poneme pale apres trditement. Ann, Inst. Pdsteur 103: 953-958, ]962. 5, Collart, P., Borel, ]., dnd Durel, P.: Recherches sur I'efficacite de Id penicillinotherdpie dU caurs de Id syphilis tdrdive experimentdle et humaine. Ann, Dermatal. Syph. 89: 488-504, ] 962. 6. Collart, P., Durel, P.: Presence et persistdnce des treponemes dans Ie L.C.R. dU cours de 1.1 syphilis experimentale et humaine apres traitement tdrdif, Ann. Dermatal. Syph. 91: 485-498, ]964. 7. Collart, P,: Etat detuel des recherches sur 1.1 persist­ance des treponemes au cours de 1.1 syphilis tdrdive­ment traitee. Marseille Med. 101: 837, ] 964. 8. Collart, P., Poggi, G., Dunoyer. M. dnd Dunoyer, F,: Quelques recherches experimentdles sur Ie com­portement de trois souches de treponemes pales (souche Nichols, souche Nichols cortisoneI.' et souche Gand). Ann. Dermatal. Syph, 95: 59-68, 1968. 9. Collart, P.: Le treponeme pale: Etude experimentale, Rev. Med. 22: ]279-1300, ] 970, 10. Collart, P., Franceschini, P., and Durel, P.: Experi­mental rabbit syphilis. Br. f. Vener Dis, 47: 380-400, ]971. ] 1. Collart, P., Pechere, J,c., Frdnceschini, Ph" and Du­noyer, P.: Persisting virulence of T. pallidum dfter incubation with penicillin in Nelson-Mayer me­dium. Br. f. Vener, Dis. 48: 29-31, IOn, ]2. Collart, P., Franceschini, Ph" Poitevin, M" Dunover, M., and Dunoyer, F,: Modified method of filtering cerebrospinal fluid dnd aqueous humour for the detection of treponemes. Proof of the persistence of their vitality in rabbits. Br, /. \fener, Dis, so: 251-250. ]974. 13. Collart, P.: Critique des elements de base sur les­quels reposent certains traitements de 1.1 syphilis. Sem. Htlp. so: 673-670, 1074. /4, Collart, P., dnd Poitevin, M,: Persistence des trepo­nemes pales apres traitement de 1.1 syphilis e'\peri­ment. lle et hum.line. Ann, Mcd, Tntern 26: 223-230, 1975. IS. Collart, P., and Poitevein, M.: Le problemI.' des trep­onemes penicillino-resist,l/lts. 51.'01. H,;p, P.lris 51: 1489-1490,1975. 16. Collart, P., and Poitevin, M.: Recherches experimen­tdles sur I'dction de 1.1 penicillinotherapie au cours de 1.1 syphilis tardive. Bull. Acad. Nat. Med, 161: 595-601, 1978. /7. Collart, P., Poitevin, M., Milovanovic, A., Herlin, A., dnd Durel, J.: Kinetic study of serum penicillin con- 81 Penicillin Ther.Jpy centr,ltions ,lfter single doses of benz.Jthine .Jnd beneth.Jllline penicillins in y,lung .Jnd old people. Br. /. VCllcr. Dis. 56: Iq80. 18. CoIIMt, 1'., ,1I1d [\Iitevin, M.: An up-to-d,lte physio­biologi(, 11 news "f the m.Jin t,lpics. I. The b.Jderio­logic. Jlquestion. Scm. H,;p. I'Mis 57: 857-8b8, 1981; II. The evolutive process of experiment.Jl syphilitic infection ,1I1d .I comp.Jrison with clinic.Jl symptoms in m.Jn. Sen!. H,;p. r.lris 57: 98q-1000, I 'l81; III. The problem of immunity. Sem. H(;p. ['.Iris 57: 10<:>3-1075. I q81; IV. The problem of the tre.Jtment by penicillin. Sen!. Hop. 1',His 57: Ilbl-1 171, 1981. 10. ColI.Jrt, P., Poitevin, M., Milovanovic, A., and Herlin, A.: Etude cinetique des concentrations seriques de penicilline apres injection d'une dose unique d'un complexe penicilline retard (benzathine penicilline ou beneth.Jmine penicilline), chez des lapins infectes pM 1,1 souche Nichols virulentI'. (In press.) 20. Del Carpio, C: Effeti di un trattamento massicio con penicillina nella sifilide sperimentale del coniglio a diversa distanza dall'infezione. Rapporti fra presenza di anticorpi immobilizzanti e persitenza dell' infe­zione. Riv. 1st. Sieroter. Ita/. 38: 166, 1963. 21. Department of Health and Human Services: Public He.Jlth Service, Center for Disease Control. Sexu.Jlly Transmitted Diseases. Statistical letter. Calendar year 129, 1979. 22. Eagle, H.: Speculations as to the therapeutic signifi­cance of the penicillin blood level. Ann. Intern. Med. 28: 260-278, 1948, 23. 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Lawton Smith, MD.. 9820 S.W. b2nd Court Miami, Florida 33156. 83