Experimental Seronegative Syphilis*

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2. Scheie, H. G.: Aspiration of soft cataracts: A new technique. Am. J. Ophth., 50:1048-1056 (Dec.) 1960. 3. Ferguson, C. : A modified instrument for aspiration and irrigation of congenital or soft cataract. Am. J. Ophth., 57 :S96-S99 (Apr.) 1964. 4. Cordes, F. D. : Failure in congenital cataract surgery. Am. J. Ophth., 43:1-21 (Jan.) 19S7. 5. Wilson, W. A. : Congenital cataracts : A review of 22 cases including the use of enzymatic zonulolysis in 15 patients. Arch. Ophth., 67:143-147 (Feb.) 1962. 6. Deweese, M. W. : A survey of the surgical treatment of congenital cataracts. Am. J. Ophth., 53 : 853-858 (May) 1962. 7. Fuchs, J. : Die Zweiwegespritze, ein neuartiges Instrument zur Absaugung weicher Stare. Klin. Mbl. Augenh., 121:592-595, 1952. 8. Fink, A. I., and Weinstein, G. W. : A modification of the Fuchs syringe. Am. J. Ophth., 58:129 (July) 1964.

E X P E R I M E N T A L SERONEGATIVE SYPHILIS* WILLIAM H.

TAYLOR, M.D.,

J. LAWTON S M I T H , M.D.,

AND J. A. SINGER,

M.D.

Miami, Florida

Seronegative ocular and neurosyphilis is a major problem in diagnosis today. This is evidenced by over 200 cases which have been detected in our hospital within the past two years.1"3 By seronegative syphilis is meant the presence of this disease in patients in whom the conventional serum reagin tests (VDRL, Kahn, Hinton, etc.) are nonreactive. Criteria for diagnosis in such cases have been: (1) a history of veneral infection, usually with inadequate treatment; (2) clinical signs of the disease; and (3) a reactive specific treponemal test. The magnitude of this problem prompted the laboratory investigation of seronegative syphilis. In a previous communication, the lesions encountered in a series of rabbits and monkeys infected with T. pallidum by direct inoculation into the cisterna magna and varying ocular sites were presented.4 This report documents for the first time to our knowledge the producton of seronegative syphilis in the untreated primate and emphasizes the sérologie responses in such animals. * From the Department of Ophthalmology, University of Miami School of Medicine, and the Venereal Disease Research Laboratory, Communicable Disease Center, Atlanta. This work was supported in part by grants NB-04604-02, NIH5TINB527705, N1003A64 and NB-05051-01, National Institutes of Health, and the Florida Lions Eye Bank, District 35-A.

Before presenting the findings in the experimental animals, explanation of the sérologie tests are in order. At the outset, it should be stated that all sérologie determinations were performed by the Venereal Disease Research Laboratory, United States Public Health Service, Atlanta, Georgia. There were three sérologie tests employed in this study: (1) the VDRL reagin test, (2) the Treponema pallidum immobilization test or TPI, and (3) the fluorescent treponemal antibody absorbed or FTA-ABS test. To appreciate the differences in both specificity and sensitivity of these tests a few milestones in serodiagnosis of syphilis will be considered. The Treponema pallidum was discovered as the causative organism in syphilis by Schaudin and Hoffman in 1905. The following year Wassermann applied the complement-fixation technique to the serum diagnosis of this disease. Multiple tests, devised during the ensuing 40 years, utilized various complement fixation and flocculation reactions. These tests were all based upon the detection of a nonspecific globulin in the patient's serum which has been called reagin. In 1941 Pangborn isolated an active principal in beef heart antigens which he termed cardiolipin. Today the majority of nontreponemal tests (as the VDRL, Kahn, Hinton, etc.) employ such cardiolipin anti-

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WILLIAM H. TAYLOR, J. LAWTON SMITH AND J. A. SINGER

gens. However, a substance similar in sérologie behavior to that found in the sera of syphilitics is also found in nonsyphilitics following febrile illnesses, immunizations and in certain collagen diseases. T h e first specific treponemal test, the T r e ponema pallidum immobilization test, was reported in 1949 by Nelson and Mayer. This test is based upon the detection of an antibody in the patient's serum which immobilizes virulent motile Treponema pallidum. T h e T P I may be inconclusive in a patient on antibiotic treatment but, with this exception, a false positive T P I is virtually unknown. A false negative reaction can occur, however, in about two thirds of patients with primary syphilis and one third of those with secondary syphilis. T h e test has shown greater sensitivity than any other available group in which the ophthalmologist is interested. T h e T P I test has certain objections. It is expensive and technically difficult to perform. Recently, a new procedure has been reported, the fluorescent treponemal antibody absorption procedure. It is technically simpler and much less expensive. Initial evaluation in patients suggests that it has greater sensitivity than any other available sérologie test for syphilis and that its specificity is at least equal to that of the T P I test. 5 The F T A - A B S should not be confused with the F T A - 2 0 0 procedure—a test of much less clinical value in the detection of late syphilis. T h e first reported results of the F T A A B S test in two small primate species are presented here. M A T E R I A L S AND M E T H O D S

In this experiment, 17 monkeys were inoculated with suspensions of virulent Treponema pallidum. In the first stage done in August, 1963, 12 owl monkeys (Aotus trivirgatus) were used. Six monkeys received the tréponèmes by inoculation into the cisterna magna. The other six animals were given the tréponèmes in varying ocular sites. T h e second stage done in January,

1964, consisted of cisternal inoculations of three owl monkeys and two squirrel monkeys (Saimiri sciurea). Of the first group of 12 monkeys, two died during the first week from complications related to anesthesia and surgical technique. O n e animal died two months later, two died at two and onehalf months, one died at five months and one died at seven months. T h e other five monkeys, currently alive, have been under continuous observation throughout the experiment. Of the second-stage animals, two owls and one squirrel monkey died six weeks after inoculation from upper respiratory infections. Preinoculation physical and eye examinations were negative in all animals. Serologie tests ( V D R L , T P I , and F T A - A B S ) were obtained at repeated intervals on all monkeys. A suspension of virulent Treponema pallidum was prepared from the testis of a rabbit with syphilitic orchitis of seven days' duration. The testis was macerated and gently agitated with 4 ml of 50-50 normal rabbit serum-saline mixture which had been previously heated to 56°C for 30 minutes. After 15 minutes' extraction and centrifugation for two minutes at 1600 rpm, darkfield count of the supernate showed approximately five million motile organisms per ml. Animals were anesthetized with Sernylan,* 0.3 ml subcutaneously, and the necks were shaved. A No. 22 pédiatrie spinal needle was used for cisternal punctures. The ocular injections were done using No. 27 needles on tuberculin syringes. In virtually each instance, the monkeys were given a volume of 0.2 ml of the treponemal suspension intracisternally, replacing an equal volume of cerebrospinal fluid which had been removed. A control animal received 0.2 ml of the sterile serum-saline mixture, but without tréponèmes, in the cisterna magna. The animals, maintained at 7 7 ° F and 8 0 % humidity, were fed only antibiotic-free monkey chow and * Supplied through the courtesy of Parke, Davis & Company.

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fresh fruit. They were examined at regular intervals, and blood was submitted for sérologie testing at intervals. RESULTS

The data here presented pertain to four of the surviving monkeys and one other monkey included because of an interesting lesion with postmortem verification. It should be noted that the lesions in experimental syphilis produced by subarachnoid inoculation have a much more virulent course than those seen after inoculations into the eye ; also, previous experiments suggest that the systemic effects of syphilis are greater in the rabbit than in the small primates studied. Three types of lesions have been noted to date in these monkeys ( 1 ) third-nerve paresis, (2) retinal lesions and ( 3 ) pupillary abnormalities. T h e animals will be considered individually.

Fig. 2 (Taylor, Smith and Singer). Owl monkey 50 died five months after intracisternal T. pallidum. Postmortem examination revealed recent subarachnoid and intracerebral hemorrhage.

*

* ·«»

OWL MONKEY (figs. 1-4) 50

This monkey received 0.25 ml (or approximate^ 1.25 million tréponèmes) in the cisterna magna. One week later physical and eye examination was normal. Eleven weeks after inoculation, ptosis and a minimally dilated pupil were present on the right. The right pupil measured 3 mm in diameter and reacted 3 + to light; the left pupil was 2 mm and reacted 4 + to light. Fundus examination after a careful dilatation was normal. It was evident that the monkey was developing a right third-nerve par-

■

-Λ. Fig. 3 (Taylor, Smith and Singer). Owl monkey 50. A cross section of the brain shows intracerebral hemorrhage below the right lateral ventricle. esis. Twelve weeks after inoculation the serum VDRL was nonreactive. However, the FTA-ABS test was already reactive. At four months after inoculation the VDRL was again nonreactive. Five months after inoculation the animal became moribund and died within several hours. Serum obtained at that time revealed a weakly reactive VDRL; both TPI and FTA-ABS tests were reactive.

Fig. 1 (Taylor, Smith and Singer). Owl monkey 50 two and one-half months after 0.25 ml T. pallidum was injected into the cisterna magna. Ptosis and a slightly larger pupil are present on the right side.

Comment. Cisternal inoculation produced primary neurosyphilis in this animal, although it remained quite healthy for over two months until a right third-nerve paresis developed. T h e mobility of the globe was considered normal but ptosis and a dilated

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WILLIAM H. TAYLOR, T. LAWTON SMITH AND J. A. SINGER reactive T P I and F T A - A B S tests. A n interesting finding was the perivascular sheathing which developed along the vessels showing hemorrhage and exudates nine months previously. OWL MONKEY 54

This animal received 0.2 ml (one million trepo-

Fig. 4 (Taylor, Smith and Singer). Owl monkey 50. The right third nerve shows a focal area of necrosis. pupil were constant until its death. At postmortem examination, there was evidence of acute subarachnoid and intracerebral hemorrhage. A focal lesion in the right oculomotor nerve was confirmed histologically. A significant sérologie finding was that the F T A - A B S test was reactive two and onehalf months prior to V D R L conversion. T h e diagnosis of syphilitic third-nerve paresis would have been missed if only a reagin test had been done during the first two months of the lesion's presence.

Fig. 5 (Taylor, Smith and Singer). Owl monkey 51 received 0.3 ml T. pallidum intracisternally. Perivascular hemorrhages and exudates were present in the left fundus at 21 weeks.

OWL MONKEY (figs. S and 6) 51

This animal received 0.3 ml (or 1.5 million tréponèmes) in the cisterna magna. One week later physical and eye examinations were normal. A diagnostic cisternal puncture revealed 167 red cells and 26 white cells per cmm. The white cells showed 15% polymorphs and 85% lymphocytes. Eleven weeks following inoculation the serum VDRL was nonreactive but the FTA-ABS test had become reactive. At 17 weeks, the VDRL was weakly reactive (0 dilutions). Twenty-two weeks after inoculation the left fundus showed three small hemorrhages and exudates were seen along several retinal vessels. At that time the VDRL was again weakly reactive (0 dilutions). A repeat test 10 days later showed a rise in reagin titer to 2.5 dils ; both the TPI and FTA-ABS tests were reactive. One month later the fundus lesions had completely resorted. Nine months after inoculation, the fundus showed perivascular sheathing (along the vessels previously lined with hemorrhages and exudates). Comment, this monkey developed retinal hemorrhages and exudates five months after cisternal inoculation. T h e diagnosis of syphilis was proven by V D R L conversion and

Fig. 6 (Taylor, Smith and Singer). Owl monkey 51. Fluorescein photograph of the left fundus at 21 weeks, showing perivascular hemorrhages and exudates.

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nemes) intracisternally. VDRL 12 weeks later was weakly reactive (0 dils.)· Twenty-seven weeks after inoculation there was definite anisocoria, the right pupil being larger than the left. Both pupils reacted well to light. No definite fundus lesions were noted. A popliteal lymph node removed 23 weeks after inoculation produced a dark-field positive lesion on the back of a normal rabbit when injected intracutaneously. Anisocoria has remained until the present. Comment. The diagnosis of syphilis in this animal was based on VDRL conversion and reactive T P I and FTA-ABS tests. The FTA-ABS was reactive six weeks prior to VDRL conversion. A positive lymph node transfer gave further confirmation of syphilis in this animal. OWL MONKEY (figs. 7 and 8) 57

This animal was given 0.01S ml of T. pallidum into the anterior chamber of the right eye (15,000 organisms) and a similar dose of saline into the anterior chamber of the left eye as a control. Six weeks after inoculation eye examination was normal. Five and one-half months after inoculation, a subhyaloid hemorrhage and an exudative macular lesion were noted in the left eye. Three weeks later, the hemorrhage was partially resorbed. Eleven months after inoculation this animal developed anisocoria which has persisted until the present time. Comment.

Repeated

VDRL, T P I

and

Fig. 8 (Taylor, Smith and Singer). Owl monkey 57. A fluorescein fundus photograph shows subhyaloid hemorrhage in the left fundus. FTA-ABS tests in this animal have given nonreactive results. It developed fundus lesions at five and one-half months and, at seven months, a popliteal node transfer to a rabbit yielded a dark-field positive lesion. OWL MONKEY 93

This animal received 0.2 ml T. pallidum intracisternally. Six weeks later, the left pupil dilated poorly with mydriatics. Two months after inoculation VDRL and T P I tests were nonreactive but the FTA-ABS test was reactive. At three and onehalf months there was definite aniscoria, the right pupil being larger than the left. At that time both TPI and FTA-ABS tests were reactive but the VDRL remained nonreactive. Six months after inoculation the VDRL was reactive to two dilutions. Comment. Conversion of specific treponemal tests before conversion of the reagin tests were again observed in this animal. The FTA-ABS became reactive four months before VDRL conversion, and the T P I was reactive two and one-half months prior to VDRL conversion. Fig. 7 (Taylor, Smith and Singer). Owl monkey 57 received 0.015 ml T. pallidum in the anterior chamber of the right eye. Five and one-half months later the left fundus showed a subhyaloid hemorrhage and an exudative macular lesion.

SUMMARY

The clinical phenomenon of seronegative syphilis has been studied in the primate. Four animals presented simultaneous sero-

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WILLIAM H. TAYLOR, J. LAWTON SMITH AND J. A. SINGER

negativity and ocular or neurologic lesions, The extreme sensitivity and specificity of the fluorescent treponemal antibody absorp-

tion procedure in the primate has been documented. 1638 N.IV. 10th Avenue (33136).

REFERENCES

1. Smith, J. L. : The Treponema pallidum immobilization test in ophthalmology. Am. J. Ophth., 57 : 973-977 (June) 1964. 2. Smith, J. L., Singer, J. A., and Reynolds, D. H. : Experimental ocular and neurosyphilis. Brit. J. Vener. Dis., 41: No. 1 (Mar.) 196S. 3. Smith, J. L., and Singer, J. A. : The fluorescent treponemal antibody absorption procedure in ophthalmology. In press. 4. Smith, J. L. : The University of Miami Neuro-Ophthalmology Symposium. Springfield, 111., Thomas, 1964. 5. Hunter, K. 1'., Deacon. W. E., and Meyer, P>. A.: An improved KTA test for syphilis: The absorption procedure (FTA-ABS). Pub. Hlth. Rep., 79:410-412 (May) 1964.

CORRELATION OF OPHTHALMODYNAMOMETRY OPHTHALMIC ARTERY PRESSURE IN T H E R H E S U S MONKEY* T H O M A S R.

HEDGES, M.D.,

J A M E S D.

AND T H O M A S W .

W E I N S T E I N , M.D., LANGFITT

WITH

N E I L F. K A S S E L L

M.D.

Philadelphia, Pennsylvania

Ophthalmodynamometric, ophthalmic artery and systemic blood pressures have been recorded simultaneously in the rhesus monkey. When pressure was applied laterally to the globe, so as not to displace the eye posteriorly into the orbit, cessation of flow in the central retinal artery could be produced without significantly altering the pressure in the ophthalmic artery. On the basis of these observations, we have concluded that ophthalmodynamometry is primarily a measure of pressure in the central retinal artery, not the ophthalmic artery. Tn addition, the effect of increased intracranial pressure on these variables has also been studied. TECHNIQUES

The technique of recording pressure directly from the ophthalmic artery by retrograde cannulation of the vessel at the supraorbital notch has been previously * From the Neuro-ophthalmology Laboratory, Department of Neurosurgery, Pennsylvania Hospital.

reported.1'2 In order to provide additional latitude in the application of the ophthalmodynamometer, a notch was made in the inferior lateral wall of the bony orbit so that the instrument could be applied perpendicular to the anteroposterior axis of the globe. Other techniques, such as lifting the globe out of the orbit by traction on the muscles, were attempted but were not nearly as satisfactory. Systemic arterial pressure was obtained from the femoral artery and intracranial pressure from a catheter in the cerebral subarachnoid space. All pressures were recorded with fluid transducers and displayed on a Sanborn polygraph. Intracranial pressure was elevated by inflation of an extradural balloon over the cerebral hemisphere contralateral to the recording catheter. The Müller ophthalmodynamometer used in these experiments is the instrument referred to in the book on ophthalmodynamometry by Weigelin and Loebstein.3 In order to convert the plunger readings into mm Hg, corrections are made according to