247

J. K. C TSUI AND OTHERS: REFERENCES

1 Alpers BJ, Drayer CS. The organic background of some cases of spasmodic torticollis.Am J Med Sci 1937; 193: 378-84.

2 Foerster O. Mobile spasm of the neck muscles and its pathological basis. J CompNeurol 1933; 58: 725-35

3 Grinker RR, Walker AC The pathology of spasmodic torticollis with a note onrespiratory failure from anaesthesia in chronic encephalitis. J Nerv Ment Dis 1933;78: 430-37.

4 Stoessl AJ, Martin WRW, Clark C, et al. PET studies of cerebral glucose metabolismin idiopathic torticollis. Neurology (in press).

5 Forgach L, Eisen A, Fleetham J, Calne DB. Studies on dystonic torticollis duringsleep. Abstract presented at 38th Annual Meeting, American Academy ofNeurology, New Orleans. Neurology (in press)

6 Lal S Pathophysiology and pharmacotherapy of spasmodic torticollis: A review. Can JNeurol Sci 1979; 6: 427-35.

7 Scott AB Botulinum toxin injection of eye muscles to correct Strabismus. Trans AmOphthalm Soc 1981; LXXTX: 734-70.

8 Scott AB. Injection treatment of endocrine orbital myopathy Docum Ophthalm 1984;58: 141-45

9 Scott AB, Kraft SP. Botulinum toxin injection in the management of lateral rectusparalysis Ophthalmology 1985; 92: 676-83.

10 Mauriello JA Jr Blepharospasm, Meige syndrome, and hemifacial spasm: Treatmentwith botulinum toxin Neurology 1985; 35: 1499-500.

11 Scott AB, Kennedy RA, Stubbs HA. Botulinum toxin injection as a treatment forblepharospasm. Arch Ophthalmol 1985; 103: 347-50

12 Tsui JK, Eisen A, Mak E, Carruthers J, Scott AB, Calne DB. A pilot study on the useof botulinum toxin in spasmodic torticollis. Can J Neurol Sci 1985; 12: 314-16.

EVALUATION OF SEROLOGICAL

CROSS-REACTIVITY BETWEEN ANTIBODIESTO PLASMODIUM AND HTLV-III/LAV

ALAN E. GREENBERG

ALEXANDER J. SULZERCHARLES A. SCHABLEWILLIAM E. COLLINS

PHUC NGUYEN-DINH

Malaria Branch, Division of Parasitic Diseases, and AIDS

Program, Center for Infectious Diseases, Centers for Disease Control,Atlanta, Georgia, USA

Summary Serum samples from 460 patients withexisting or previous Plasmodium infections,

high antimalarial antibody titres, and no apparent risk ofexposure to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV)were assayed for HTLV-III/LAV antibody; only 1 sample,from a 21-year- old African woman, was strongly reactive byenzyme-linked immunosorbent assay (ELISA) and positiveby western blot. Conversely, no sample from 100 HTLV-III/LAV-positive American homosexual men was stronglyreactive for antibodies to the four Plasmodium species thatinfect human beings by an indirect fluorescent antibodytechnique, or for antibodies to Plasmodium falciparum by anELISA technique. Thus, exposure to Plasmodium does notresult in HTLV-III/LAV seropositivity, and HTLV-III/LAV antibodies are not strongly cross-reactive with malarialantigens.

IntroductionTESTING for antibodies to the human T-lymphotropic

virus type III/lymphadenopathy-associated virus (HTLV-III/LA V) is valuable in surveillance of the acquiredimmunodeficiency syndrome (AIDS). The specificity of theenzyme-linked immunosorbent assay (ELISA), the testmost commonly used for HTLV-III/LAV antibodyscreening, is therefore very important. Some reports havesuggested an association between infection with

Plasmodium, the causative agent of malaria, and HTLV-III/LAV seropositivity.1,2 Of 250 healthy adults from ruralZaire, 31 were HTLV-III/LAV-positive by ELISA, andanother 30 were on the borderline; HTLV-III/LAVseropositivity correlated with high titres of antibodies toPlasmodium falciparum.1 In a study from Venezuela, thefrequency of HTLV-III/LAV seropositivity was higher inpatients with P falciparum and P vivax infections than in

healthy blood donors in the same area.2 No evidence ofclinical AIDS was found in either of these populations.Serological cross-reactivity between antimalarial antibodiesand HTLV-III/LAV is one possible explanation of thesefindings.To determine whether antibodies to Plasmodium are

cross-reactive with HTLV-III/LAV, we studied serumsamples from patients who had well-documented infectionswith one agent but no apparent risk of exposure to the other.

Patients and Methods

Patients with Plasmodium InfectionsWe selected serum samples from 460 patients with existing or

previous Plasmodium infections and no apparent risk of exposure toHTLV-III/LAV. All had high antimalarial antibody titres as

determined by a standard indirect fluorescent antibody technique. 3The patients were selected from six different populations. The

first group was 78 American travellers whose samples werecollected in 1964-1974 during or after acute malarial infections withvarious Plasmodium species. The earliest known case of HTLV-I I I/LAV seropositivity in the United States occurred in 1978.4

In a previous study, samples from 9 of 16 neurosyphilitics withinduced P falciparum and P malariae infections in 1962-1963 wereweakly reactive on an HTLV-III/LAV ELISA test, and the serumfrom 1 was strongly reactive. Samples from 4 of these patients wereweakly positive on western blot for the p24 band of HTLV-III/LAV. We selected 31 serum specimens collected during theacute infections of 4 of these patients and tested them for HTLV-III/LAV antibody.We also analysed specimens collected in 1985 from two African

and two South American populations with chronic exposure tomalaria and high antimalarial antibody titres. The samples werefrom 57 African children (aged 1-5-8 years) with severe

P falciparum infections; 183 parasitaemic, symptom-free Africanchildren and adults (0-5-55 years) with chronic exposure to

P falciparum, P ovale, and P malariae; 59 South American childrenwith chronic exposure to P falciparum, P vivax, and P malariae; and52 South American Indian adults living in an area where P vivax isendemic. The African populations came from outside the knownhigh-prevalence areas of HTLV-III/LAV seropositivity in CentralAfrica.

Patients with HTLV-III/LAV InfectionsWe selected specimens that had been collected in 1982-1984

from 100 American homosexual men. All samples were repeatedlyreactive to HTLV-III/LAV by ELISA and positive by westernblot. 75 of the men had well-documented AIDS, and 25 weresymptom-free. Medical records indicated that none of these

patients had acute malarial infections, although blood smears werenot examined specifically for Plasmodium parasites.

Malaria SerologyAll serum samples were assayed for antibodies to Plasmodium by a

standard indirect fluorescent antibody technique.3 In a previousstudy of positive and negative control sera, the sensitivity andspecificity of this test were both greater than 95°" when a cut-offtitre of 1/64 was used.s The specificity approaches 100’B,, however,if 1/1024 is used as the cut-off titre for P falciparum and 1/256 as thecut-off titre for P vivax, P ovale, and P malariae, Thus, titres of lessthan 1/64 are considered non-reactive; 1/1024 or greater(P falciparum) or 1/256 or greater (P vivax, P ovale, and P malariae)strongly reactive; and intermediate titres weakly reactive.

Samples from the 100 HTLV-III/LAV-positive patients werealso assayed for antibodies to P falciparum by a previously describedELISA technique.6 An optical density value of 03 is the standardcut-off used in our laboratory, but the specificity of the test

approaches 100" if the cut-off is 1 ’0. Thus, specimens with opticaldensity values of less than 03 are considered non-reactive; 03-10weakly reactive; and greater than 1.0 strongly reactive.

248

TABLE 1—HTLV-III/LAV SEROLOGY IN 460 PATIENTS WITH EXISTING OR PREVIOUS MALARIAL INFECTIONS

PF = P falciparum, PV = P vivax, PO = P ovale, PM = P malariae; ND = not done.*111/183 African sera analysed for antibodies to P ovale and P malariae.

HTLV-III/LAV SerologyWe used a commercially available ELISA kit (Abbott

Laboratories HTLV-III antibody test; North Chicago, Illinois) toassay all specimens for antibodies to HTLV-III/LAV. A samplewas considered positive only if repeatedly reactive by ELISA(sample/kit optical density ratios greater than 1 -0) and positive byWestern blot (reactive with either the p24 or the gp41 band).’ In aprevious study in our laboratory, the specificity of the AbbottELISA kit was shown to be greater than 99-8% when a cut-off ratioof 1 -0 was used; only 1.9% of samples that were weakly reactive byELISA (optical density ratios 1.0-5.9) were positive by Westernblot, but 86.7% of sera that were strongly reactive by ELISA(optical density ratios greater than 6-0) were positive by Westernblot.8

Results

Because of extensive cross-reactivity among antibodies tothe four Plasmodium species in the indirect fluorescentantibody test and the transmission of several Plasmodiumspecies in areas where malaria is endemic, malaria

serological results are rarely monospecific. Therefore, wecalculated the number and percentage of specimens thatsatisfied our criteria for species-specific reactivity.

460 patients with a variety of malaria serological profileswere tested for HTLV-III/LAV antibody (table I). Serumfrom a 21-year-old African woman was strongly reactive byELISA (optical density ratio 13-2, with an optical densityvalue > 2-0) and positive by Western blot (both the p24 andthe gp41 band were present). Samples from 6 patients wereweakly reactive by ELI SA (optical density ratio, value: 1 17,0 143,1-27,0-155,1-28,0-193,1-50,0-183; 1-82,0-222,2-37,0-290), but all were negative by Western blot. Serum fromanother patient was weakly reactive by ELISA (1 -33,0-202),but there was not enough serum for Western blot analysis.None of the 100 HTLV-III/LAV-positive patients was

strongly reactive for antibodies to Plasmodium species by theindirect fluorescent antibody test or for antibodies to

Pfalciparum by ELISA (table II). 21 patients’ samples wererepeatedly weakly reactive to P falciparum by the indirectfluorescent antibody test (19 at 1/64,2 at 1/256), and 17 were

TABLE II-MALARIA SEROLOGY IN 100 AMERICAN HOMOSEXUALMEN INFECTED WITH HLTV-111/LAV

weakly reactive by ELISA (optical density values0-31—0-60). Only 1 sample, however, was weakly reactiveby both techniques. 5 samples were weakly reactive toP vivax, 1 to P ovale, and 3 to P malariae.

Discussion

This study shows that antibodies to Plasmodium are notcross-reactive with HTLV-III/LAV. All the patients whohad well-documented Plasmodium infections, highantimalarial antibody titres, and no apparent risk of

exposure to HTLV-III/LAV were seronegative for thevirus. The only sample positive for HTLV-III/LAVantibody was collected in 1985 from a 21-year-old Africanwoman. Since HTLV-III/LAV seropositivity and AIDShave been identified in heterosexually active Africanadults9-12 this patient may have been exposed to HTLV-111;LAV.

HTLV-III/LAV antibodies in patients with or withoutAIDS did not cross-react strongly with malarial antigensand, although 37 samples were weakly reactive to

P falciparum by one of the tests used, only 1 was weaklyreactive by both tests. Though unlikely, past Plasmodiuminfections could account for these findings, since the

patients’ travel histories were not available. Alternatively,the polyclonal stimulation of B cells and increasedimmunoglobulin secretion that has been described in AIDSpatients 13 might cause the weak reactivity.HTLV-III/LAV-antibody screening is becoming

increasingly important in public health practice and inscientific investigations, and there has been concern aboutthe specificity of the assay in patients who have been exposedto malaria. The US Public Health Service recommends thatall individuals at high risk for HTLV-III/LAV infection(including homosexual men, intravenous drug users,

prostitutes, haemophiliacs, and people from countrieswhere there is apparently substantial heterosexualtransmission of HTLV-III/LAV) should be offeredvoluntary testing.14 Furthermore, all blood donations in theUSA are screened for HTLV-III/LAV antibody," andAmerican military recruits are tested routinely. Positive

HTLV-III/LAV serological results in people who havetravelled to or lived in malaria-endemic areas should not beattributed to cross-reactivity with antibodies to Plasmodium.We thank Ms Peggy Stanfill, Mr Albert Turner, Mr Larry Wells, Ms Faye

Cowart, and Ms Lois Norman for technical assistance; Dr Kirk Miller, DrGary Campbell, Dr Thomas Quinn, Dr Jonathan Mann, Dr Carlos

Campbell, and Dr James Curran for helpful discussions and editoria. assistance.

Correspondence should be addressed to A. E. G., Malaria Branch, C-’’.Centers for Disease Control, Atlanta, GA 30333, USA.

249

A. E. GREENBERG AND OTHERS: REFERENCES

1 Biggar R, Gigase P, Melbye M, et al. ELISA HTLV retrovirus antibody reactivityassociated with malaria and immune complexes in healthy Africans. Lancet 1985; ii:520-23

2 Volsky D, Yin Tang Wu, Stevenson M, et al. Antibodies to HTLV-III in Venezuelanpatients with acute malarial infections. N Engl J Med 1986; 314: 647-48.

3 Sulzer AJ, Wilson M, Hall EC. Indirect fluorescent antibody tests for parasiticdiseases Am J Trop Med Hyg 1969; 18: 199-205.

4 Curran JW, Morgan WM, Hardy AM, et al. The epidemiology of AIDS: currentstatus and future prospects. Science 1985; 229: 1352-57

5 Sulzer AJ, Wilson M. The fluorescent antibody test for malaria. CRC Crit Rev ClinLab Sci 1971, 2: 601-09.

6 Spencer H, Collins WE, Chin W, Skinner J. The enzyme-linked immunosorbentassay ELISA) for malaria. Am J Trop Med Hyg 1979; 28: 927-32.

7 Tsang VCW, Peralta JM, Simons AR. Enzyme-linked immunoelectrophoresistransfer blot techniques (EITB) for studying the specificities of antigens andantibodies separated by gel electrophoresis. In: Landgone JJ, Vunakie HV, eds.Methods in enzymology immunochemical techniques, Part E. New York:Academic Press, 1983 377-91.

8 Ward JW, Grindon AJ, Feorino PN, et al Laboratory and epidemiologic evaluation of

an enzyme immunoassay for antibody to human T-lymphotropic virus type III.JAMA 1986; 256: 357-61.

9. Kreiss J, Koech D, Plummer F, et al. AIDS virus infection in Nairobi prostitutes. NEngl J Med 1986; 314: 414-18.

10. Piot P, Quinn T, Taelman H, et al. Acquired immunodeficiency syndrome in aheterosexual population in Zaire. Lancet 1984; ii: 65-69.

11. Clumeck N, Robert-Guroff M, Van De Perre P, et al. Seroepidemiological studies ofHTLV-III/LAV antibody prevalence among selected groups of heterosexualAfricans. JAMA 1985; 254: 2599-602

12. Van De Perre P, Clumeck N, Carael M, et al. Female prostitutes: a risk group forinfection with human T-cell lymphotropic virus type III. Lancet 1985; ii: 524-26.

13. Lane HC, Masur H, Edgar LC, et al. Abnormalities of B-cell activation and

immunoregulation in patients with the acquired immunodeficiency syndrome. NEngl J Med 1983; 309: 453-58.

14. Centers for Disease Control. Additional recommendations to reduce sexual and drugabuse-related transmission of human T-lymphotropic virus type III/Lymphadenopathy-associated virus. MMWR 1986; 35: 152-55.

15. Centers for Disease Control. Provisional public health service inter-agencyrecommendations for screening donated blood and plasma for antibody to the viruscausing acquired immunodeficiency syndrome. MMWR 1985; 34: 1-5.

Preliminary Communication

UNEXPECTED MOBILISATION OF LEADDURING CISPLATIN CHEMOTHERAPY

A. M. EL-SHARKAWI1S. COBBOLD3C. J. EVANS4

D. R. CHETTLE5

W. D. MORGAN2

M. B. M. JAIB4L. J. SOMERVAILLE5

M. C. SCOTT5

Departments of Radiotherapy and Oncology,1 Medical Physicsand Clinical Engineering,2 and Medicine,3 Singleton Hospital,

Swansea; Department of Physics, University College Swansea;4 andMedical Physics Group, Department of Physics, University of

Birmingham5

Summary During an investigation by X-ray fluor-escence of platinum uptake in the kidney

after chemotherapy with cisplatin, lead was found to haveaccumulated in the kidney in four subjects. The averagekidney lead burden in one case exceeded 800 µg/g. Althoughtwo of the subjects had been occupationally exposed to lead,the other two had not. The tibia lead burden was also high inthe two subjects in whom it was measured. The origins ofthis mobilised lead and the implications for cisplatinnephrotoxicity are discussed.

INTRODUCTION

CISPLATIN is widely used in cancer chemotherapy.12There are, however, several side-effects, of which

nephrotoxicity is the major dose-limiting factor.3,4 To

investigate this nephrotoxicity in more detail, and to obtaincertain pharmacodynamic information5 directly, theSwansea In-Vivo Analysis Research Group (SIVARG) hasdeveloped an X-ray fluorescence technique6 to measure theuptake of platinum, both in the kidney and in some tumoursites. A similar principle has been used by other groups forin-vivo measurements of lead in finger bone7,8 and tibia,9,10of mercury in the head and wrist,li and of cadmium in thekidney.’2

.

METHODS

The technique involves irradiating the kidney with 122 keV and136 keV gamma rays from a cobalt-57 source (7 mCi; 259 MBq) andseeking emitted X-rays with a hyperpure germanium detector withits axis at 90° to the incident gamma-ray beam. Because of the high

attenuation of the incident and detected photons, the overlyingtissue affects the measurement sensitivity and accuracy. The depthof the kidney is therefore determined by ultrasound so thatattenuation corrections can be made. For an overlying tissue depthof 35 mm, a skin dose of 1-8 mGy (180 mrad), and a measurementtime of 2000 s, the sensitivity (defined as two standard errors of thenet peak counts) is 37 Ilgjg. It should be noted that with a cobalt-57source activity of 20 mCi, the sensitivity can be reduced to 20 Ilgjgfor an incident skin dose of 5 mGy (500 mrad) and the samemeasurement time. Elements with similar atomic numbers, andhence X-ray energies, are detected at the same time, but they havedifferent, though similar, measurement sensitivities.A pilot study in twenty patients who have given their informed

consent to the procedure is in progress. The results of this study, inwhich platinum retention and distribution will be related to thetherapeutic regimen and indices of nephrotoxicity, will be presentedin due course. This report concerns the unexpected findings in fourof the first ten patients of substantial concentrations of lead in thekidneys.

PATIENTS AND RESULTS

Patient A

This 66-year-old man had worked as a painter since theage of 14. His work had been both domestic and industrial,including painting ships for which lead paint is widely used.In December, 1985, 2 days after he had received a secondcourse of chemotherapy, the platinum content of his rightkidney was measured. A small peak was observed in thespectrum, corresponding to a platinum concentration of30 µg/g. However, the spectrum was dominated by the largeK&agr;1 and KiX2 peaks of lead; on the assumption that theseemanated from the kidney, these peaks were subsequentlyanalysed to yield a lead concentration in the kidney of813 ± 48 µg/g tissue.

Such a massive lead burden is quite unprecedented, evenin the most severe circumstances of lead intoxication. 13Indeed, the level was so high that it was received withdisbelief. Careful checks were carried out on the apparatus.The cisplatin compound was tested for contamination, sincetraces of lead had been detected in its vial. However, sincethe measurement had been carried out identically for otherpatients in whom no lead peaks were observed, theconclusion remained that patient A showed a very largeconcentration of lead in the volume of tissue containedwithin the fields of view of the X-ray source and thedetector, most probably the kidney. A blood sample takenthe next day showed a lead level of 25 µg/d1, at the top of thenormal range.