Clioquinol

* Background.-A mysterious disease spread over Cuba from 1991 to 1993, the cause of which has not been fully established. Major symptoms were the same in patients with the Cuban disease as in patients with subacute myelooptico-neuropathy (SMON), which had occurred in Japan nearly 30 years ago and from which Inoue-Melnick virus (IMV) type 1 was first isolated. We investigated the presence of IMV in cerebrospinal fluid samples of patients with this epidemic neuropathy in Cuba.

Methods.-The established method for detecting IMV in MRC-5 fibroblasts was used throughout the study, which was conducted in two independent laboratories, one in Buffalo, NY, and the other in Kyoto, Japan. The cerebro

spinal fluid samples of 20 patients and four controls were provided by Cuban researchers.

Results.-All cerebrospinal fluid specimens from 20 Cuban patients tested were positive for IMV type 2 (100%), and four cerebrospinal fluid specimens from Cuban controls were negative for IMV (0%). These clear-cut results were identical in both laboratories.

Conclusion.-Our study indicates an important association of IMV type 2 with patients of this Cuban epidemic. The etiologic role of IMV type 2 in the epidemic neuropathy in Cuba, along with weak cytopathic effect viruses isolated by the Cuban group, remains to be elucidated.

(Arch Pathol Lab Med. 1998;122:520-522)

A severe epidemic of a mysterious disease swept over Cuba from 1991 to 1993, and appeared to end in the summer of 1993 after more than 51000 patients had been affected.'1-8This disease is now called epidemic neuropathy in Cuba1,4,8Its manifestation as optical and peripheral neuritis-like symptoms was akin to that of the subacute myelo-optico-neuropathy (SMON), which spread in Japan more than 30 years ago.9-11 Inoue et al first isolated a novel virus from both the cerebrospinal fluid (CSF) and feces of patients with SMON in Japan, and the isolate was named SMON virus.12-16 Later, Melnick et al also isolated the same or similar viruses with different antigenicity, from the CSF of patients with multiple sclerosis and other neurologic disorders.l7-19 Subsequently, these viruses were called Inoue-Melnick virus (IMV), and three serotypes have been identified: the original SMON virus is now IMV type 1 (IMV-1), and the other two isolates from Houston, Tex, are IMV type 2 (IMV-2) and IMV-intermediate.20 The serotypings were done by neutralization assays in the MRC-5 human fibroblast cultures with reference antisera made in rabbits against IMV-1 or IMV-2.20 The cytopathic effects (CPEs) of IMV in these cells are weak and require experienced eyes to distinguish. Because of difficulty in handling these viruses, the nature of IMV has not been fully characterized, but it appears to be a herpesvirus; it is of medium size, contains DNA, is enveloped, and is sensitive to phosphonoacetic acid.16 It was reported that there are no cross-reactions by neutralization between IMV and known human herpesviruses, including HHV-6.21

A Cuban group of researchers recently reported that enteroviruses, Coxackie A9, and related agents with weak CPE have been isolated from the CSF of 84% of patients with epidemic neuropathy in Cuba." They suggested these viral agents played a role in this disease epidemic along with other factors. We have now isolated IMV-2 from the CSF of patients with epidemic neuropathy in Cuba. The results of this study are the subject of this report.

MATERIALS AND METHODS

Clinical Specimens

Cerebrospinal fluid samples from patients with epidemic neuropathy in Cuba, along with samples from controls, were collected by Cuban doctors and stored at -70 deg C at the Institute of Tropical Medicine, Havana, Cuba. Samples from 20 patients and four controls (surgery patients without any symptoms of epidemic neuropathy) were kindly provided by G. Kouri, MD, and M. G. Guzman, MD, of the Institute. Unfortunately, detailed information on individuals from whom the CSF specimens were taken, such as clinical conditions, age, sex, and geographic locations, were not available to us. The CSF specimens were shipped from Havana, Cuba, to Buffalo, NY, and subsequently to Kyoto, Japan, in air-tight containers packed in dry ice. This international collaborative study was approved by the administration of Roswell Park Cancer Institute, Buffalo, NY.

Isolation of IMV

Established procedures were used to isolate IMV,2223 by inoculating a 1:10 dilution of each CSF sample after three cycles of quick freezing and thawing. Isolation and titration of IMV in CSF samples were performed by inoculating serial 10-fold dilutions of the specimens into tube cultures of MRC-5 human fibroblasts maintained in Eagle's minimum essential medium plus 2% fetal bovine serum, 0.1% bicarbonate, and antibiotics including an antimycoplasma agent. The tube cultures were incubated for 7 days at 36 deg C in a roller tube apparatus. The CPEs were observed on days 6 and 7 after inoculation of virus. The CPEs were weak, and experienced eyes were needed to distinguish the IMV CPEs.22,23 Briefly, the CPEs appeared mostly on the edge of the cell sheet, which must be examined at a low magnification (4 x 10). The fibroblasts became elongated and slightly thickened with increasing space between the cells. These effects followed a disruption of the parallel cell patterns and criss-crossing of the thickened fibroblasts. Such areas of CPE continued along the border of cell sheets, gradually moving inward. The center part of the cell sheet did not become involved. Infectivity titers of IMV in CSF were calculated by the Reed-Muench method24 and were expressed as median tissue culture infective dose (TCID^sub50^)/0.1 mL. Subcultures were made by inoculating fresh tube cultures with the culture fluid from tubes that received the highest dilutions of CSF and showed CPEs characteristic of IMV. In negative cases, at least two blind passages were made prior to declaring the culture negative for IMV

Neutralization of IMV

For identification and serotyping, neutralization tests were performed with two reference rabbit antisera against either prototypes of IMV-1 (SMON-S strain) or IMV-2 (Baylor-6 strain), according to established criteria.20 These reference antisera were prepared in Japan and had high neutralizing antibody titers to the homologous type of IMV.

Sensitivity Test to Phosphonoacetic Acid

The sensitivity of each isolate to phosphonoacetic acid, an inhibitor of herpesviruses, was examined. This test could distinguish IMV from enteroviruses.25 The IMV isolates in MRC-5 cells usually had a titer of 6.5 log^sub10^TCID^sub50^/O.1 mL. When the growth of isolates was suppressed by more than 4.5 log,^sub10^TCID^sub50^ in the presence of 20 (mu)g/mL of phosphonoacetic add, the isolates were judged as sensitive to the acid.

RESULTS

To date, a total of 24 CSF specimens have been shipped from Cuba, including CSF samples from 20 patients with epidemic neuropathy in Cuba and four specimens from Cuban controls. Virus isolation, direct titration, and identification of IMV isolates were conducted independently in two separate laboratories, one in Buffalo (M.I.) and one in Kyoto (YN.). Results are shown in Table 1 and Table 2. All 20 CSF specimens from Cuban patients with neuropathy were clearly positive for IMV (100%). Furthermore, all the isolates were typed as IMV-2 and were sensitive to phosphonoacetic acid at both laboratories (Table 1). Concentrations of infectious IMV-2 in CSF specimens were directly titrated, and geometric mean titers of IMV in patient CSF samples were 3.59 and 3.65 log^sub10^TCID^sub50^/0.1 mL at the Buffalo and Kyoto sites, respectively (Table 2). No IMV was detectable in CSF samples from the four Cuban controls tested.

COMMENT

Many causes have been considered to be responsible for the outbreak of epidemic neuropathy in Cuba, especially nutritional deficiencies.1-8 The role of infectious agents such as viruses cannot be excluded, as has been discussed previously.1,4,8 The results we report here clearly demonstrate a high prevalence of IMV-2 among patients with epidemic neuropathy in Cuba, and moderately high titers of IMV-2 were consistently found in CSF specimens from the Cuban patients. The direct or causative role of IMV in this Cuban disease still needs to be investigated.

Recently, a group of Cuban researchers reported that viruses were isolated in CSF samples from 105 of 125 (84%) Cuban patients with epidemic neuropathy.8 Five of the viruses were identified as typical Coxsackie A9. The remaining 100 isolates, so-called CPE-L type of viruses, showed weak but slowly progressive CPE in Vero cells, and all were transmissible. As IMV is able to grow but unable to produce CPE in Vero cells (YN. and YK.I., unpublished data), the Cuban isolates may be distinct from IMV-2.

Three Cuban isolates were given to us for further investigation. We found that these Cuban isolates contained IMV-2 in addition to enterovirus-like agents. However, to fully characterize the nature of these viruses, more investigations will be needed at the genomic level.

It is very interesting to point out that Inoue et al isolated IMV-1 from the CSF of almost 80% of SMON patients tested in Japan many years ago.l2-16 Subacute myelo-opticoneuropathy appears to share many symptoms with this Cuban disease. The SMON epidemic in Japan was considered to be the result of toxic effects of clioquinol, an antidiarrheal drug,26 but this conclusion was later challenged by an epidemiologist in England.27 It is extremely important to explore the molecular biology of IMV and to eventually develop simple and reliable assay systems for IMV and its antibodies. A breakthrough in this area of IMV research is absolutely essential for further investigation of this virus, which appears to be associated with many important human diseases.

Recent studies indicate that IMV is a reasonably common virus, at least in the area of Buffalo, NY Seroepidemiologic studies have revealed that Buffalo appears to be an endemic area of IMV, and most people there are exposed to the virus before 15 years of age.28 On the other hand, no IMV antibodies are detectable in Japanese children younger than 10 years.29,30 Also, IMV can be isolated from the CSF of many patients with multiple sclerosis in Buffalo (M.I., unpublished data) and from other IMV-associated diseases, such as colorectal carcinoma.23,31 One may speculate that epidemic neuropathy in Cuba, as well as SMON in Japan, might occur in areas where the majority of people have never been exposed to IMV in childhood. This hypothesis could be tested by seroepidemiologic studies in the Cuban population before and after the epidemic.

We thank Pedro Mas, MD, Maria G. Guzman, MD, and Gustavo Kouri, MD, Institute of Tropical Medicine (Pedro Kouri), Havana, Cuba, for supplying precious patient specimens. We also thank the Government of Cuba; the Pan American Health Organization; Joseph L. Melnick, PhD, Division of Molecular Virology, Baylor College of Medicine, Houston, Tex, and James Karr, PhD, of Roswell Park Cancer Institute, Buffalo, NY, for their special encouragement and support in making this study possible; and Arlene R. Collins, PhD, Department of Microbiology, State University of New York at Buffalo, for critical reading of this manuscript. Travel grants were provided by the Pan American Health Organization and the Ministry of Public Health, the Government of Cuba; anonymous donations for the Inoue-Melnick virus studies are greatly appreciated.

References

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2. Lincoff NS, Odel JG, Hirano M. "Outbreak" of optic and peripheral neuropathy in Cuba?JAMA.1993;270:511-518.

3. Tucker K, Hedges TR. Food shortages and an epidemic of optic and peripheral neuropathy in Cuba. Nutrition Rev 1993;51349-357.

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6. Borrajero I, Perez JL, Dominguez C, et al. Epidemic neuropathy in Cuba: morphological characterization of peripheral nerve lesions in sural nerve biopsies. J Neurol Sci. 1994;127:68-76.

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9. Sobue I, Ando K, Takagaki T, lida M, Yamamura Y, Matsuoka Y. Myelo-neuropathy with abdominal disorders in Japan. Neurology. 1971;21:168-173. 10. Kono R. Introductory review of subacute myelo-optico-neuropathy (SMON) and its studies done by the SMON Research Commission. Jpn J Med Sci Biol.1975;28:1-21.

11. Shimada Y. Subacute myelo-optico-neuropathy (SMON). Asian Med J. 1971;14:339-349.

12. Inoue YK, NishibeY, Nakamura Y.Virus associated with S.M.O.N. in)apan. Lancet. 1971;i:853-854.

13. Inoue YK, Nishibe Y, Kimura T, Shimada T, Yamada G. Antibody response in cases of subacute myelo-optico-neuropathy against the associated virus. Lancet. 1972;ii:705-706.

14. Nishibe Y, Kimura T, Inoue YK. Properties of virus associated with subacute myelo-optico-neuropathy. Arch ges Virusforsch. 1973;40:189-197. 15. Bessho H, Nishibe Y, Inoue YK. Morphology of SMON virus. Arch ges Virusforsch. 1974;45:370-372.

16. Inoue YK. Inoue-Melnick virus and associated diseases in man: recent advances. Prog Med Virol.1991 ;38:167-179.

17. Melnick JL, Seidel E, Inoue YK, Nishibe Y. Isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis. Lancet. 1982;i:830-833.

18. Melnick JL, Wang SS, Seidel E, Muchinik G, Zhang LB, Lanford RE. Characterization of IM virus, which is frequently isolated from cerebrospinal fluid of patients with multiple sclerosis and other chronic diseases of the central nervous system. J Virol.1984;52:739-744.

19. Melnick JL, Seidel E, Wang SS, et al. Persistence of Inoue-Melnick virus and antibody in cerebrospinal fluid. J Clin Microbiol.1985;22:651-653. 20. Inoue YK, Nishibe Y, Melnick )L. Classification of Inoue-Melnick virus into three antigenic types. J Clin Microbiol. 1984;20:118-119.

21. Dale JK, Straus SE, Ablashi DV et al. The Inoue-Melnick virus, human herpesvirus-6, and chronic fatigue syndrome. Ann Intern Med. 1989;110:92-93. 22. Nishibe Y, Inoue YK, Melnick JL. Factors influencing the assay of InoueMelnick virus. J Virol Methods.1987;17:293-298.

23. Ito M, Rodriguez-Bigas MA, Evans MJ, Petrelli NJ. Isolation of Inoue-Melnick virus from colorectal carcinoma tissues. Cancer. 1993;71:1960-1963. 24. Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg.1938;27:493-497.

25. Nishibe Y, Inoue YK. Effects of phosphonoacetic acid on subacute myelooptico-neuropathy virus in vitro and in vivo. J Med Virol. 1978;2:225-229. 26. Tsubaki T, Homma Y, Hoshi M. Neurological syndrome associated with clioquinol. Lancet. 1971;i:696-697.

27. Mead TW. Subacute myelo-optico-neuropathy and clioquinol: an epidemiological case-history for diagnosis. Br] Prev Soc Med. 1975;29:157-169.

28. Ito M. Seroepidemiology of Inoue-Melnick virus in the general population of Buffalo, New York. J Med Virol.1995;47:83-86. 29. Nishibe Y, Inoue YK. Reappraisal of modified neutralization test for InoueMelnick virus. J Virol Methods. 1993;42:345-350. 30. Inoue YK, Nishibe Y, Kimura T, Melnick JL. Antibody survey for InoueMelnick virus infections in Osaka and Houston. Am J Epidemiol.1987;125:536538.

31. Inoue YK, Nishibe Y, Nakamura Y. Direct isolation of Inoue-Melnick virus from human colorectal carcinoma tissues but not from stomach carcinoma tissues. J Med Virol. 1991;35:250-252.

Accepted for publication February 18, 1998. From Roswell Park Cancer Institute, Buffalo, NY (Dr Ito); Institute for Virus Research, Kyoto University, Japan (Dr Nishibe); and Katsura, Kyoto, Japan (Dr Inoue). Dr Ito is now with the Department of Microbiology, State University of New York at Buffalo.

Reprint requests to Department of Microbiology, State University of New York at Buffalo, Buffalo, Buffalo, NY 14214 (Dr Ito).

Copyright College of American Pathologists Jun 1998
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