A case of acute hepatitis E in Victoria.

OBJECTIVE: To report a case of acute hepatitis E in Victoria, confirmed by laboratory investigations. CLINICAL FEATURES: A 10-year-old boy presented for medical attention with a seven-day history of anorexia and jaundice, 17 days after arriving from Pakistan. The diagnosis of acute hepatitis E was suspected after exclusion of the known causes of viral hepatitis, and was further established by specific antibody testing and identification of hepatitis E virus-like particles in a faecal sample collected three weeks after the onset of illness. INTERVENTION AND OUTCOME: The patient was managed at home, treated symptomatically and made a complete recovery. CONCLUSION: In patients who arrive from countries where hepatitis E is endemic, and who develop non-A, non-B, non-C viral hepatitis, hepatitis E should be considered as a possible diagnosis.

Cultivation of pathogenic treponema in tissue cultures of SflEp cells.

Recently, the successful in vitro cultivation of the Nichols strain of Treponema pallidum was achieved. Afterward, attempts were made to cultivate three other strains of T. pallidum and two strains of T. pertenue. The cultivation of the KKJ, Mexico A, and Bosnia A strains of T. pallidum was somewhat successful; the average increases were 10.8, 9.1, and 7.5-fold, respectively. The range of growth for each of these strains varied dramatically from experiment to experiment. The KKJ strain varied from 14.4 to 8.0-fold; the Mexico A strain from 12.8 to 5.4-fold; and the Bosnia A strain from 11.3 to 3.6-fold. However, the attempts to cultivate the Gauthier and the FB strains of T. pertenue were unsuccessful. The average increases were 1.7 and 1.9-fold, respectively. Although the maximum growth observed was about threefold with either of these strains of T. pertenue, over 50% of the treponemes remained motile for 10 d. These results suggest that although each of these strains of T. pallidum and T. pertenue has been shown to be genetically identical, they are very diverse biologically even among strains of the same species.

Enumeration of Treponema pallidum cells cultivated in vitro by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to enumerate Treponema pallidum cells. The assay could detect from 2 X 10(7) to 4 X 10(8) treponemes per ml. Reactive rabbit serum and goat anti-rabbit immunoglobulin G (peroxidase conjugate) were used in the assay. Optimum results were obtained when 2,2'-azino-di(ethylbenzthiazolinesulfonic acid) was used as the dye for the enzyme reaction and the reactions were allowed to run for 45 min. Interestingly, assays in which in vivo-cultivated T. pallidum was used produced lower absorbance values than those in which T. pallidum was cultivated in vitro.

Further studies on replication of virulent Treponema pallidum in tissue cultures of Sf1Ep cells.

A number of parameters aimed at optimizing culture conditions for both Sf1Ep cells and Treponema pallidum have been investigated. Optimum temperature for replication of T. pallidum ranged between 33 and 35 degrees C. At 33 degrees C, replication occurred in the presence of atmospheric oxygen concentrations of less than 0.3 to 10%, the optimum range being 1.5 to 5%. No replication occurred in the presence of 12.5% oxygen. When both temperature and oxygen concentrations were varied between 33 and 35 degrees C and 1.5 to 5%, respectively, little differences in replication were noted. Although variation in the oxygen concentration within each temperature group had little or no effect on replication, it did have an effect on motility, which remained greater in the 5% oxygen concentration after 9 to 12 days of cultivation. Optimum concentration of fetal bovine serum in the culture medium was 20%, although replication occurred in concentrations ranging from 5 to 30%. If carefully screened, calf serum could be substituted for fetal bovine serum. Testis extract was an essential component of the culture medium. Although extract obtained from an adult rabbit--either normal or T. pallidum infected--was slightly superior, replication of T. pallidum occurred when rat or hamster testis extract was substituted.

Cultivation of virulent Treponema pallidum in tissue culture.

In a series of seven experiments, the virulent Nichols strain of Treponema pallidum was shown to attach and replicate on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1Ep) growing in conventional monolayer cultures under an atmosphere of 1.5% oxygen. Five days after inoculation of 10(6)T. pallidum, the number of treponemes had increased to between 8 x 10(6) and 2.59 x 10(7). The viability of harvested organisms ranged from 86 to 97%. The number of T. pallidum continued to increase, generally reaching a plateau between days 9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 x 10(8) irrespective of the inoculum, which ranged from 10(6) to 10(8)T. pallidum. Concurrent deoxyribonucleic acid assays were performed on the cultures containing T. pallidum to obtain further evidence of replication. Significant increases in treponemal deoxyribonucleic acid were observed when the inocula ranged from 10(6) to 10(7), with the greatest increases, as might be expected, being in the former group. There was also excellent correlation in the amount of deoxyribonucleic acid per treponeme; the averages for the 10(6), 2.5 x 10(6), and 10(7) groups were 3.46 x 10(-14), 3.28 x 10(-14), and 2.79 x 10(-14) g per treponeme, respectively (3.14 +/- 0.72 x 10(-14) g per treponeme). In each experiment, organisms were harvested from the group inoculated with 10(6)T. pallidum after 7 days of incubation to test for virulence. In all instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms. Scanning electron microscopy revealed that during the course of replication many microcolonies of treponemes formed on the surface of the cells.