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In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities

Although most patients with cutaneous T cell lymphomas, including mycosis fungoides (MF) and its leukemic variant, the Sezary syndrome, are seronegative for antibodies to the human T cell lymphotropic viruses (HTLV-I/II), it has recently been shown that > 95% of such patients harbor proviral DNA sequences related to the region of the HTLV genome that encodes the transregulatory/transforming gene, tax. However, the demonstration of HTLV sequences, even after amplification by polymerase chain reaction (PCR), has not been universally successful, and some investigators continue to question this observation. In an effort to resolve this controversy, we have compared published methodologies that have been less successful with techniques currently used in this laboratory. Major differences were found in (a) the nature of the cells used [freshly isolated versus cultured peripheral blood mononuclear cells (PBMC)] and (b) the methods used to prepare samples for PCR (whole cell lysates versus DNA extracts). PBMC from 10 different MF patients and the healthy daughter of 1 of the patients were subjected to comparative analyses. While all of the PBMC lysates were positive, the DNA extract from only one of these individuals revealed HTLV tax sequences. Studies were also conducted comparing cell lysates and DNA extracts of cultured cells derived from tax sequence-positive PBMC from seven different MF patients. The cells from four of the seven were shown to have retained tax sequences after varying times in culture, when whole-cell lysates were used as targets for PCR amplification and Southern analysis, whereas none of the DNA extracts were positive. It appears that the use of whole-cell lysates instead of DNA extracts and the use of fresh instead of cultured cells greatly enhance the ability to detect HTLV-1 tax sequences in specimens from MF patients

Despite anecdotal literature that Sezary cells express the CD4+ CD7- immunophenotype, no formal validation has been published establishing the use of this immunophenotype for clinical or experimental purposes. Consequently, the only method presently available for Sezary cell identification is nuclear contour analysis, a labor-intensive procedure not generally available at most major medical centers. In this study, the accuracy of CD4+ CD7- subset quantitation for the identification of Sezary cells was examined. The study found that the percentage of CD4+ CD7- cells is elevated in many Sezary syndrome/MF patients relative to normal, healthy individuals. In addition, CD4+ CD7- enumeration correlates with enumeration by nuclear contour analysis in most patients (11 of 15) with elevated CD4/CD8 ratios. The CD4+ CD7- subset also correlates with the expression of other aberrant immunophenotypes, such as CD3low or CD4low. Lastly, CD4+ CD7- subset quantitation correlates with the number of clonal T lymphocytes, as measured using V beta-specific T-cell receptor monoclonal antibodies. The study found this method to be exceptionally accurate, with two caveats: (1) the absence of an expanded CD4+ CD7- subset in patients with a normal CD4/CD8 ratio is uninformative; and (2) in approximately 25% of patients with an elevated CD4/CD8 ratio, the Sezary cells are CD7+

Infection with the human T-cell lymphotropic virus types I and II (HTLV-I/II) usually is determined by tests that detect antibodies to the viral structural proteins. However, recent studies revealed that patients with mycosis fungoides have proviral DNA sequences related to the HTLV transactivating-transforming gene tax, without having antibodies to the virus. These results raised the possibility that the prevalence of HTLV infection in the general population of the United States also may be underestimated. To reassess the prevalence of HTLV-I/II infection effectively, a population at increased risk for infection (i.e., a cohort of injection drug users [IDUs]) was studied. Paired sera and peripheral blood mononuclear cells (PBMCs) from 81 IDUs were subjected to testing by Western blot analysis for antibodies to the viral structural proteins gag and env and by polymerase chain reaction (PCR) Southern analysis to detect gag, pol and tax proviral DNA sequences. Western blot assays showed 1 of 81 IDUs to be positive for HTLV-I, 14 to be positive for antibodies to HTLV-II, and 3 to be HTLV-serotype indeterminate. When whole-cell lysates of PBMCs from these individuals were subjected to PCR and Southern analysis. 39 of 81 were found to have HTLV-related sequences. A total of nine IDUs were found to be infected with HTLV-I, a figure nearly 10 times higher than that estimated by serology alone. Bio-molecular analysis showed HTLV-II-specific proviral sequences in 21 IDUs. Three individuals were seropositive for HTLV-II but lacked PCR evidence of gag, pol and tax sequences. Thus, the overall prevalence of HTLV infection among this cohort was 59% (43 of 81) (i.e., more than twice the frequency predicted by serology, 18 of 81 or 22%). These results indicate that it may be necessary to incorporate biomolecular as well as serological methodologies to identify all persons infected with these retroviruses