Noninvasive Detection of Antibodies to Human T-Cell Lymphotropic Virus Types 1 and 2 by Use of Oral Fluid

Abstract

Human T-lymphotropic viruses type 1 and 2 (HTLV-1/2) are prevalent in endemic clusters globally, and HTLV-1 infects at least 5 to 10 million individuals. Infection can lead to inflammation in the spinal cord, resulting in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or adult T cell leukemia/lymphoma (ATL). Obtaining venous blood for serological screening, typically performed using enzyme immunoassays (EIAs), is invasive, sometimes socially unacceptable, and has restricted large-scale seroprevalence studies. Collecting oral fluid (OF) is a noninvasive alternative to venesection. In this study, an IgG antibody capture EIA was developed and validated to detect anti-HTLV-1/2 IgG in OF. OF and plasma specimens were obtained from seropositive HTLV-1/2-infected patients attending the National Centre for Human Retrovirology (n = 131) and from HTLV-1/2-uninfected individuals (n = 64). The assay showed good reproducibility and high diagnostic sensitivity (100%) and specificity (100%) using both OF and plasma. The Murex HTLV I+II commercial assay was evaluated and did not detect anti-HTLV-1/2 IgG in 14% (5/36) of OF specimens from seropositive donors. The reactivities of OF and plasma in the IgG capture correlated strongly (r = 0.9290) and were not significantly affected by delayed extraction when held between 3°C and 45°C for up to 7 days to simulate field testing. The use of OF serological screening for HTLV-1/2 infection could facilitate large-scale seroprevalence studies, enabling active surveillance of infection on a population level.

Keywords: ELISA; HTLV-1; HTLV-2; IgG; diagnostics; gingival crevicular fluid; human T-cell leukemia virus; immunoassays; immunodiagnostics; oral fluid.

FIG 1

Comparison of paired OF and plasma samples, expressed as S/CO values. (Left) One hundred seropositive (n = 131 paired samples) and 13 seronegative individuals in the IgG-capture assay. (Right) Thirty-six seropositive and 8 seronegative individuals in the Murex HTLV I+II EIA. Black dots, samples from seropositive individuals; red dots, samples from seronegative individuals. Solid horizontal bars show median. The horizontal dashed line indicates the S/CO at 1.00, above which samples are considered reactive.

FIG 2

Correlation of 131 paired OF and plasma samples from 100 seropositive individuals, expressed as S/CO values, in the IgG-capture assay (left), and 36 paired OF and plasma samples from seropositive individuals in the Murex HTLV I+II EIA (right). Spearman’s rank correlation coefficient (r_s) is shown. P value indicates degree of statistical significance.

FIG 3

Comparison of OF (left) and plasma (right) samples analyzed by IgG-capture assay and Murex HTLV I+II EIA. The horizontal dashed line indicates the S/CO at 1.00, above which samples are considered reactive.

FIG 4

Analytical sensitivity of the IgG capture assay based on 2-fold serial dilutions of anti-HTLV-1/2 antibody-containing plasma (A) and OF (B) in transport medium and that of antibody-containing plasma in plasma pooled from seronegative donors (C) until the limit of detection. The horizontal dashed line indicates the S/CO at 1.00, above which samples are considered reactive.

FIG 5

Variation of S/CO values over time in IgG capture assay. Samples obtained from 24 individuals, with an average of 3.3 months between the two time points. (A) plasma and (B) oral fluid. Horizontal dashed line indicates the S/CO at 1.00, above which samples are considered reactive. P value indicates degree of statistical significance.

FIG 6

Comparison of S/CO values of plasma (A) and oral fluid (B) samples from HTLV-1-infected patients presenting with a clinical phenotype of HTLV-1-associated myelopathy (HAM/TSP) (n = 34), adult T cell leukemia/lymphoma (ATL) (n = 10), or asymptomatic carriage (AC) of infection (n = 35). Solid horizontal bars show median. P value indicates degree of statistical significance.

FIG 7

Comparison of S/CO values between anti-HTLV-1/2 antibody-containing OF samples submitted to delayed processing at various temperatures (3°C, room temperature [RT], 37°C, and 45°C) for 24 h (24h) or 7 days (7d) and samples extracted on the day of collection. Each temperature condition was evaluated by five different paired OF specimens for 24 h and by 6 paired samples for 7 days. The horizontal dashed line indicates the S/CO at 1.00, above which samples are considered reactive. P value indicates degree of statistical significance.