ABSTRACT.
This retrospective case–control study examined the prevalence of HTLV-I and its association with tuberculosis among urban clinic patients in Haiti. Prevalence of HTLV-I among tuberculosis cases was 2.1% and among controls was 2.4%. Prevalence of HLTV-I was higher in females than males (odds ratio [OR] 2.45, P = 0.020). HTLV-I prevalence in those ≥ 50 years was 8.4% compared with 1.3% in those < 50 (OR 6.74, P < 0.001). We found no association between HTLV-I and tuberculosis in this population.
INTRODUCTION
Human T-cell lymphotrophic virus-I (HTLV-I) is the most common of the HTLV family of retroviruses.1,2 HTLV-I infection leads to myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma in a small percentage of those infected. It may be associated with tuberculosis, although studies examining this association have reached different conclusions.3–9
We sought to determine the prevalence of HTLV-I and its association with tuberculosis among patients at an urban clinic in Haiti. We hypothesized that those with tuberculosis would have a higher prevalence of HTLV-I infection than those without HTLV-I infection. If an association exists, then enhanced tuberculosis preventive strategies may need to be considered for people with HTLV-I, similar to strategies that already exist for people living with HIV.
METHODS
This is a retrospective case–control study nested within two larger cohorts at the Haitian Group for the Study of Kaposi’s Sarcoma and Opportunistic Infections (GHESKIO) in Port-au-Prince, Haiti. GHESKIO is the largest tuberculosis and HIV treatment facility in the Caribbean.
Two cohorts were analyzed for this study. Cohort 1 was a longitudinal case–control study conducted in 2007–2008 and 2011–2012 examining serum antibody titers and urine metabolite concentrations in participants with tuberculosis (cases) and participants without tuberculosis (controls). Cohort 2 was a cross-sectional study conducted in 2014–2019 examining biomarkers associated with latency and persistence of Mycobacterium tuberculosis among participants with and without active tuberculosis. Both studies included participants living with HIV. Participants in these studies consented to have sera banked for use in future research.
Cases were defined as treatment naive adults ≥ 18 years old with a new diagnosis of pulmonary tuberculosis based on clinical symptoms, chest radiography and microbiologic evidence of infection (positive AFB smear, positive Xpert (Cepheid, Sunnyvale, CA) or positive culture). Controls were defined as adults aged ≥ 18 years with no clinical symptoms consistent with tuberculosis. Controls were matched to cases retrospectively by grouping participants based on HIV status and similar age, in 5-year increments. Banked sera from 1,701 participants were then tested for the presence of HTLV antibodies.
Sera were collected from participants at time of enrollment in the primary study. Deidentified sera were shipped on dry ice to New York, New York, and subsequently stored at –80°C for future use.
Sera were thawed before use. All serum samples were tested with the Avioq HTLV-I/II Microelisa System (Avioq Inc., Durham, NC) per the manufacturer’s instructions. Serum samples were diluted 1:5 then incubated for an hour at 37°C in wells precoated with HTLV-I lysate, HTLV-II lysate, and recombinant HTLV–p21E antigen. Horseradish peroxidase served as the enzyme conjugate and the substrate was a tetramethylbenzidine solution. The reactions were stopped using 2N sulfuric acid before measuring absorbance of the wells. Absorbance was measured at a wavelength of 450 nm, subtracting the absorbance at 620 nm that served as a reference. Positive and negative controls were included on each test plate. Samples were deemed positive for HTLV antibodies if the sample absorbance was greater than the defined cutoff value of 0.363. Positive samples were retested with Avioq HTLV-I/II Microelisa System before confirmatory testing per the manufacturer’s instructions. Samples resulting as indeterminate were retested.
Samples that were positive for HTLV antibodies based on the Avioq HTLV-I/II Microelisa System were then confirmed with the MP Diagnostics HTLV Blot 2.4 (MP Biomedicals, Irvine, CA). This assay confirms the presence of HTLV and differentiates HTLV-I from HTLV-II through identification of unique viral envelope proteins. Twenty microliters of each sample were tested according to the manufacturer’s instructions. Positive and negative controls were included for quality control on each testing date.
Participant characteristics were abstracted from the study databases of the primary studies and the GHESKIO electronic medical record. HTLV-I/II positivity was defined by positive ELISA with positive confirmatory Western blot. HTLV negative was defined as negative by ELISA or negative by Western blot. Indeterminate samples were positive by ELISA and were unable to be subtyped by Western blot but not negative by Western blot. These were included with the HTLV-I group as part of the analysis. HTLV sensitivity analysis was performed by classifying HTLV “indeterminate” subtypes based on Western blot as negative.
Data were analyzed using STATA version 15 (College Town, PA). We assessed the association of HTLV-I positivity and tuberculosis for our observational data using conditional logistic regression by grouping participants with the same HIV status and 5-year age intervals in the analysis.
This study was approved by all relevant institutional review boards.
RESULTS
The study population included 1,701 participants, 863 cases and 838 controls. Females comprised 46% (n = 398) of cases and 69% (n = 576) of controls (P < 0.001) (Table 1). Sixteen percent (n = 141) of cases and 19% (n = 155) of controls were HIV-positive. Due to the unequal number of cases and controls, the percentage of HIV in each group was different, but not significantly so.
Table 1.
Participant characteristics
| TB cases, N = 863 (%) | TB controls, N = 838 (%) | P value | |
|---|---|---|---|
| Sex* | |||
| Female | 398 (46.4) | 576 (68.9) | |
| Male | 459 (53.6) | 260 (31.1) | < 0.001 |
| Age, median (IQR) | 27 (20, 40) | 32 (24, 42) | † |
| HIV status | † | ||
| Positive | 141 (16.3) | 155 (18.5) | |
| Negative | 722 (83.7) | 683 (81.5) | |
| CD4 Count at enrollment for HIV-positive participants, median (IQR)‡ | 301 (163, 537) | 535 (456, 761) | 0.241 |
| Income | < 0.001 | ||
| < 5,000 HGD | 333 (38.6) | 186 (22.2) | |
| > 5,000 HGD | 339 (39.3) | 357 (42.6) | |
| Unknown or missing | 191 (22.1) | 295 (35.2) | |
| Education | < 0.001 | ||
| Primary completed or less | 328 (38.0) | 207 (24.7) | |
| Some secondary or more | 414 (48.0) | 468 (55.9) | |
| Unknown or missing | 121 (14.0) | 163 (19.5) |
IQR = interquartile range.
Sex missing for eight participants.
Matched based on this characteristic.
Ninety-six HIV-positive participants had CD4 data available.
Among 1,701 participants, sera from 41 (2.4%) participants were positive for HTLV antibodies. Of these, 38 samples were HTLV-positive by Western blot testing, one sample was unable to be tested, and two samples were negative despite positive antibodies. Of the 38 HTLV-positive samples based on Western blot, 31 were confirmed HTLV-I, two had both HTLV-I and HTLV-II infection, and five could not be subtyped using the MP Diagnostics HTLV Blot 2.4 assay. There was no difference in risk of tuberculosis among those with HTLV when “indeterminate” samples were classified as “negative” compared with those without HTLV. Thirty-eight HTLV positive participants were included in the final analysis.
Prevalence of HTLV-I among tuberculosis cases was 2.1% and among controls 2.4%. In a post hoc analysis specifically looking at age and sex, prevalence of HTLV-I in those aged ≥ 50 years was 8.4% compared with 1.3% in those < 50 (odds ratio [OR] 6.74, P < 0.001). When examining by decile, HTLV-I prevalence increased significantly from participants in their fortieth decade to those in their 50th decade: < 40, 1.43%; 40 to 49, 1.24%; 50 to 59, 9.15%; 60 to 69, 7.14%; ≥ 70, 6.67%. Prevalence of HLTV-I was higher in females than males (OR 2.45, P = 0.020). This was true across cases, controls and age groups.
Cases did not demonstrate increased odds of being HTLV-I positive. There was no association between HTLV-I and tuberculosis detected in this study.
DISCUSSION
In this retrospective case–control study of 1,701 participants, there was no significant difference in risk of tuberculosis among those with and without HTLV-I infection. To the best of our knowledge, this is the first study to examine the association between HTLV-I and tuberculosis in Haiti. Cases and controls were recruited from the same clinic setting. HIV and age, both of which are known to be associated with HTLV-1, were controlled in the matching process to avoid confounding the potential relationship between HTLV-I and tuberculosis. Post hoc analysis demonstrated higher odds of HTLV-I in older age and among females, which is consistent with other studies.10,11
The overall HTLV-I prevalence of 2.3% demonstrated here is lower than areas of high endemicity such as Japan or Brazil, where prevalence can be much higher.10 Although the prevalence was lower than expected, this did not limit our ability to detect a significant difference between cases and controls in this study. This rate is also lower than historical studies performed in Haiti when HIV prevalence was higher. Among patients with symptoms concerning for HIV presenting to a rural Haitian hospital between 1988 and 1989, HTLV-I antibodies were 4 times more frequent in HIV-positive compared with HIV-negative individuals, with an overall HTLV-I prevalence of 11.2%.12 It may be the high prevalence of HIV contributed to the concurrently high prevalence of HTLV-I at that time given commonalities in transmission. Although antiretroviral therapies for HIV are not effective against HTLV-I, the improved prevention strategies and public health campaigns that contributed to the decline of HIV may have also contributed to decreased prevalence of HTLV-I.
Previous studies examining the relationship between HTLV and tuberculosis have reached different conclusions. The strongest evidence in favor of an association comes from a meta-analysis including three cross-sectional and three case–control studies demonstrating people with HTLV-I had 2 times higher odds of tuberculosis compared with people without HTLV-I.13 The increased risk was no longer statistically significant after removal of two studies3,4 from the analysis due to potential confounding with socioeconomic status. Interestingly, in the four remaining studies, only one5 included people living with HIV. This case–control study based in Guinea-Bissau demonstrated higher odds of tuberculosis in HIV-positive individuals with HTLV-I with no increased risk in HIV-negative individuals. Cases were adults hospitalized with tuberculosis and matched to population-based controls, controlling for age and gender but not HIV status. Because we matched cases to controls based on HIV status, this may have contributed to the different findings demonstrated in our study.
There is concern that HTLV-I may cause more immunological alterations than previously thought. HTLV-1 impairs the innate immune response through blunting the IFN-1 pathway, decreasing TNF levels as well as altering macrophage, monocyte, and natural killer cell function.14,15 The innate immune response is critical to preventing as well as controlling M. tuberculosis infection once it has occurred and preventing progression to symptomatic disease.16 HTLV-1’s impact on innate processes may contribute to an increased risk of M. tuberculosis. The adaptive immune system is also affected by HTLV-1 infection; whereas depletion of CD4+ T cells is the hallmark of HIV infection, HTLV-I is associated with spontaneous proliferation of CD4+ T cells.17,18 It is unknown if such dysfunction may then lead to disease.
On the basis of our findings, HTLV-I is not associated with tuberculosis per se but may impact other health outcomes. The prevalence of HTLV-1 found in this study is lower than highly endemic areas; this may have limited the ability to detect an association between HTLV-1 and tuberculosis. HTLV-I prevalence in Haiti has decreased since the peak of the HIV epidemic, suggesting that public health measures to reduce HIV may impact HTLV-I prevalence.
ACKNOWLEDGMENTS
We acknowledge the dedication of the GHESKIO staff to their mission of improving the health of their patients and community.
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