Although HAART can successfully reduce plasma HIV-1 viral load to undetectable levels in most HIV-1-infected patients, HIV-1 viruses cannot be eliminated completely due to various reservoirs in the body, including cellular reservoirs (resting CD4+ T cells, monocytes and macrophages, etc.) and anatomical reservoirs/compartments (central nervous system, genital tract, etc.) . In the 1980s, researchers isolated HIV viruses in tears, cornea, aqueous humor, conjunctiva, retinal vascular endothelium and even in contact lenses [2–4]. After the advent of HAART era, there were also observations of higher viral load in intraocular fluid than that in plasma, suggesting that the ocular organ potentially could be a viral sanctuary . Here, we report that HIV-1 viruses existed in tears of patients even under effective HAART therapy, which suggested that the lacrimal gland and/or other tear-associated tissues could be new reservoirs/compartments for HIV-1.
We performed a cross-sectional study recruiting 21 HIV-1-infected participants in the Department of Infectious Disease, Peking Union Medical College Hospital. In long-term HAART group, 16 participants with successful viral load suppression for at least 3 months were recruited. We recruited two antiretroviral therapy-naive participants and three participants with drug-resistant virus infection as two different types of control with high plasma viral load, and five healthy volunteers were also included. All participants underwent ophthalmic examinations in the Department of Ophthalmology, Peking Union Medical College Hospital and did not reveal any clinical signs and symptoms. Written informed consents were obtained from all the participants and the study was approved by the Ethics Committee of Peking Union Medical College Hospital.
Tear samples (reflex tears or psychic tears) were collected into sterile tubes from patients’ eye directly. For qualitative HIV-1 viral assay, total RNA from tear samples were extracted and reverse transcription PCR (RT-PCR) was performed targeting LTR and integrase. Primers sequences were as follows: 5′-CCCTCAGATGCTGCATATAAGCAGC-3′ and 5′-GCACTCAAGGCAAGCTTTATTG-3′ for LTR; 5′-TAAGACAGCAGTACAAATGGC-3′ and 5′-TACTGCCCCTTCACCTTTCCA-3′ for integrase. For quantitative HIV viral assay, plasma and tear HIV-1 RNA viral load was measured by either bDNA Analyzer System 340 (Siemens, Munich, Germany) or the COBAS Ampliprep/TaqMan 48 (Roche, Indianapolis, Indiana, USA) according to the manufacturer's instructions. CD4+ T-cell counts were determined by flow cytometer analysis (three-color EPICS-XL flow cytometer, Beckman-Coulter Inc., Miami, Florida, USA).
Statistical analyses were performed using SPSS for Windows (Statistical Package for Social Sciences, version 16.0; SPSS Inc., Chicago, Illinois, USA). Normal variables were displayed as the mean ± SD and nonnormal variables as the median and interquartile range according to a Kolmogorov–Smirnov test. An independent samples t-test was used to compare the means of normal variables, and the analysis was two-tailed, when P < 0.05 was considered significant difference.
The basic characteristics of participants are displayed in Table 1. In long-term HAART group, the duration of successful viral suppression was 27.2 ± 14.3 months. The median plasma viral load was 32 564 copies/ml (13 032, 282 265) and CD4+ T-cell count was 131 ± 117 cells/μl in baseline. In tear-sampling time point, most participants had good viral response with undetectable plasma viral load and good immunological response with CD4+ T-cell count 334 ± 162 cells/μl.
Qualitative RT-PCR targeting LTR and integrase showed positive results for all participants (Table 1). Quantitative assay targeting Gag and Pol showed that the tear viral load was 5189 ± 7263 copies/ml in 16 long-term HAART participants even though the plasma viral loads were undetectable for quite a long time, and there was no difference from the other five controls who had tear viral load 4477 ± 5276 copies/ml (P = 0.842). As the amount of tear sample was very small, not all viral detection assays were performed for each sample. Mocks using either ddH2O or tears of volunteers showed no positive results in all types of assays. The viral load in tear sample was not correlated with age, sex, baseline plasma viral load and CD4+ T-cell count, the duration of viral suppression or the tear-sampling time point plasma viral load and CD4+ T-cell count (data not shown).
Our data supported the previous observations that HIV viruses exist and survive in tears . We perfomed both qualitative and quantitative analyses covering different fragments of HIV-1 genome; thus, the possibility that the detected HIV-1 sequences were only genomic RNA debris was low. Together with the fact that HIV viral load was quite high in tear sample, despite that HIV virus had been suppressed successfully in blood for a long time, we showed clear evidences that the lacrimal gland as well as other tear-associated tissues could serve as new reservoirs/compartments of HIV-1 virus. Another important meaning of this study is that precautions should be taken when doing any eye examination in spite of the infectivity of HIV virus under HAART treatment was unknown.
However, our data also raise many questions to be further answered. How did HIV viruses get into the tears? It was reported that CD4+ T cells, macrophages and dendritic cells could infiltrate into the lacrimal gland, cornea and the conjunctiva in Sjögren's syndrome and other eye diseases. These findings provide the possibility that these cells could release HIV viruses in tears after infection [6,7]. Why did HIV fail to be suppressed in tears under HAART? Could the antiviral drugs distribute into tears effectively? Could the enzymes in tears suppress or block antiviral drug activity ? What is the genetic information of HIV virus in tears? Further experiments are needed to find out the subtype, tropism and drug resistance information, and so on. Does HIV tear load correlate with any parameter? We did not observe whether the viral load in tears had any correlation with age, sex, baseline plasma viral load and CD4+ T-cell count, the duration of viral suppression or the concurrent plasma viral load and CD4+ T-cell count. Does HIV viral load in tears correlate with clinical ophthalmic problems? Many HIV-infected individuals have keratoconjunctivitis, dry eye and other eye problems [9,10], whereas none of our participants had any clinical ophthalmic manifestations. It seems that the ophthalmological manifestations are more likely to correlate with intraocular viral load [5,11], and not tears.
This work was supported by the National Natural Science Foundation of China (81071372); National Key Technologies R&D Program for the 11th Five-year Plan (2008ZX10001–006); and Key Clinical Program of the Ministry of Health 2010–2012.
Y.H. designed the study and performed quantitative HIV-1 viral load tests in tears and plasma; N.W. did the statistical analyses and wrote the manuscript; W.Z. performed qualitative experiments proving viral existence in tears; Y.L. and L.Z. recruited patients, obtained the written consents and collected tear samples; J.Y. supervised eye examinations for all patients; Z.Q. and J.X. tested CD4+ T-cell count; and T.L. supervised all aspects of this study, including study design, execution, interpretation of data and manuscript preparation. Y.H. and N.W. contributed equally to this paper.
The authors thank Huijuan Kou and Mengyu Zhou, Peking Union Medical College, for their help with revising the manuscript.
Conflicts of interest
There are no conflicts of interests for all authors.
1. Haggerty CM, Pitt E, Siliciano RF. The latent reservoir for HIV-1 in resting CD4+ T cells and other viral reservoirs during chronic infection: insights from treatment and treatment-interruption trials
. Curr Opin HIV AIDS
2. Fujikawa LS, Salahuddin SZ, Palestine AG, Masur H, Nussenblatt RB, Gallo RC. Isolation of human T-lymphotropic virus type III from the tears of a patient with the acquired immunodeficiency syndrome
3. Tervo T, Lahdevirta J, Vaheri A, Valle SL, Suni J. Recovery of HTLV-III from contact lenses
4. Ablashi DV, Sturzenegger S, Hunter EA, Palestine AG, Fujikawa LS, Kim MK, et al. Presence of HTLV-III in tears and cells from the eyes of AIDS patients
. J Exp Pathol
5. Pathanapitoon K, Riemens A, Kongyai N, Sirirungsi W, Leechanachai P, Ausayakhun S, et al. Intraocular and plasma HIV-1 RNA loads and HIV uveitis
6. Ogawa Y, Kuwana M, Yamazaki K, Mashima Y, Yamada M, Mori T, et al. Periductal area as the primary site for T-cell activation in lacrimal gland chronic graft-versus-host disease
. Invest Ophthalmol Vis Sci
7. Forrester JV, Xu H, Kuffova L, Dick AD, McMenamin PG. Dendritic cell physiology and function in the eye
. Immunol Rev
8. Ohashi Y, Dogru M, Tsubota K. Laboratory findings in tear fluid analysis
. Clin Chim Acta
9. Geier SA, Libera S, Klauss V, Goebel FD. Sicca syndrome in patients infected with the human immunodeficiency virus
10. Verma N, Kearney J. Ocular manifestations of AIDS
. P N G Med J
11. Hsu WM, Chiou SH, Chen SS, Shyong MP, Ho CK, Chen SJ, et al. The HIV RNA levels of plasma and ocular fluids in AIDS patients with ophthalmic infections