Oral cytology in human immunodeficiency virus ( HIV ) : An immune marker ?

Manipal College of Dental Sciences, Mangalore Affiliated to Manipal University, India. Department of Oral Medicine and Radiology, Manipal College of Dental Sciences, Mangalore, Affiliated to Manipal University, India. Department of General Medicine, Kasturba Medical College, Mangalore Affiliated to Manipal University, India Department of Oral Pathology, Manipal College of Dental Sciences, Mangalore Affiliated to Manipal University, India.


INTRODUCTION
Human immunodeficiency virus (HIV) infection is characterized by progressive and continuous impairment of the immune system function, with varying rate of progression among patients.Antiretroviral treatments (HAART) have been shown to be effective in arresting immune system impairment and prevention of disease progression.The CD4 T-lymphocyte (CD4) count is considered the best laboratory marker of progression of HIV disease (Fahey et al., 1990).Many HIV-associated oral disorders occur early in HIV infection, not infrequently infrequently as the presenting sign or symptom.Thus, early detection of associated oral disease should, in many cases, result in earlier diagnosis of HIV infection (Ranganathan et al., 2000).Cytology, a simple, painless and inexpensive method has become a preferred method for early diagnosis of certain lesions.HIV affects primarily the immune cells but there are studies (Montaner et al., 1992) which have concentrated on the cervical and gastrointestinal epithelium when investigating the various mechanisms of HIV infection across mucosal surfaces.

SUBJECTS AND METHODS
The present study included a group of forty HIV-infected patients seen at a clinic for HIV in Kasturba Medical College, Mangalore, India, for the diagnosis or follow up of this infection.All patients had been diagnosed with HIV infection by enzyme-linked immunosorbent assay (ELISA), and the diagnosis was confirmed by Western blot analysis.Informed consent was received from all patients.The patients seen at this clinic were first evaluated by physicians who specialized in HIV infection; these physicians determined from the patient's medical history if they met the criteria for inclusion in the study.The patients selected were referred to the laboratory, where they were examined following an established clinical and microbiological protocol.The clinical protocol consisted of a medical history, social history and general physical examination.The general examination included careful scrutiny of the oral cavity.Oral smears were collected from normal appearing tongue and buccal mucosa of 40 HIV infected (Study Subjects) and cells were analyzed for cell yield, cell cohesion, fungal growth and other atypical features.Oral cytological analyses of our subjects were compared with their CD4 counts.The cytological smears obtained from the HIV positive patients were evaluated based on factors like cell yield, cell cohesion, atypical features (increased nuclear cytoplasmic ratio and pleomorphism), presence of fungal organisms, bacterial colonies, the degree of inflammation and the type of inflammatory cells seen.The CD4 lymphocyte counts of each of the 40 cases was noted and categorized as those below 500 cells/mm 3 and above 500 cells/mm 3 .

Ethics committee
Ethical approval for the study was obtained from Institutional Ethics Committee of Manipal College of Dental Sciences, Mangalore.Informed consent was obtained from all study subjects before obtaining oral smears.

RESULTS
A total of 24 cases showed CD4 counts less than 500 cells.The CD4 lymphocyte counts were correlated with each of the cytological factors.It was observed that the lesser the CD4 lymphocyte count, the greater the cell yield (chi-square=4.393, p=0.111) (Figure 1).Cohesive clusters of cells were seen to be maximum in patients with CD4 counts less than 500 (chi-square=3.706,p=0.295).Increased nuclear cytoplasmic ratio was seen in 80% (n=12) cases with lower CD4 counts.On comparing the inflammatory component with CD4 lymphocyte counts, it was seen that the inflammatory cells were either cells were either absent or meagerly present and consisted mainly of acute inflammatory cells in HIV cases with CD4 counts less than 500 cells.Oral candida was commonly seen among subjects with lesser CD4 counts (Figure 2).

DISCUSSION
CD4+ T-lymphocyte counts are considered the markers for HIV disease progression, but not very affordable and hence its use gets limited in developing countries.The use of total lymphocyte counts (TLC) has been advocated to predict and to stage HIV disease in some countries (Montaner et al., 1992;WHO, 1993).In this study, cost was not a limiting factor and CD4 counts are preferred to be used as a lab marker.
Our earlier study had revealed that HIV induces significant changes in oral epithelium, detectable by microscopy and cytomorphometry Jagad et al., 2014).In this study, a close association between the patient's immune state and the presence of cell yield were found with an increase in cell yield as CD4 lymphocyte counts decreased.In HIV-infected patients, oral candida (OC) was a frequent finding and has been associated with a less favorable immune status and more accelerated deterioration, similar findings were reported in other studies also (Dodd et al., 1991;Nielsen et al., 1994).Phelan et al. (1997) found that a CD4¹ count of less than 200 or the presence of OC at the time of data collection increase the risk of death or development of AIDS, and such prognosis was much worse if both factors were pre-sent at the onset of evaluation.This relationship between OC, oral cytological alterations with reduced CD4 lymphocyte counts allows us to interpret these changes as an indirect marker of the immune status of HIV-infected patients when a CD4 count is unavailable (Burcham et al., 1991).
Some authors have recommended using the percentage of CD4 lymphocytes rather than CD4 counts, as the main indicator of immune function in HIV-infected patients, because the latter exhibited more variability in consecutive samples and had a poorer prognostic value for the development of AIDS (Malone et al., 1990).
Our results demonstrate that in patients with HIV, the degrees of immunosuppression determine the oral epithelial changes and a greater probability of presenting OC.Consequently, the presence of OC and other HIVassociated oral lesions could be clinical markers of the disease progression defined by oral cytological changes and the latter may be a useful complementary indicator for monitoring antiretroviral therapy in HIV-infected patients.

Conclusion
Emphasis should be placed on the importance of cytological examination of the oral cavity in all medical follow-up examinations of HIV-infected patients, not only because of the need to diagnose and treat associated oral diseases, but also because the presence of these changes provides indirect information about the patient's immune state.