Rapid Detection of HIV-1 and 2 Antibodies by using Oral Fluid: A Multicentric Pilot Study in India

Rapid Detection of HIV-1 and 2 Antibodies by using Oral Fluid: A Multicentric Pilot Study in IndiaNallan CSK Chaitanya,pinakapani R, Pavan Kumar, Rajeshwari Annigeri, G Raghu Rama Rao, Amareswar, Veena RamannaJournal Of Indian Academy Of Oral Medicine And Radiology 2011.23[3]204-207

Presented By Dr. Priyadershini A. Kasture

Introduction:

Detection of HIV antibodies is done by the standard blood tests. Rapid tests provide results in few minutes. The OraQuick rapid HIV1/2 antibody test using oral fluid has not been tried in resource-limited settings in India. The aim was to evaluate the efficacy of the OraQuick test kit using oral fluid for HIV antibody detection in patients attending the dental hospitals

Human immunodeficiency virus type 1 and 2 are the etiological agents of AIDS.A global summary of AIDS epidemic by WHO in 2008, estimated 33.4 (31.1 to 35.8) million people are living with HIV-1 worldwide, while about 2 million have already died in 2008.There could be still unknown, undiagnosed cases probably living with HIV virus. These estimates may mask the dynamic nature of this evolving epidemic in relation to temporal changes, geographic distribution, magnitude, viral diversity and mode of transmission.

. Cao H, Walker BD. Immunopathogenesis of HIV-1 infection. Clin Dermatol 2000;18(4):401-10.2. UNAIDS report on the global AIDS epidemic 2008.http://data.unaids.org/pub/Global Report/2008/JC1511_ GR08_ExecutiveSummary_en.pdf

According to NACO GUIDELINES(March 2007)

Whom to test for HIV-One needs to be able to correctly suspect and screen a client at risk for HIV status. No Mandatory HIV testing should be imposed as a precondition for employment OR for providing health care services and facilities. Not all Patients for Surgery(Operation) should be subjected to HIV testing Routine Mandatory HIV testing should not be under taken for the benefit of Health care workers. regardless of patients infection status every patients blood /body fluid is considered to be positive/infectiousOn accidental exposure to any patients Body fluid/potentially infectious material, Health care worker should take necessary PEP (Post Exposure Prophylaxis) ideally within 2 hours but certainly within 72 hours.

Negative HIV status of patient reported by a Laboratory, does not give liberty to HCW to be careless(regarding their own safety while providing their heath care service to patient ) and ignore the principles/guidelines of Universal Safety Precaution ,as it can be a case of False Negative HIV status reporting during Window period where the routine screening tests (aimed at detecting presence of HIV antibodies) are unable to detect the HIV status of asymptomatic patients .

HIV testing of every ANC women/patient is recommended(but not mandatory) so as to determine the risk of transmission from mother to foetus and further to take action to prevent it (PPTCTCs) .

OraQuick test kit

[To detect HIV infection in Asymptomatic Individual(ICTCs, PPTCTCs) ] 3 test(rapid) kit required

A1 (Comb AIDS Rs) (Sensitivity 100% , Specificity 98. 7%)

A1+ A1- Report Negative A2 (SD Bioline HIV1/2 3.0 ) (Sensitivity 100% , Specificity 99.8%)

A1+ A2+ A1+ A2-

A3 (HIV Tridot) A3 (Sensitivity 99.6% for HIV1, 100% for HIV2, and Specificity 100%)

A1+ A2+ A3 + A1+ A2+ A3- A1+ A2- A3 + A1+ A2- A3-Report Positive Indeterminate Indeterminate Report Negative

Interpretation: A1 Non-Reactive = HIV Antibody Test Negative . A1 Reactive+ A2 Non-Reactive +A3 Non-Reactive= HIV Antibody Test Negative (A1 being highly sensitive). A1 & A2 Reactive+A3 Non-Reactive / A1 & A3 Reactive+A2 Non-Reactive = HIV Antibody Test Indeterminate . A1 A2 A3 Reactive = HIV Antibody Test Positive (A2 & A3 being highly specific).

A Serum sample , showing result of the above three different ELISA or Simple & Rapid HIV Antibody test as HIV Antibody Test Positive or HIV Antibody Test Indeterminate , should be retested by Supplemental ( Western Blot assay or Indirect Immunofluorescence ) test.

Screening (E/R/S) testsa) ELISAb) Rapid tests - Dot blot assays - HIV spot and comb tests - Fluorometric microparticle technologiesc)Simple tests -Also based on ELISA principle but takes 1-2 hours - Particle agglutination(gelatin, RBC, latex, microbeads)Supplemental tests a). Western blot assay b).Immunofluorescence testConfirmatory testsa).Virus isolation-Cocultivation with lymphocytes and Interleukin-2b).Detection of p24 antigenc). Detection of viral nucleic acid - In situ hybridization - Polymerase chain reaction

The window period is the time from infection until a test can detect any change.Average window period for HIV-1 is 25 days & HIV-2 is 16 days.

ELISA- wet-lab type analytic biochemistry assay[EIA] to detect the presence of an antigen in blood sample.

Western Blot- protein immunoblot used to detect specific proteins in a sample of solid tissue or its extract.

Rapid HIV-test kitBio- saliva HIV test is a non-invasive, accurate way to test for HIV-1/2 antibodies without blood needle or lancets.It tests the HIV antibodies not the disease.Test kit is having a swab and a device with a test strip.After taking swab of oral fluid,then device is placed in a vial that holds an enzyme solution which reacts to any antibody-antigen binding.If positive strip shows red line, if no line then its negative.Require 20 min for one test.

Repeated tests should be done for confirmation.Ora Sure-uses oral mucosal transudate present in buccal and gingival area.OraQuick-saliva test kit86% accuracyRapid HIV test results from the OraQuick are slightly more accurate than blood samples.www.usatoday.com, www.aidsmeds.com, www.home-hiv-tests.com

The demographic features of spread of HIV in Asia are different from elsewhere. India stands second in the world with respect to people living with HIV. UN estimates project that Indias adult HIV prevalence will peak at 1.9% in 2019. About 2.3 million people are living with HIV infection by 2007. There is a declining trend in the epidemic in South Indian states.

Rajendran R, Sivapathasundaram B. Viral infections of oral cavity. Shaferss textbook of oral pathology (5th ed). Elsevier publications 2006;488-98. NACO guidelines: Annual HIV Sentinel Surveillance Country Report 2006. http://www.nacoonline.org National_AIDS_Control_Program/Surveillance

The early detection assumes paramount importance at the present scenario. Diagnosis of HIV infection is based on the detection of specific antibodies, antigens or both. Serological tests are employed for the screening purposes. The CDC recommends that diagnostic HIV testing and opt out HIV screening be a part of routine clinical care in all health care settings, while maintaining the individual concerned for not opting the testing for optimal clinical and preventive care.Sierra S, Kupfer B, Kaiser R. Basics of the virology of HIV-1 and its replication. J Clin Virol 2005;34(4):233-44.Hahn EK. Incorporating the CDC recommendations for adolescent HIV screening into practice. Journal for Nurse Practitioners 2009;5(4):265-73.Rothman RE, Merchant RC, Talan DA, Moran GJ, Pinner R.Update on emerging infections from the centers for disease control and prevention. CDC update 2007;49(5):575-77.

Technological advances in the diagnosis of HIV infection provide the clinicians with greater opportunities to reduce HIV transmission rates.9 It estimates that increased awareness of serological status will decrease the number of new infections mainly by behavior modification of HIV-positive people and treatment that decreases viral loading in infected individuals. 9

Doughty M, Locksmith GJ. New rapid diagnostic tests for HIV infection. Ob/Gyns 2003;10(3):131-34.

To date, only traditional ELISA was available for detection of anti-HIV antibodies in serum. Overtime there have been improvements of performance of ELISAs from usage of viral lysates to usage of recombinant antigens, synthetic peptides or combination of these both.This has led to better sensitivity and specificity of these assays. The ELISA more recently has been employed to detect HIV antibodies in whole blood, urine and salivary samples.The Westernblot remains a gold standard in HIV antibody conformation. None of the test samples had shown false-positive and false- negative results when compared with the reference tests.Urassa W, Godoy K, Killewo J, Kwesigabo G, Mbakileki A Mhalu F, et al. The accuracy of an alternative confirmatorytrategy for detection of antibodies to HIV-1: Experience from a regional laboratory in Kagera, Tanzania. J Clin Virol 1999;14(1):25-29.King SD, Winter SH, Bain BC, Brown WA, Johnston JN, Delk AS. Comparison of testing saliva and serum for detection of antibody to human immunodeficiency virus in Jamaica, West Indies. Journal of Clinical Virology 2000;19(3):157-61.

DiscussionRapid point of care HIV tests greatly aid in knowing sero- status by providing faster and accurate results in minutes. This can be used in areas with limited laboratory resources and they provide convenience in testing on site results by allowing the delivery of definite negative or provisional reactive results greatly enhancing interventional programs.There are rapid HIV tests using oral fluid and whole blood. The efficacy of these tests has been evaluated individually and also by comparing both the tests. Holgun A, Gutirrez M, Portocarrero N, Rivas P, Baquero M. Performance of OraQuick advance rapid HIV-1/2 antibody test for detection of antibodies in oral fluid and serum/plasma in HIV-1+ subjects carrying different HIV-1 subtypes and recombinant variants. J Clin Virol 2009;45(2):150-52.24. Mnard D, Maro A, Mandeng MJ, Doyemet P, Koyazegbe T, Evaluation of rapid HIV testing strategies in under equipped laboratories in the Central African Republic. Journal of Virological Methods 2005;126:75-80.

In this study, the OraQuick test demonstrated high sensitivity and specificity of 100% with oral fluid specimens as claimed by the manufacturer. These findings are in accordance with a previous study done in rural population in India, although there were conflicting reports of lower sensitivity and specificity of oral fluid-based tests elsewhere with high incidence of false-positive and false-negative results.This study shows that OraQuick test using oral fluid seemed to be of patients preference than the blood-based test as it was a painless, simple and noninvasive procedure. It was also the test administrators preference over the conventional blood-based tests as it was rapid, safe with least occupational exposures, easier sample collection and simple to perform.Mylonakis E, Paliou M, Lally M, Flanigan TP, Rich JD. Lab testing for infection with the HIV: Established and novel approaches. Am J Med 2000;109(7):568-76.Reynolds SJ, Muwonga J. OraQuick advance rapid HIV-1/2 antibody test. Expert Rev Mol Diag 2004; 4(5):587-91.

This was also the test administrators preference over the conventionalblood-based tests as it was rapid, safe with least occupational exposures, easier sample collection and simple to perform. 28,29 The test offers an immense benefit in pregnant patients without prenatal care and also in case of occupational exposures. Dentists and emergency care providers are most commonly prone for infections from contact with patients blood and oral fluids. Due to lack of adequate sterilization measures and detection facilities, there is a greater chance of not only acquiring infection but also spreading it among other patients unwittingly.Thus, incorporating this simple to perform test kit, using noninvasive means, can greatly reduce the risk of transmitting the infection. 31. Vernillo AT, Caplan AL. Routine HIV testing in dental practice: Can we cross the rubicon? J Dent Educ 2007;71(12): 1534-39.32. White DA, Cheung PT, Scribner AN, Frazee BW. A comparison of HIV testing in the emergency department and urgent care. J Emerg Med 2009;20

There is a more recent study on this oral fluid-based test kit which demonstrated its utility for detecting infections due to HIV-1 subtypes and recombinants.

Although the present study demonstrated the usefulness of the oral fluid-based OraQuick rapid HIV-1 and 2 antibody test, the lies in its inability to specify whether the sample contained HIV-1 or HIV-2 antibodies specifically, HIV-2 being less prevalent than HIV-1.

Holgun A, Gutirrez M, Portocarrero N, Rivas P, Baquero M. Performance of OraQuick advance rapid HIV-1/2 antibody test for detection of antibodies in oral fluid and serum/plasma in HIV-1+ subjects carrying different HIV-1 subtypes and recombinant variants. J Clin Virol 2009;45(2):150-52.

conclusionAlthough the present study demonstrated the usefulness of the oral fluid-based OraQuick rapid HIV-1 and 2 antibody test, the disadvantage lies in its inability to specify whether the sample contained HIV-1 or HIV-2 antibodies specifically, HIV- 2 being less prevalent than HIV-1.

We conclude that this test kit is efficacious as an effective screening device of HIV antibody detection; further studies are nm warranted in larger group of population involving the dental set-up, emergency care units, pregnant patients and high-risk groups directed at its diagnostic accuracy and to introduce it as a routine office screening procedure.

Review Of The Literature

In four separate studies, we compared the accuracy of the rapid test performed on whole blood and oral fluid specimens with the results of conventional HIV tests.

Oral fluid and whole blood from persons of unknown HIV status recruited from clinics, labor and delivery units, and outreach venues were tested with the OraQuick Advance rapid HIV-1/2 antibody test. Kevin P Delaney, Bernard M Branson, Apurva Uniyal, Peter R Kerndt, Patrick A Keenan, Krishna Jafa, Ann D Gardner, Denise J Jamieson, Marc Bulterys AIDS 09/2006; 20(12):1655-60

Sensitivity and specificity were compared with results of the enzyme immunoassay (EIA) and Western blot algorithm used by the study sites. OraQuick sensitivity was 99.7% with whole blood and 99.1% with oral fluid from 327 persons who were HIV antibody positive by the conventional algorithm.

OraQuick specificity was 99.9% with whole blood and 99.6% with oral fluid from 12 010 HIV-negative persons.

EIA specificity was 99.7%. A cluster of 16 false-positive oral fluid tests occurred in one study, in which specificity was lower (99.0%) than in the other three studies (99.6-99.8%).

In diverse settings in four studies, the OraQuick test showed high sensitivity and specificity for HIV antibody in whole blood and oral fluid specimens.

Slightly more false-positive and false-negative results occurred with oral fluid than with whole blood, but performance with both specimen types was similar to, or better than, that of conventional EIAs.

To date, no data had been published on the use of OraQuick Advance Rapid HIV-1/2 Test (OraQuick) in the UK. He reported preliminary findings of an ongoing evaluation of OraQuick in UK genitourinary (GU) medicine clinics.

A total of 820 samples from patients in high-risk groups for HIV were tested with OraQuick and results were compared with standard HIV antibody testing.

Kevin P Delaney, Bernard M Branson, Apurva Uniyal, Peter R KerndtAIDS 09/2006; 20(12):1655-60. 6.41

HIV prevalence (enzyme immunoassay [EIA]) was 5.73%, sensitivity of OraQuick was 93.64% (95% CI 82.46-98.66%), specificity 99.87% (99.28-100%), positive predictive value 97.78% (88.27-99.94%) and negative predictive value 99.61% (98.87-99.92%).

This included three false-negatives considered to be due to observer error and now rectified by further training. It increased test sensitivity to 100%.

The observed test performance of OraQuick compares well with EIA and with other rapid tests.

They also believed that simple, non-invasive antibody detection tests such as OraQuick can increase HIV testing and diagnosis in UK GU medicine and community settings.

David T.W. Wong, Salivaomics, JADA 2012;143(10):19S-24SFood and Drug Administration (FDA)approved OraQuick Advance Rapid HIV-1/2 Antibody Test (OraSure Technologies, Bethlehem, Pa.) is diagnostic for HIV infection.

It claims only to screen for the presence of antibodies to HIV-1 in saliva. These antibodies suggest HIV-1 infection, but the assay is not robust enough to prove it.

A confirmatory test (OraSure HIV-1 Western Blot,OraSure Technologies) is required to actually diagnose HIV-1 infection.

The two tests are designed to work together, with a rapid, convenient and inexpensive saliva-based screening step followed by a validated, accurate, laboratory based Western-blot assay for confirmation.

Saliva and its constituents, together with the tests noninvasiveness, were better suited for disease screening and risk assessment than for diagnostic purposes.

In addition to HIV, for which there is an FDA-approved autoantibody detection test, infectious diseases of local origin (caries and periodontal diseases) and systemic origin (human papillomavirus and hepatitis C virus) will be detectable in saliva.

H. Blake1, P. Leighton and S. Sharma, Saliva Testing As a Practical Tool for Rapid HIV Screening, 2011,www.intechopen.com,627-42WHO have identified four distinct categories of HIV testing: Diagnostic testing, Voluntary counselling and testing (VCT), Routinised testing in specific setting, and Mandatory testing.

Diagnostic HIV testing is testing undertaken where signs and symptoms related to an HIV infection are observed in any individual.

Testing is carried out to ensure timely clinical diagnosis, and to ensure the provision of adequate clinical support and services.

People with certain diseases, such as tuberculosis and any other sexually transmitted disease, are also tested for HIV infection on a regular basis.

There are several limitations in that oral fluid assays may be unlikely to detect those in early stages of HIV infection or with reduced viral load, and have shown altered accuracy in pregnancy; however, suchlimitations also apply to other rapid assays.

Research has suggested that in adults the most important factors in HIV testing are test accuracy, time to results and privacy of results.

Studies have also suggested that patients express a preference for oral testing over venepuncture sampling since it is rapid and less invasive, although preferences may vary in different settings.

It seems that saliva specimens can be easily collected under difficult field conditions with minimal training and provide a valuable alternative to testing blood for HIV-seroprevalence studies.

Salivary testing for HIV may be a convenient and potentially accurate epidemiological tool.

But it should be used with caution since single test systems may be less appropriate to diagnose HIV infection in an individual without follow-up testing.