A03 Diagnoses, classification and staging of HIV disease


A diagnosis of HIV infection requires the practical application of tests differing in methodology as well as testing purpose. Generally, tests are done for three reasons: individual diagnosis, protection of blood or tissue product safety, and public health surveillance. According to the objective of testing, the most appropriate test is chosen based on convenience, test characteristics, and very often the population testing is targeted at.

After HIV successfully establishes infection, a chronic life-long state of disease ensues, characterised by progressive impairment of cell-mediated immunity. The final outcome for most, if not all, of the patients is the development of major complications and death. As expected, clinical presentations vary mainly with the extent of immunodeficiency as indicated by the CD4 count.[Box 3.1] However, local incidence and prevalence of disease, chemoprophylactics and vaccination received by the patient also contribute to the particular clinical profile of a population. Schemata for classifying HIV infection and its staging have been developed by national and international organisations. In practice, these are adapted, and at times, modified for use in other countries.

This chapter deals with the diagnosis of HIV infection using serological and/or virologic tests, followed by a discussion on the staging/classification systems currently in use in Hong Kong.

Box 3.1. Spectrum of opportunistic infections associated with CD4 cell ranges

CD4/μL Opportunistic Infections
200-500 Pneumococcal & other bacterial infections
Pulmonary TB
Herpes zoster
Oral thrush
<200 PCP
Extrapulmonary TB
<100 Toxoplasmosis
Fungal infection – cryptococcosis, Talaromyces marneffei infection
Oesophageal candidiasis
<50 Disseminated CMV
Disseminated Mycobacterium avium complex

Serologic testing for HIV

Presence of antibody to HIV proteins is now well accepted as indicative of HIV infection except in special circumstances such as after receiving an experimental vaccine. Certain clinical conditions may also rarely result in false-positive HIV screening tests. Accurate laboratory diagnosis of HIV infection relies on testing algorithms that maximise overall sensitivity and specificity by employing a sequence of tests in combination. In general, a highly sensitive test is used for screening, followed by a highly specific test for confirmation.

HIV antibody assays by ELISA, WB and HIV-1/HIV-2 differentiation immunoassay

Antibodies to HIV proteins may be detected by various methodologies; traditionally the standards are enzyme-linked immunosorbent assays (ELISAs) for screening and Western blot (WB) for confirmation. ELISA is highly sensitive but has been fraught with the major issues of false positives resulting from insufficient specificity and false negatives mainly from low levels of antibody in very early infection, the so-called window period.[Box 3.2] With each successive generation of assays, the rate of false positives has fallen and the window period shrunk significantly. This improvement owes itself to the replacement of viral lysate by recombinant antigens (2nd generation), the use of double-antigen sandwich to enhance capture of IgM and IgG antibodies (3rd generation), the incorporation of p24 antigen detection assay (4th generation) and the provision of separate results for HIV-1, HIV-2 antibodies and antigens in the newest assay. The incorporation of p24 antigen detection assay has reduced the average window period to around 2 weeks. The sensitivity is >99.5% and specificity >99% to both B and non-B subtypes for the current ELISA tests. However, a theoretical second window period may still exist, during which p24 falls and HIV-antibody remains at low, undetectable levels.

Box 3.2. Major causes of false-positive and negative HIV ELISA[1]

False positive
Antibodies to HLA antigens
Multiple transfusions
Recent influenza immunisation
Improper specimen handling (e.g. heating)
Receipt of experimental HIV vaccine
Manufacturing, human or laboratory error

False negative
Recent infection (window period)
Advanced HIV infection (rare)
HIV-2 (applies only to test kits specific for HIV-1)
Unusual HIV-1 serotype
Manufacturing, human or laboratory error

In a low prevalence community like Hong Kong, there would be an unacceptably high rate of false positive if ELISA were used alone for diagnosis. To improve specificity, the standard algorithm of HIV diagnosis is the tandem use of ELISA followed by confirmatory tests such as WB.[Algorithm 3] Most laboratories re-test the same sample with another ELISA test when a positive result is obtained, before it is subjected to confirmation by WB. The most commonly used criterion of a positive WB result is the presence of any two bands corresponding to p24, gp 41 and gp120/160. In some laboratories in Hong Kong, recombinant immunoblot assay (RIBA) or HIV-1/ HIV-2 antibody differentiation immunoassay (refer to the paragraphs below) is used for confirmation, which has comparable performance to WB.

WB the presence of only one band to a panel of antigens amounts to an indeterminate result, with single p24 reactivity being the most common. This has rarely been associated with non-Hodgkin’s disease and autoimmune diseases such as systemic lupus erythematosis, Sjogren’s syndrome and Hashimoto’s thyroiditis. Indeterminate results have also been reported with samples from patients with polyclonal gammopathy, haemolysis, rheumatoid factor, and some infections, such as HTLV-1. Since an early evolving HIV infection is also a possibility, longitudinal follow-up with reassessment of risk of HIV infection and repeat testing is required. Consideration may also be given to performing a virologic test for diagnosis. Confirmation of an HIV-2 positive test is similarly by WB or line immunoassay with HIV-2 specific antigens. Of note, studies showed that the HIV-1 specific WB has been interpreted as positive for HIV-1 in 46% to 85% of specimens from persons infected with HIV-2, resulting in incorrect or delayed diagnosis.[2][3]

HIV-1/HIV-2 antibody differentiation immunoassay is another option for confirmation after initial positive ELISA test. In 2014 the Centre for Disease Control and Prevention (CDC) in the United State (US) updated its recommendations for HIV confirmation by replacing the use of WB with HIV-1/HIV-2 antibody differentiation immunoassay, with the aim to better differentiate HIV-1 and HIV-2 infection.[4] The test is a lateral flow immunochromatographic assay which looks for antibodies binding to specific HIV-1 and HIV-2 antigens, e.g. p31, p24, gp41 and gp160 for HIV-1 and gp36 and gp140 in HIV-2. It has comparable sensitivity and specificity with the conventional WB. It has an advantage of faster turn-around time, easier to operate and better identification of HIV-2 infection.[5][6][7] Specimens tested negative in the antibody confirmatory assay after initial positive screening ELISA tests should undergo nucleic acid amplification tests (NAT) for detection of HIV-1 RNA to rule out acute HIV infection.[4]

It is noted that World Health Organization (WHO) endorses alternative algorithms for use in resource-limited settings, where a positive ELISA result is confirmed by one ELISA tests targeting different HIV antigens in tested population of HIV prevalence of greater than 5% or two ELISA tests in tested population of HIV prevalence of less than 5%.[8]

Rapid tests

A wide variety of rapid tests including dipstick assays, flow-through membrane assay and lateral flow membrane assays are available in the market. Some are intended for point-of-care use. At least eleven rapid HIV tests are approved by the US FDA, some of which are available in Hong Kong. Most of these tests are presented as strips or cartridges incorporating reagents which require no additional equipment. They are usually easy to use and can be carried out by health care workers who has received appropriate training. Results are available from 5 to 20 minutes and are often interpreted visually. The Oraquick® Advance Rapid HIV-1/2 Antibody test (Orasure Technologies) is appropriate for point-of-care setting with whole blood or oral fluid and is used by the Department of Health and Hospital Authority. Similar rapid tests are also available from other manufacturers, some of which are Ag-Ab combination tests which could reduce the window period. Clinical settings for which rapid tests might be useful include diagnosis for pregnant women with unknown HIV status at delivery and management of occupational exposures to HIV. For all rapid tests, strict adherence to manufacturers’ instructions is imperative to assure accurate results. The claimed sensitivity of Oraquick® is 99.3% and specificity 99.8%, but performance is somewhat lower in the field, especially with oral fluid.[9]

Nevertheless, it must also be noted that:

  • Rapid test is but one format of screening which requires confirmation by supplementary tests for diagnosis. Phlebotomy is still required for those with positive rapid test results.
  • It is controversial if rapid tests should be recommended for self-test purpose at home in Hong Kong, in particular with oral fluid, due to the lack of counselling opportunity, and the inferior performance with oral fluid. Further evidences and experiences are needed to establish a recommendation on the use of HIV self-test kit locally.

HIV p24 antigen assay

The p24 protein is a product of the gag gene and resides in the viral core. Prior to the availability of viral load testing, p24 was used for monitoring disease activity as well as diagnosis. It is specific as a diagnostic test but falls short on sensitivity, as a negative test is not uncommon in genuine HIV infection. In general, the level of p24 rises early soon after infection but subsequently falls as specific antibody develops, only to rise again in the advanced stage of AIDS. Nevertheless, its presence generally precedes that of antibody and may potentially shorten the window period if tested positive. It is now incorporated into newest generation ELISA antibody tests. Isolated p24 test is not recommended for diagnostic purpose.

Alternative samples – urine and oral fluid

ELISA and WB are usually performed on serum or plasma specimens. As a result, venipuncture is required, which by itself may be a deterrent to testing. Furthermore, individuals with poor venous access will pose difficulties. Under these circumstances, oral fluid and urine testing are useful. HIV antibodies, mainly IgG, are present in a lower concentration in these fluids than in serum. Thus, although the same general algorithm can be used, with immunoassays followed by WB, it is necessary to employ antibody capture assays to ensure adequate sensitivity. In Hong Kong, testing for HIV antibody using urine is carried out in methadone treatment centres. Urine and oral fluid tests may also be suited for screening programmes and epidemiologic surveillance by providing an acceptable alternative to phlebotomy.

Virologic tests for HIV

Both HIV RNA and proviral DNA are amenable to assay. The major application of RNA assay in HIV medicine is in the measurement of viral load which is essential to gauge the efficacy of antiretroviral therapy. Proviral DNA is incorporated in host cells, of which the peripheral blood mononuclear cells (PBMC) are the most accessible for testing. Both proviral DNA and HIV RNA may be tested for diagnosis. HIV isolation in lymphocyte culture is also diagnostic but has a long turnaround time and places extraordinary demands on biosafety facilities and expertise. These virologic tests are mainly used for diagnosis in infants and patients in the window period. NAT is also used in algorithms for further reduction of the window period in donated blood.

Diagnosis of HIV in perinatally exposed infants

In infancy and up to the age of 18 months, presence of placentally transferred HIV antibody from the mother precludes antibody testing for diagnosis. Virologic tests are preferred in this period, which are able to diagnose HIV in most infants by the age of 1 month and almost all by 6 months. Of note, cord blood is not advisable for testing because of potential contamination.

Diagnosis of acute HIV infection

Many of the considerations in paediatric infection are applicable to diagnosis of acute HIV infection. In the window period, antibody testing is usually negative or indeterminate. Diagnosis is thus possible only with virologic tests. With the new generation ELISA tests, the window period is reduced to around 2 weeks. Making a diagnosis of acute HIV infection informs proper management and rules out other mimicking syndromes. Infectivity is high in the seroconversion period which accounts for 10%-50% of all new HIV-1 transmissions and counselling on measures to reduce transmission should be given. Early antiretroviral treatment initiation on diagnosis if not contraindicated is beneficial in terms of a better preservation of immunity and CD4 count recovery, earlier virological suppression and overall fewer AIDS-related and non-AIDS related death.[10] It is therefore important to use more sensitive test for diagnosis of early HIV infection.

Indeterminate HIV antibody test results are not uncommon and it is a clinical decision as to whether a virologic test should follow. The presence of features compatible with acute HIV infection, especially after a high risk exposure, will suggest acute infection and repeat testing in a week is advisable. Alternatively, virologic tests can be used.

Use of NAT in blood donor screening

Blood borne pathogens in donated blood may escape detection if only serologic markers are used for screening. With the concurrent use of NAT, this window period for HIV detection is reduced to as short as 6 days. Although similar methodologies are used as in HIV viral load testing, NATs used in blood banking are qualitative tests optimised for sensitivity. They are implemented as standard in many developed countries, including Hong Kong, to assure the safety of blood supply. Despite its benefit in the detection of early HIV infection, it is not recommended for routine testing because of the possibility of false negatives e.g. in elite controllers and false positives results,[11] and also that most conventional NAT only detects common HIV-1 and HIV-2 viruses. Its cost-effectiveness balance is uncertain and will likely depend on the incidence of HIV in a population.[12] It must be noted that for clinical diagnosis, NAT complements HIV antibody test, and is rarely used in isolation.

Principles of the diagnostic HIV test[HK Guidelines 3A]

The HIV test as used for individual diagnosis in both opt-in and opt-out settings is underpinned by informed consent and confidentiality. There is no role for mandatory testing in Hong Kong. Pre-employment screening is not exempt from informed consent and, as a hiring policy, has to be very carefully justified. The law specifically protects the rights of those infected. To ensure consent is informed, discussion before and after testing is usually required. However, this does not need to be tedious or overly complex. The focus should be to identify and address the patient’s concerns. Where there are uncertainties, a readily available resource for consultation is the AIDS Hotline run by the Department of Health (2780 2211).[Box 3.3] In general, verbal consent suffices.

As the pillar of the doctor-patient relationship, confidentiality is paramount. It should not be compromised by the advent of electronic medical records. Confidentiality should still be safeguarded while identifying at-risk partners for HIV testing. This process of partner counselling and referral is voluntary and requires a patient’s trust in his/her health care provider to be successful. In Hong Kong, this is recommended to be done in the designated HIV clinics. HIV testing programmes are covered in Chapter B5.

Although HIV infection is not a notifiable disease, all medical practitioners are encouraged to report newly diagnosed HIV infection/AIDS to the Department of Health in an anonymous manner. The reporting form, DH2293, is downloadable from https://www.aids.gov.hk/english/surveillance/form.html

Box 3.3. Common themes of discussion in diagnostic HIV test

Theme Remarks
The HIV test, its benefits and consequences The purpose of testing, testing characteristics such as sensitivity and specificity, the voluntary nature of testing, and the principle of confidentiality should be explained. All questions raised by the subject should be conscientiously answered. He/she should be assured that no discrimination in medical care would result.
Risk assessment and knowledge of how HIV can be prevented These are important as a public health measure to reduce the prevalence of high-risk behaviour. Especially for those who test positive, they should be made aware of the need to inform others at risk, such as their sexual partners and those who shared their injecting equipment.
Importance of obtaining test results In the conventional two-step testing approach where subjects are seen twice (for testing and for retrieving results), a proportion may not return. This problem may be partially addressed by the use of rapid test.
Meaning of test results A positive rapid test result would need to be confirmed; the existence of a window period means that the subject who has exhibited risk behaviour recently should be retested later despite a negative result. Retesting is advised in about three months. For those at high risk, retesting earlier than 3 months should be considered.
Referral for HIV care* and support Referral and its mechanism shall be made known to the tested person, in the event a positive result is obtained. Referral to other medical specialists and psychologists may also be necessary.
Further information General information source includes that provided by the Department of Health (AIDS hotline: 2780 2211; http://www.aids.gov.hk)

*As of 2019, there are three designated HIV clinics in Hong Kong

  • Integrated Treatment Centre (Tel: 2116 2888), 8/F and 9/F Kowloon Bay Health Centre, 9 Kai Yan St, Kowloon Bay, Kowloon
  • Special Medical Clinic, Queen Elizabeth Hospital (Tel: 3506 6571), 6/F Ambulatory Care Centre, 30 Gascoigne Road, Kowloon
  • Infectious Disease Special Medical Clinic (Tel: 6461 0613), 4/F, Block K, Princess Margaret Hospital, 2-10 Princess Margaret Hospital Road, Lai Chi Kok, NT

Classification and staging of HIV/AIDS

Hong Kong Scientific Committee on AIDS

In Hong Kong, the current classification schema of adult HIV infection was prepared by the Scientific Committee on AIDS in 1993, which was adapted from that proposed by US CDC in the same year.[Box 3.4][HK Guidelines 3B] It assigns a stage of infection by combining the clinical manifestations (category A, B or C) and current CD4 count (1, 2 or 3, representing a count of ≥500/μL, 200-499/μL or <200/μL respectively). AIDS (C1, C2 and C3) is defined by 24 AIDS indicator conditions, regardless of the CD4 count. HIV-infected patients are classified based on the most advanced stage they have reached. Although not specifically required, it is desirable that the reporting physician include laboratory documentation of HIV infection.

Box 3.4. Classification for HIV infection and surveillance case definitions for AIDS in adults and adolescents in Hong Kong

CD4 categories (A)
Asymptomatic, acute (primary) HIV or PGL
symptomatic, not (A) or (C) conditions
AIDS-indicator conditions
(1) ≥500/μL A1 B1 C1
(2) 200-499/μL A2 B2 C2
(3) <200/μL A3* B3* C3
Category A Asymptomatic HIV infection
Acute HIV infection with accompanying illness or history of acute HIV infection
PGL, persistent generalised lymphadenopathy
Category B (includes but not limited to) Bacillary angiomatosis
Candidiasis, oropharyngeal (thrush)
Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy
Cervical dysplasia (moderate or severe)/cervical carcinoma in situ
Constitutional symptoms, such as fever (38.5°C) or diarrhea lasting >1 month
Hairy leukoplakia, oral
Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome
Idiopathic thrombocytopaenia
Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess
Peripheral neuropathy
Category C: AIDS indicator conditions Candidiasis of bronchi, trachea, or lungs
Candidiasis, esophageal
Cervical cancer, invasive
Coccidiodomycosis, disseminated or extrapulmonary
Cryptococcosis, extrapulmonary
Cryptosporidiosis, chronic intestinal (>1 month’s duration)
Cytomegalovirus (CMV) disease (other than liver, spleen or nodes)
Cytomegalovirus retinitis (with loss of vision)
Encephalopathy, HIV-related
Herpes simplex: chronic ulcer(s) (>1 month’s duration); or bronchitis, pneumonitis, or oesophagitis
Histoplasmosis, disseminated or extrapulmonary
Isosporiasis, chronic intestinal (>1 month’s duration)
Kaposi’s sarcoma (KS)
Lymphoma, Burkitt’s (or equivalent term (BL))
Lymphoma, primary, of brain
**Mycobacterium tuberculosis; extrapulmonary or pulmonary/cervical lymph node (only if CD4 <200/μl)
Pneumonia, recurrent
**Talaromyces marneffei infection, disseminated
Mycobacterium, other species or unidentified species, disseminated or extrapulmonary
Pneumocystis pneumonia
Progressive multifocal leukoencephalopathy (PML)
Salmonella septicemia, recurrent
Toxoplasmosis of brain
Wasting syndrome due to HIV
*A low CD4 alone is not an AIDS defining condition in Hong Kong for surveillance purpose
**AIDS defining conditions which differ from the US

US Centers for Disease Control and Prevention (CDC) classification

Classification by the US CDC has undergone successive iterations. In 1982 and before the discovery of its causative agent, the agency had developed a case definition for AIDS. Updates from 1985 to 1987 included newly identified disease manifestations and classified HIV infection into acute infection, asymptomatic infection, persistent generalised lymphadenopathy, and symptomatic infection. In 1993, it was revised into a dual clinical (category A, B and C) and immunologic (three CD4 count strata) system. The definition of AIDS was also extended to include a low CD4 count <200/μL per se without AIDS-defining condition (i.e. Stages A3 and B3). This was controversial and never adopted in Hong Kong.

In 2008, CDC combined the case definitions of HIV and AIDS into a single one for HIV disease. It requires laboratory confirmation of HIV infection and defines three stages according to clinical criteria and the CD4 count. This is in recognition of the continuum between asymptomatic infection and AIDS. AIDS is also de-emphasised as a surveillance event as, with effective treatment, HIV infected patients do not necessarily develop AIDS as was previously the case. Most recently in 2014,[13] stage 0 is added to the classification system to identify patients with early infection which may previously be classified into other stages due to a low CD4 count.[Box 3.5] Patients may progress and later be classified into stages 1, 2 and 3 six months after the initial diagnosis depending on the CD4 count and whether an opportunistic infection is diagnosed.

Box 3.5. HIV staging by US CDC

Stage Laboratory evidence Clinical evidence#
Stage 0^ Confirmed HIV infection + a negative or indeterminate HIV test result within 180 days before the first confirmed positive HIV test result of any type regardless of the CD4 count With or without presence of any AIDS-defining conditions
Stage 1 Confirmed HIV infection + CD4 ≥500/μL (or CD4 ≥26%*) and No AIDS-defining condition
Stage 2 Confirmed HIV infection + CD4 200-499/μL (or CD4 14-25%*) and No AIDS-defining condition
Stage 3 (AIDS) Confirmed HIV infection + CD4 <200/μL (or CD4 <14%*) or AIDS-defining condition
Stage unknown Confirmed HIV infection + unknown CD4 count and % and unknown AIDS-defining condition
*If CD4 count and % do not correspond, choose the more advanced stage
#AIDS-defining condition overrides CD4 criteria
^If the criteria for stage 0 are met, the stage is 0 regardless of the criteria for other stages (CD4 count and clinical evidence)

World Health Organization (WHO) classification

Early case definitions of AIDS by WHO closely followed those of CDC. In 1990, it proposed a staging system. It consisted of a clinical axis of four stages, ranging from stage 1 (asymptomatic, PGL), stage 2 (early or mild disease), stage 3 (intermediate or moderate disease), to stage 4 (late or severe disease). Conditions listed as stage 4 largely corresponded to the AIDS-defining illnesses of CDC. There was a parallel performance scale and an optional laboratory axis of three strata of CD4 or total lymphocyte count. In combination, they staged disease in a similar fashion as that of the Hong Kong classification or 1993 CDC system. Laboratory confirmation of HIV infection was desirable but not essential.

The last revision in 2007 continues with four clinical stages, i.e. HIV infection (Stages 1 and 2), and advanced HIV disease (Stages 3 and 4 (AIDS)). These stages form the basis of WHO recommendations on chemoprophylaxis and treatment. Clinical and definitive diagnostic criteria are now given for stage-defining clinical events. A parallel immunologic schema classifies CD4 count, if available, into four stages: non-significant (≥500/μL), mild (350-499/μL), advanced (200-349/μL) and severe (<200/μL or <15%). Laboratory confirmation of HIV infection is required. Consistent with the trend to de-emphasise AIDS, ‘advanced HIV disease’ suffices for reporting purpose.[14]


Although classification of HIV and AIDS originates from surveillance considerations, it has been successfully used for clinical purpose. Staging greatly assists estimation of prognosis and decisions on chemoprophylaxis and treatment. Classification schemata also allow cross comparison between clinical cohorts. Nevertheless, there are limitations. First, the different AIDS defining illnesses are not the same in reflecting one’s health status. For instance, pulmonary tuberculosis [Chapter D23] and Kaposi’s sarcoma (KS) [Chapter E30] may occur relatively early and carry a fairly good prognosis; however, CMV retinitis [Chapter D20] and disseminated Talaromyces marneffei infection [Chapter D24] do not occur until there is severe immunodeficiency and hence poor outlook. Second, while staging is not reversible by definition, the progressively downhill course of HIV disease is now commonly reversed by the use of HAART.[Chapter C10] This undermines the clinical significance of staging. The clinician should bear this in mind when communicating with patients and their families, as to most people AIDS is still taken to mean irreversible, terminal HIV disease.

Algorithm 3. General protocol for laboratory diagnosis of HIV infection


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HK Guidelines

  1. Scientific Committee on AIDS and STI. Principles of consent, discussion and confidentiality required of the diagnostic HIV test. Hong Kong: CHP, 2011. Available from: APPENDIX II: X8 and link
  2. Scientific Committee on AIDS. Classification system for HIV infection and survelliance case definition for AIDS among adolescents and adults in Hong Kong. Hong Kong: Department of Health, 1995. Available from: APPENDIX II: X21 and link