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Stratégies du diagnostic biologique de l'infection due au VIH chez les sujets âgés de plus de 18 mois (à l'exclusion du dépistage sur les dons de sang et chez les donneurs d'organes ou de tissus) - Laboratory diagnosis of HIV Infection 2000 - Guidelines

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Posted on Jan 01 2000 Laboratory markers of HIV infection Laboratory diagnosis strategies in relation to clinical situations Guidelines concerning prescription of laboratory tests and communication of the results Posted on Jan 01 2000

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Professor François DENIS, virologist, Hôpital Dupuytren, Limoges I.3. Level of evidence of the guidelines A further screening test is performed on the second sample ; it is not necessary to perform a Testing for p24 Ag or HIV-RNA is the second choice, if a conclusion cannot be drawn from
Dr. Jean-Michel DESCAMPS, specialist in internal medicine, Centre Hospitalier Général, Niort confirmation test routinely. interpretation of serology tests (see algorithm below).
Unless indicated otherwise, the guidelines are based on professional agreement.Dr. Patrice DOSQUET, project manager for ANAES, Paris
This guideline for a diagnostic strategy is based on professional agreement and requires aProfessor François FREYMUTH, virologist, Centre Hospitalier Universitaire, Caen Viral culture combined with testing for proviral DNA cannot be used in routine practice and is
Dr. Syria LAPERCHE, laboratory analyst, French National Blood Transfusion Service, Paris - Project Manager modification of the current wording of the nomenclature for laboratory testing procedures. reserved for specific clinical situations (suspected variant, persistent atypical serological profile) and
II. LABORATORY MARKERS OF HIV INFECTIONMme Rose-Marie LEBLANC, pharmacist and pathologist, Bordeaux for laboratories equipped to perform such tests.
Dr. Pascale MAISONNEUVE, laboratory analyst, Agence Française de Sécurité Sanitaire des Produits de Santé, II.5. Two methods should be used for the screening test
Saint-Denis II.1. Laboratory markers tested for in routine practice
III.2.2. General algorithmFor a screening test for HIV antibodies, the current wording of the nomenclature for laboratory testProfessor Patrice MASSIP, specialist in infectious disease, Hôpital Purpan, Toulouse
The laboratory markers tested for in a blood sample in routine practice are: procedures requires that two methods should routinely be used on the same sample, at least one ofDr. François PREVOTEAU DU CLARY, general practitioner, Paris The algorithm below is designed for laboratory diagnosis of infection when the presumed exposure• HIV antibodies (HIV Ab), using serological screening and confirmation methods;Dr. Christian RABAUD, specialist in infectious disease, Centre Hospitalier Universitaire, Vandœuvre-lès-Nancy them being a mixed ELISA method. The present guidelines do not call this into question. Even if the to HIV occurred more than three months previously. It is based on professional agreement.• p24 antigen (p24 Ag), tested for by enzyme immunoassay (ELISA);Professor Christine ROUZIOUX, virologist, Hôpital Necker-Enfants Malades, Paris performances of HIV infection screening methods have been improved, particularly for screening for
Dr. Catherine TAMALET, virologist, Hôpital de la Timone, Marseilles • HIV RNA (HIV-RNA), tested for by molecular biology methods. early infection, it is recommended that two methods still be used on the same sample when carrying III.3. Strategy for laboratory diagnosis if there are clinical signs suggesting primary infectionDr. Chantal VERNAY-VAÏSSE, dermatologist, Anonymous Free Screening Centre, Bouches du Rhône
Testing for proviral DNA and isolating the virus by viral culture are not commonly performed and are out a screening test. with HIVDr. Alain WAJSBROT, general practitioner, Avignon
only carried out in laboratories equipped to perform such tests. The requirement to use two methods in a screening test has been retained out of a concern to minimise
III.3.1. Laboratory markers which can be used
the number of false negatives obtained during screening, particularly if the prevalence of seropositive
II.2. Marker kinetics during the early phase of HIV-1 infectionREADING GROUP During primary infection with HIV, the HIV antibodies, indirect markers of infection, are absentsubjects exceeds 0.1% in the population studied. This recommendation may be reviewed once a national
A diagrammatic representation of the kinetics of the viral markers tested for in routine practice during during the very early phase which follows contamination (see Fig. 1). It is therefore recommendedprospective study designed to evaluate the relevance of using two methods for screening testing compared
Dr. Patrick ALVIN, paediatrician, Hôpital de Bicêtre, Le Kremlin-Bicêtre
the early phase of HIV-1 infection is shown in Fig 1. Times to appearance of the various markers are that screening for HIV antibodies, which must be carried out in all cases, be combined with testingwith just one has been completed. The working group hopes to see this study begun in the near future.Dr. Jean-Pierre AUBERT, general practitioner, Paris
given as mean guideline data, obtained using the best available techniques to detect each marker. for a direct marker of viral replication, either p24 Ag or plasma HIV-RNA.Professor Alain BERGERET, company medical officer, Lyon The combination recommended is to perform two mixed screening methods, one of which must be an
These times are subject to variation depending on the performance of the methods used and theDr. François BONNAL, specialist in internal medicine, Centre Hospitalier de la Côte Basque, Bayonne ELISA method. ELISA testing for p24 Ag is performed more easily and more commonly than testing for plasma HIV-
Dr. Vincent CALVEZ, virologist, Hôpital Pitié-Salpêtrière, Paris immune response of the infected subject.
RNA, which requires a molecular biology method. Although p24 Ag is detected later than HIV-RNA,
Dr. Hélène CHAPOULART, obstetrician and gynaecologist, Bordeaux II.6. Applicability of combined screening methods and only transitorily during the very early phase of infection (see Fig. 1), testing for p24 Ag is stillDr. Bernard COADOU, general practitioner, Bordeaux
HIV Ab Like, simple screening methods the current combined screening methods should be employed indicated for diagnosing early infection when testing for HIV-RNA is not available locally. TheDr. Joël COGNEAU, general practitioner, member of ANAES ‘ Scientific Council, Chambray-lès-Tours
Dr. Christophe COMPAGNON, dermatologist, Marseilles exclusively in the context of screening for infection. They should not be used for testing for just p24 laboratory analyst should routinely perform a neutralisation test on each screened sample which is
Dr. Anne-Marie COUROUCÉ, laboratory analyst, French National Blood Transfusion Service, Paris Ag. Specific methods should be used to test for p24 Ag. positive for p24 Ag, in order to confirm the specificity of the reaction observed.
Professor Elisabeth DUSSAIX, virologist, Hôpital Paul-Brousse, Villejuif
In comparative studies, it appears that the combined screening tests currently available are often ableDr. Joseph FAGOUR, general practitioner, Fort-de-France Algorithm (Part 1) Presumed exposure occurred more than three months previouslyHIV-1-RNA to detect infection earlier than the simple screening methods (a mean of 2 to 4.8 days earlier); but thisDr. Sylvie FERRARA, general practitioner, Ajaccio
Dr. Jean-Marc FRANCO, general practitioner, Saint-Pierre observation has been inconsistent. It is therefore premature to recommend the routine inclusion of
Dr. Gilles GRATEAU, specialist in internal medicine, Hôtel-Dieu, Paris combined screening methods in the two methods which are used for the screening test.
Dr. Anne GRUSON, laboratory analyst, member of ANAES’ Scientific Council, Arras
Dr. Philippe HOFLIGER, general practitioner, Nice II.7. Confirmation testMarker detection
Dr. Michel JANOWSKI, specialist in internal medicine, Centre Hospitalier Intercommunal, Montreuil threshold p24 Ag The confirmation test for HIV infection remains WB or IB. The criteria for interpreting HIV-1-WBProfessor Michel KAZATCHKINE, immunologist, Hôpital Broussais, Paris
D1 Time (days) are summarised in the annex.Dr. Alain LAFEUILLADE, specialist in internal medicine, Hôpital Chalucet, Toulon INFECTION 8-17 20-4512-26
Dr. Anne LAPORTE, epidemiologist, Health Monitoring Service, Saint-Maurice
II.8. Distinction between infection with HIV-1 and HIV-2Dr. Michèle MANIEZ-MONTREUIL, laboratory analyst, Nord-Pas-de-Calais Blood Transfusion Centre, Lille Figure 1. Relative of kinetics of viral markers during the early phase of infection with HIV-1.
Dr. Sophie MATHERON, specialist in infectious disease, Hôpital Bichat-Claude-Bernard, Paris It is necessary to distinguish between HIV-1 infection and HIV-2 infection in the confirmation test
Dr. Christophe MICHON, specialist in infectious disease, Centre Hospitalier de la Région Annecienne, Annecy
because of the differences in pathogenicity of the two viruses, in particular because of the slowII.3. Terminology for tests to detect HIV antibodiesProfessor Jean-Michel MOLINA, specialist in infectious disease, Hôpital Saint-Louis, Paris
progress of infection with HIV-2, and also the natural resistance of HIV-2 to certain antiretroviralDr. Anne MYARA, laboratory analyst, Hôpital Saint-Joseph, Paris Screening test: test intended to detect HIV antibodies, without determination of specificity. The
agents and the current unavailability of tests to quantify plasma RNA for HIV-2.Professor Dominique PEYRAMOND, specialist in infectious disease, Hôpital de la Croix-Rousse, Lyon
methods used in screening for HIV antibodies are
Josiane PILLONEL, epidemiologist, Health Monitoring Institute, Saint-Maurice
• ELISA; II.9. Diagnosis of infection with a variant of HIV-1Dr. Bernard POLITUR, general practitioner, Cayenne
• agglutination;Dr. Nerina PROFIZI, laboratory analyst, Centre Hospitalier Intercommunal, Toulon The pathologist may suspect infection with a variant of HIV-1 in various circumstances:
Professor Jacqueline PUEL, virologist, Hôpital Purpan, Toulouse • the so-called “rapid test” methods, on various types of media (nylon membrane, plastic membrane
• discrepancies in the results obtained by screening methods;Dr. Claude SICHEL, general practitioner, Carnoux-en-Provence etc).
• incomplete WB profile. Professor Alain SOBEL, immunologist, Hôpital Henri-Mondor, Créteil
Mixed screening method: method which simultaneously detects HIV-1 and HIV-2 antibodies Dr. Jean-François VIVES, specialist in internal medicine, Centre Hospitalier Antoine Gayraud, Carcassonne A definitive diagnosis of infection with a variant of HIV-1 is made using specific tests which are
(HIV-1/-2 antibodies).
performed only in laboratories equipped to perform these tests.
Simple screening method: method which detects HIV-1/2 antibodies without simultaneously
GUIDELINES detecting p24 Ag.
III. LABORATORY DIAGNOSIS STRATEGIES IN RELATION TO CLINICAL
Combined screening method (as opposed to simple screening method): method which simultaneously SITUATIONS
I. INTRODUCTION detects HIV-1/-2 antibodies and p24 Ag.
III.1. Clinical situations determining the laboratory diagnosis strategy
Confirmation test: test determining the specificity of HIV-1 or HIV-2 antibodies present in the serumI.1. Subject of the guidelines
studied. The method used is either a Western Blot (WB) or immunblot (IB). Doctors face three main clinical situations, each leading to a specific strategy for laboratory diagnosis:
The guidelines concern strategies for laboratory diagnosis of HIV infection in subjects aged over 18 • presumed exposure to HIV more than three months previously;A positive screening test should always be completed by a confirmation test. Seropositivity is only
months, excluding the screening of blood donations and screening of organ or tissue donors. They do • presence of clinical signs suggesting primary infection with HIV;established when the confirmation test result is positive.
not concern treatment strategy. • possible exposure to HIV, whether or not in a professional context, within the last three months,
but no clinical signs suggesting primary infection. II.4. Results from two separate samples must be available before HIV infection is
I.2. For whom the guidelines established
III.2. Laboratory diagnosis strategy when the presumed exposure to HIV occurred more
The guidelines are intended for all doctors and laboratory analysts who may have to establish a If the screening test is positive, it is recommended that the confirmation test be performed on the same than three months previously
diagnosis of HIV-induced infection. They are intended to assist doctors in choosing a diagnostic sample, so that the doctor can be alerted more rapidly to the fact that infection is present. However,
III.2.1. Laboratory markers that can be used strategy and in interpreting the laboratory results in relation to both the clinical circumstances of the if the confirmation test is positive, a second sample must be taken to eliminate any accidental error.
diagnosis and also the performance of the tests now available. HIV infection can only be definitely confirmed by a positive result on the second sample. The first choice of diagnostic strategy is based on testing for HIV antibodies.
Amplitude
Sample no. 1
Test for HIV antibodies using two methods M1 and M2 (M1/M2) (a)
M1 neg/ M2 neg M1 pos/ M2 neg M1 pos/ M2 pos
WB or IB (b)
POSITIVE NEGATIVE or INDETERMINATE
NO
INFECTION
See Algorithm - (part 2)
Seropositivity probable
Sample no. 2
Test for HIV Ab (M1/M2) (c)
M1 pos/M2 pos M1 pos/M2 neg M1 neg/M2 neg
INFECTION 1) Identification error
WITH HIV 2) Tubes mixed up
Sample no.3
Control M1/M2 and/or WB or IB (d)
(a) HIV antibody testing is routinely performed using two different methods, at least one of which should be a mixed ELISA
(b) If the confirmation test is WB, the first method used should be a HIV-1-WB in view of the marked predominance of prevalence of infection caused by HIV-1 compared with infection caused by HIV-2 in France;
If the laboratory performing the confirmation test is not the one that performed the screening test, the screening test should be repeated (2 methods);
Criteria for interpreting HIV-1-WB are given in the annex.
(c) Testing for HIV antibodies on the second sample may be performed using different methods from those used to test the first sample. A mixed ELISA must always be included.
(d) If the result for sample no. 3 is identical to the result for sample no. 2, this confirms the hypothesis that there has been an identification error or that the tubes for sample no. 1 were mixed up Algorithm (Part 2) III.3.2. Laboratory diagnosis strategy IV. GUIDELINES CONCERNING PRESCRIPTION OF LABORATORY TESTS AND avec la participation de
COMMUNICATION OF THE RESULTSIn the presence of clinical signs suggesting primary infection, the following tests should be performed
when the subject first goes to see a doctor: The ordering of laboratory tests to detect HIV infection is always an essential stage in the
• HIV antibodies; consultation. In addition to this, time should be taken to listen, explain and offer preventive advice.
• plasma HIV-RNA or p24 Ag. These tests are significant for the individual, but they also have a societal dimension relating to
control of the epidemic. The ordering of tests should never be treated as a minor matter.If the results of these tests are negative, and in the absence of antiretroviral treatment, the absence of
HIV infection is confirmed by the absence of HIV antibodies three months after the clinical signs The prescribing doctor should tell the patient that they will be given the test results at the next
were observed. consultation, which should be timed in accordance with the local situation with regard to carrying out
tests. The importance of the consultation at which results will be given should be emphasised.If the test for plasma HIV-RNA or p24 Ag is positive and if antiretroviral treatment has been started,
the kinetics of appearance of HIV antibodies may be delayed longer than usual. At the present time Whatever the result, the laboratory should send it to the doctor who ordered the test. The
these kinetics are not well known. A strategy for laboratory diagnosis may be specified when more is interpretation should be made by the laboratory analyst, after contact with the doctor ordering the test,
known about the kinetics, particularly when the results of monitoring of cohorts of subjects treated if necessary. The patient may be told by the laboratory analyst that the results have been
with antiretroviral agents have been published. communicated to the doctor. The laboratory analyst should not tell the patient the results directly. It
is the prescribing doctor who must give the results to the patient during a specific consultation. This
III.4. Strategy for laboratory diagnosis in the event of possible exposure to HIV, whether
enables the doctor to repeat advice about prevention of transmission of HIV infection, or to explain
professional or not, within the last three months, but in the absence of clinical signs
the other diagnostic tests which might be necessary, or if HIV infection has been confirmed, to begin
suggesting primary infection
the long-term management and monitoring of the patient. CLINICAL PRACTICEThe patient may be seen within 48 hours of possible exposure to HIV. In these circumstances, once
the risk of contamination has been evaluated, prophylactic antiretroviral therapy may be prescribed. V. UPDATING OF THE GUIDELINES GUIDELINES
The diagnostic strategy should take account of whether or not such therapy has been prescribed. STRATEGIES FOR
These guidelines should be revised, probably within a maximum of two years, to take account of
The patient may be seen later than this and the presumed date of exposure may be taken as a guide
diagnostic advances, the registration of diagnostic methods and the results of clinical studies currently
when prescribing laboratory tests.
in progress. ABORATORY IAGNOSISL D
III.4.1. In an exposed subject
VI. ANNEX. PROCEDURE TO OBSERVE AND INTERPRETATION OF HIV-1 WB
In all cases HIV antibodies should be tested for from the time of the first consultation. Table I OF NFECTIONS IN UBJECTS AGEDHIV I S
summarises the diagnostic strategies recommended and already published in France.
Procedure and interpretation
If the source subject status is HIV negative, laboratory monitoring of the exposed subject is not
Definite positive OVER 18 MONTHSnecessary when the initial screening test is negative, unless there is a suspicion that seroconversion is
A minimum of 2 env antibodies • Second sample asked for immediately to check that there has been no
in progress in the source subject. (gp120 and gp160) error in sampling or contamination of the first sample. (EXCLUDING SCREENING OF BLOOD DONATIONS
AND • if there is a strong reaction on proteins derived from the gag and/or
Table 1. Strategy for laboratory diagnosis of HIV infection in the event of possible exposure within AND SCREENING OF ORGAN OR TISSUE DONORS)1 gag or pol antibody pol genes compared with the proteins derived from the env genes, per-
the previous three months. form HIV-2 serology tests and consider HIV-1 group O infection.
Probable positive
Exposed subject who has not received Exposed subject who has received a) 1 p24 antibody • Further sample required 1 to 2 weeks later:
antiretroviral prophylaxis antiretroviral prophylaxis AND - If change is observed: HIV-1 seroconversion January 20001 gp160 antibody - If no change and HIV-2 WB is negative : probable false positiveInitial tests when the subject • screening for HIV antibody • screening for HIV antibody
(unusual) or group O HIV-1 (rare profile).has been seen during the 48-hour • plasma p24 Ag or HIV-RNA • plasma p24 Ag or HIV-RNA
- If no change and HIV-2 WB positive: HIV-2 seropositivity (rare profile).period following exposure (if multiple exposures during previous (if there have been multiple exposures
b) 2 env antibodies (gp120 and gp160) • Further sample required 1 to 2 weeks later:two months) during previous two months)
- If sample negative: contamination of first sample or identification error
Week 4 Between 3 and 6 weeks after exposure : - If change observed: HIV-1 seroconversion (rare profile)
• screening for HIV antibody - If no change and HIV-2 WB positive : HIV-2 seropositivity (rare profile)
• plasma p24 Ag or HIV-RNA
This profile may also be observed in late stage AIDS.
Week 8 Between 3 and 6 weeks after the end of Patterns to be monitored
treatment: Isolated gp160 antibodies • Perform HIV-2 serology, especially if the 2 screening methods are clear
• screening for HIV antibody Isolated p24 antibodies (+/- p55 Ab) positives
• plasma p24 Ag or HIV-RNA Isolated p34 antibodies (+/-p24 Ab) • Ask for another sample 1 to 2 weeks later.
• If no change and HIV-2 negative: false positive reaction or HIV-1 variantWeek 12 3 months after exposure:
(unusual).• screening for HIV antibody
MANAGEMENT COMMITTEENegativeWeek 16 3 months after the end of treatment:
p17 antibodies An absence of reaction for WB combined with clearly positive results for• screening for HIV antibody Dr. Jean-Pierre AUBERT, general practitioner, ParisOther patterns not listed here screening methods suggests the start of seroconversion. A further sample
Professor Francis BARIN, virologist, Hôpital Bretonneau, Tours6 months* No antibodies taken 1 to 2 weeks later is necessary in this case.
Professor Stéphane BLANCHE, paediatrician, Hôpital Necker-Enfants Malades, Paris
Professor Françoise BRUN-VEZINET, virologist, Hôpital Bichat-Claude-Bernard, Paris* Testing for HIV antibody six months after presumed exposure is a legal obligation in the case of an accident at work. Proteins derived from env genes: gp 160, gp120, gp41; gag genes: p55, p24, p18; pol genes: p68, p34
Professor François FREYMUTH, virologist, Centre Hospitalier Universitaire, Caen
Dr. Pascale MAISONNEUVE, Agence Française de Sécurité Sanitaire des Produits de Santé, Saint-Denis
II.4.2. Source subject
Dr. Liliane MARMIÉ, general practitioner, Paris
If an antiretroviral prophylaxis treatment is envisaged, it is important to try to discover the status of Professor Jean-Michel MOLINA, specialist in infectious disease, Hôpital Saint-Louis, Paris
The full report: ISBN: 2-910653-71-4 (Net price: 15,24 € - 100,00 FF) is available fromTechniques for measuring HIV-RNA can give false positives values close to the detection threshold, and Dr. Anne MYARA, laboratory analyst, Hôpital Saint-Joseph, Paristhe source subject with regard to HIV infection. If the source subject was seronegative at the time of
Agence Nationale d’Accréditation et d’Évaluation en Santé (ANAES) Professor Daniel SERENI, specialist in internal medicine, Hôpital Saint-Louis, Pariscertain viruses are not detected (HIV-2, HIV-1 variants). A diagnosis of infection with HIV cannot be the exposing incident, it may not be necessary to start antiretroviral therapy in the exposed subject, or
Service Communication et Diffusion
made from carrying out these tests alone. Measurement of plasma HIV-RNA is therefore only one it may be possible to stop it. When the HIV status of the source subject is not known, particularly after
159, rue Nationale – 75640 Paris Cedex 13 – FRANCE WORKING GROUPelement in the series of clinical and laboratory tests involved in diagnosing HIV-1 infection. In addition, exposure at work, it is recommended that the source subject undergo an HIV antibody screening test,
Send a written order together with a cheque made out tothe reagents used to detect HIV-RNA are not currently registered as reagents specific to diagnosis of HIV provided their consent is obtained, if their state of consciousness so permits. A rapid unitary test Dr. Claude BACHMEYER, specialist in internal medicine, Hôpital Laennec, Creil
« l’Agent comptable de l’ANAES »infection, but as reagents used to monitor HIV-RNA levels in cases of known infection. method may be used when ELISA techniques cannot be performed in an emergency setting to help in Professor Francis BARIN, virologist, Hôpital Bretonneau, Tours
Professor Françoise BRUN-VEZINET, virologist, Hôpital Bichat-Claude-Bernard, Paris - deciding whether the exposed subject should be given prophylaxis. In all cases, the result obtained The full report can be downloaded for free from the ANAES website:
The fact that plasma HIV-1-RNA or p24 Ag have not been detected cannot exclude exceptional cases Working Group Chairmanusing a rapid unitary method should subsequently be verified. www.anaes.fr or www.sante.fr
of symptomatic primary infection with HIV-2 or a variant of HIV-1. Dominique COSTAGLIOLA, epidemiologist, INSERM, Paris
WB or IB
Sample no. 1
NEGATIVE or INDETERMINATE
Plasma p24 Ag or HIV-RNA *
POSITIVE NEGATIVE
p24 Ag neg plasma HIV-RNA neg
Suspected recent infection 1) Suspected recent infection 1) HIV-2
2) HIV-2 3) False positive ELISA
3) False positive ELISA 4) Variant HIV-1
4) Variant HIV-1
Sample no. 2
HIV Ab (M1/M2) (a) AND
WB or IB AND p24 Ag or HIV Ab (M1/M2) AND WB or IB HIV Ab (M1/M2) AND WB or IB
plasma HIV-RNA
M1 neg/M2 neg M1 pos/M2 pos M1 pos/M2 pos M1 neg/M2 neg M1/M2 unchanged M1 neg/M2 neg
WB or IB neg Change in WB or IB Change in WB or IB WB or IB neg WB or IB unchanged WB or IB neg
Ag or RNA neg p24 Ag pos or neg
HIV-RNA pos
1) Identification error 1) Identification error 1) HIV-2 (b) 1) Identification error
2) Tubes mixed up Recent infection 2) Tubes mixed up 2) False positive ELISA (c) 2) Tubes mixed up
Sample no. 3
Control M1/M2 Control M1/M2 Control M1/M2
and/or WB or IB (e) and/or WB or IB (e) and/or WB or IB (e)
(a): Testing for HIV antibodies on the second sample may be performed using different methods from those used for the test on the first sample. A mixed ELISA must always be included.
(b): A HIV-2-WB or IB test is required to confirm HIV-2 infection. A third sample is not usually required.
(c): Confirmation of a false positive ELISA requires further ELISA tests to be performed, sometimes on a third sample.
(d): A third sample is usually required for confirmation of infection with variant HIV-1 in view of the specific retrovirus cultures and PCR tests, carried out in a laboratory equipped to perform these tests.
(e): If the result for sample no. 3 is identical to the result for sample no. 2, this confirms the hypothesis that there has been an identification error or that the tubes for sample no. 1 were mixed up