Diagnosis of HIV Infection in children

by

Diagnosis of HIV Infection in children

by

Associate Professor Kulkanya Chokephaibulkit

Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University.

Associate Professor Kulkanya Chokephaibulkit

Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University.

Topic To Be DiscussedTopic To Be Discussed

WHO Testing ProtocolWHO Testing ProtocolExperiences with dry blood spot techniqueExperiences with dry blood spot techniqueEntry points for testingEntry points for testing–– immunizationimmunization–– growth monitoringgrowth monitoring–– feeding centerfeeding center–– IMCIIMCI

Informed consent for testing in children/minorsInformed consent for testing in children/minorsRecommendation for resourceRecommendation for resource--limited settingslimited settings

Benefit of Making Early Diagnosis

Benefit of Making Early Diagnosis

Efficiently monitor of PMTCT program Efficiently monitor of PMTCT program

-- lead to improvement of the program lead to improvement of the program

Facilitate medical Rx, improve outcomeFacilitate medical Rx, improve outcome

-- PCP prophylaxisPCP prophylaxis

-- ARVARV

Society & Mental benefit Society & Mental benefit (especially for uninfected (especially for uninfected results)results)

-- Family make proper plan for the child and Family make proper plan for the child and caretakerscaretakers

Diagnosis of HIV Infection in Children

Diagnosis of HIV Infection in Children

In >18 months of age In >18 months of age -- diagnose by Antidiagnose by Anti--HIV serologyHIV serology

In <18 months of ageIn <18 months of age-- Maternal antibody may persist up to 18 monthMaternal antibody may persist up to 18 month--oldold

-- Clinical symptoms can guide, but not reliable, and may Clinical symptoms can guide, but not reliable, and may overlap with other problemsoverlap with other problems

-- Require detection of the virus to confirm infectionsRequire detection of the virus to confirm infections

-- DNADNA--PCRPCR

-- RNARNA--PCRPCR

-- p24 Agp24 Ag

WHO Recommendations Methods for Establishing HIV Infection in Infants and Children

WHO Recommendations Methods for Establishing HIV Infection in Infants and Children

Method of Method of diagnosisdiagnosis

Recommendations for useRecommendations for use Strength of recommendation/level Strength of recommendation/level of evidenceof evidence

VirologicalVirologicalmethodsmethods

To diagnose infection in infants and children To diagnose infection in infants and children aged under 18 months; initial testing is aged under 18 months; initial testing is recommended from 6 weeks of agerecommended from 6 weeks of age

HIV DNA (A(I))HIV DNA (A(I))HIV RNA (A(I))HIV RNA (A(I))U p24 U p24 agag (CII)(CII)

To diagnose HIV infection in mother or To diagnose HIV infection in mother or identify HIV exposure of infantidentify HIV exposure of infant

A (I)A (I)

HIV HIV antibody antibody testingtesting

To diagnose HIV infection in children aged To diagnose HIV infection in children aged 18 months or more18 months or more

A (I)A (I)

To identify HIVTo identify HIV--antibody positive children antibody positive children aged under 18 months and support a aged under 18 months and support a presumptive clinical diagnosis of severe HIV presumptive clinical diagnosis of severe HIV disease to allow initiation of ARTdisease to allow initiation of ART

A (IV)A (IV)

To exclude HIV infection where HIV antibody To exclude HIV infection where HIV antibody negative in children aged under 18 months negative in children aged under 18 months who are HIV exposed and never breastfedwho are HIV exposed and never breastfed

A (I)A (I)

To exclude HIV infection where HIV antibody To exclude HIV infection where HIV antibody negative in children aged under 18 months negative in children aged under 18 months who are HIV exposed and discontinued who are HIV exposed and discontinued breastfeeding for more than 6 weeksbreastfeeding for more than 6 weeks

A (IV)A (IV)

Anti-HIV SerologyAnti-HIV Serology

Should be tested by 2 different HIV testsShould be tested by 2 different HIV testsIf negative If negative (and asymptomatic)(and asymptomatic) >>can exclude infection in any age>>can exclude infection in any age

-- 74% of HIV74% of HIV--exposed uninfected infants exposed uninfected infants serosero--revert by revert by 9 mo, and 96% by 12 mo.9 mo, and 96% by 12 mo.

-- Breast feeding may cause infection later >> Need to Breast feeding may cause infection later >> Need to DD’’C BF >6 wk before excluding infection C BF >6 wk before excluding infection

If positive:If positive:-- Beyond 18 mo => confirm infection Beyond 18 mo => confirm infection ⇒⇒ Do not use Do not use ““combicombi”” test, it may pos beyond 18 mo.test, it may pos beyond 18 mo.

-- Younger than 18 mo =>unable to interpret Younger than 18 mo =>unable to interpret

Seronegative result can be “false” in some advanced cases

Seroconversion SensitivitySeroconversion Sensitivity

HIV combination Ag/Ab kits

(4th generation)

HIVAb kits3rd generation

Roche

No. Age Ab/Ag I Ab/Ag II Ax Vi PA 1 1 yo +50.43 5.8 ND ND3 1 yo +5.84 -0.36 -0.65 ND4 1 yo +1.3 -0.23 ND 0.0175 1 yo +30 -0.43 +1.4 ND6 1 yo +22.5 -0.61 -0.61 ND8 1 yo +8.25 -0.5 -0.86 ND9 17 mo +2.72 +2.31 -0.43 0.14

18 mo +2.06 +1.35 -0.43 Determine +2 18 mo +2.10 -0.29 -0.34 ND7 1 yo ND ND +2.25 134 2+

18 mo +1.15 -0.19 -0.38 ND

Fourth generation Third generation

4th generation serologic test

may be too sensitive to

diagnose HIV infection in

infants

Virological Tests(PCR for DNA / RNA, p24 Ag)

Virological Tests(PCR for DNA / RNA, p24 Ag)Suggest to test at 6-8 wk of age

- Repeat on a separate sample if possible, may be at 4 mo (US suggests : HIV is excluded if at least 2 negative PCR (at >1 mo, and > 4 mo)

Caveat :

Should confirm virological test with serology

Some non-subtype B or group “O” can be false negative PCR (newer assays are better)

Special SituationsSpecial Situations

Diagnosis in breast feeding infantsDiagnosis in breast feeding infants

>>>> Need to quit BF >6 wk before testing, as Need to quit BF >6 wk before testing, as infection occur at any time via breast milk infection occur at any time via breast milk

>>>> If quit > 6 wk, interpret the results as usualIf quit > 6 wk, interpret the results as usual

Negative serology in symptomatic infantsNegative serology in symptomatic infants

>>>> Recheck with Recheck with virologicalvirological testtest

Discrepant PCR result (+ Discrepant PCR result (+ →→ --, or , or -- →→ +)+)

>>>> confirm with serology at 9confirm with serology at 9--18 mo.18 mo.

WHO: Management of HIV Infection in Infants / Children WHO: Management of HIV Infection in Infants / Children

WHO: Management of HIV Infection in Infants / Children WHO: Management of HIV Infection in Infants / Children

Diagnosing HIV infection in infants and children less than18 months of age with unknown HIV exposure

Diagnosing HIV infection in infants and children less than18 months of age with unknown HIV exposure

Diagnosing HIV infection in infants and children less than 18 months of age with ongoing breastfeeding

Diagnosing HIV infection in infants and children less than 18 months of age with ongoing breastfeeding

Diagnosing HIV infection in infants and children less than 18 months of age with an initial negative HIV virological test and presenting with signs/symptoms of HIV at follow-up visit

Diagnosing HIV infection in infants and children less than 18 months of age with an initial negative HIV virological test and presenting with signs/symptoms of HIV at follow-up visit

Diagnosing HIV

infection in infants and

children aged 18

months or more

Diagnosing HIV

infection in infants and

children aged 18

months or more

Non breastfed childNon breastfed child Breastfed childBreastfed child

Diagnostic virological test from 6 week of ageDiagnostic virological test from 6 week of age

Negative test resultNegative test result Positive test resultPositive test result Negative test resultNegative test result

Child is uninfectedChild is uninfected Child is infectedChild is infected Children remains at risk of acquiring HIV infection until complete cessation of breastfeeding

Children remains at risk of acquiring HIV infection until complete cessation of breastfeeding

Refer for HIV treatment and care including initiation of ART

Refer for HIV treatment and care including initiation of ART

Child develops signs or symptoms suggestive of HIVChild develops signs or symptoms suggestive of HIV Child remains wellChild remains well

Diagnostic HIV testing (9-18 mo)Diagnostic HIV testing (9-18 mo) Routine follow-up testing as per national

programmerecommendations

(9-18 mo)

Routine follow-up testing as per national

programmerecommendations

(9-18 mo)

Virology test availableVirology test available Virology test not readily availableVirology test not readily available

Virology test positiveVirology test positive

Child is infectedChild is infected

HIV antibody test (18 mo)HIV antibody test (18 mo)

HIV antibody positive, Presumptive severe HIV diseaseHIV antibody positive, Presumptive severe HIV disease

Refer for assessment for HIV treatment and care including initiation of ARTRefer for assessment for HIV treatment and care including initiation of ART

Not breasted

Breasted

WHO Recommendations Methods for Establishing HIV Infection in Infants and Children < 18 mo

WHO Recommendations Methods for Establishing HIV Infection in Infants and Children < 18 mo

Does DNA-PCR Equal RNA-PCR?Does DNA-PCR Equal RNA-PCR?

Most of the time RNAMost of the time RNA--PCR is as sensitive PCR is as sensitive (90(90--100%) especially by 2100%) especially by 2--3 mo, because all of 3 mo, because all of perinatalperinatal infection have very high viral load by 2infection have very high viral load by 2--3 3 month of agemonth of age

RNARNA--PCR may be PCR may be ““undetectableundetectable”” from the effect of from the effect of perinatalperinatal ART & neonatal prophylaxis, and may be, ART & neonatal prophylaxis, and may be, ART in breast milkART in breast milkp24 Ag may be affected by p24 Ag may be affected by perinatalperinatal ARTART

DNADNA--PCR is not affected by PCR is not affected by perinatalperinatal ART and ART ART and ART in breast milkin breast milk

Rate of Positivity in HIV-Infected InfantsRate of Positivity in HIV-Infected Infants

AgeAge DNADNA--PCRPCR RNARNA--PCRPCR

2 weeks2 weeks 38%38% 63%63%

4 weeks4 weeks 71%71% 100%100%

77--8 weeks8 weeks 100%100% 100%100%

Sensitivity and Specificity of RNA and DNA polymerase chain reaction (PCR)Sensitivity and Specificity of RNA and DNA polymerase chain reaction (PCR)

AgeAge RNARNA--PCRPCR 95% CI95% CI DNADNA--PCRPCR 95% CI95% CI

SensitivitySensitivityBirthBirth2 months2 months6 months6 months

25/53 (47%)25/53 (47%)47/47 (100%)47/47 (100%)35/35 (100%)35/35 (100%)

3333--61619292--1001009090--100100

20/53 (38%)20/53 (38%)47/47 (100%)47/47 (100%)35/35 (100%)35/35 (100%)

2525--52529292--1001009090--100100

SpecificitySpecificityBirthBirth2 months2 months6 months6 months

100/100 (100%)100/100 (100%)Not evaluatedNot evaluated100/100 (100%)100/100 (100%)

9696--100100

9696--100100

329329--329 (100%)329 (100%)325/325 (100%)325/325 (100%)282/282 (100%)282/282 (100%)

9999--1001009999--1001009999--100100

Young NL. JAID 2000;24:401-7.

Other TestsOther Tests

HIVHIV--cultureculture-- Not better than PCRNot better than PCR-- Need facilities, more expensive, take longer timeNeed facilities, more expensive, take longer timeHIVHIV--IgMIgM: non: non--specific and cross react with RFspecific and cross react with RFHIVHIV--IgAIgA: not sensitive in younger than 6 mo. : not sensitive in younger than 6 mo. P24 Ag by Immune Complex Dissociation AssayP24 Ag by Immune Complex Dissociation Assay-- Highly specific but less sensitiveHighly specific but less sensitive((senssens = 81%, spec = 100% at 15 d = 81%, spec = 100% at 15 d --3 mo)3 mo)NEJM 1993;328:297NEJM 1993;328:297--302.302.

Experiences With Dry Blood Spot Technique: AdvantagesExperiences With Dry Blood Spot Technique: AdvantagesSmall volume required

Ease of sample collection, storage and shipment

Noninfectious transport medium

Safety/ handling exposure

Stability of sample – stable in room T > 1 month

Allows for centralization of testing facilities

Facilitates systemic, unbiased surveillance

Experiences With Dry Blood Spot Technique: LimitationsExperiences With Dry Blood Spot Technique: LimitationsLive viral isolates can not be determined

from DBSSample processing is more difficult,

require more steps

Lymphocytes subsets can not be measured

It is difficult to obtain long PCR fragment

(>1.2 kb) DBS – based genetic screening

Performance characteristics between whole blood and DBS for HIV-1 infant diagnosis and

viral load monitoring

Performance characteristics between whole blood and DBS for HIV-1 infant diagnosis and

viral load monitoring

CharacteristicsCharacteristics Whole bloodWhole blood DBSDBSStability of Stability of samplessamples

-- Up to 4 days at 2Up to 4 days at 2--2525ººCC -- Up to at least 3 months at Up to at least 3 months at room temperatureroom temperature

Sample Sample collectioncollection

-- More difficult especially in More difficult especially in infant age less than 1 yearinfant age less than 1 year

-- Easy to collect sample in Easy to collect sample in young infantyoung infant

Transportation Transportation of specimenof specimen

-- Require cold chain storage Require cold chain storage to ensure specimen integrityto ensure specimen integrity

-- Can be transport at room Can be transport at room temperature but avoid from temperature but avoid from heat and humidityheat and humidity

Assay to be Assay to be usedused

-- InIn--house or commercial kit house or commercial kit -- Standard procedure, less Standard procedure, less complexitycomplexity

-- InIn--house or commercial kit house or commercial kit -- Modified procedure, more Modified procedure, more complexcomplexityity

Volume of blood Volume of blood samplesample requiredrequired

-- At least 100At least 100µµLL -- At least 10At least 10µµLL

FDA Approved Filter papersFDA Approved Filter papers

Whatman- BFC 180

Scheicher & Schuell- Grade 903

HIV-1 proviral DNA Detection in Whole Blood and DBS by Multiplex DNA-PCR (In house) and Commercial Standard

Method Amplicor HIV-1 test

HIV-1 proviral DNA Detection in Whole Blood and DBS by Multiplex DNA-PCR (In house) and Commercial Standard

Method Amplicor HIV-1 test

Sample with known HIV statusHIV-1 proviralDNA detection in : Positive (50 cases) Negative ( 30 cases)

WB by Amplicor

WB by Multiplex

S&S IsoCode

Whatman

42/47* (89.4%) 28/28** (100%)

47/50 (94%) 30/30 (100%)

47/50 (94%) 50/50 (100%)

50/50 (100%) 50/50 (100%)

Sensitivity Specificity

* Low DNA template 3 cases **Low DNA template 2 cases Uttayamakul S. J of Virological Methods 2005;128:128-134

Prospective Field-Collected Dried Blood Spot DNA & RNA PCR for Infant Diagnosis

and Viral load Monitoring of HIV-1 Infection in Thailand

Young NL1-2, Chokephaibulkit K3, Chotpitayasunondh T4, Chaowanachan T1, Teeratkul A1, Jetsawang B1,

Neeyapun K1, Simonds RJ 1-2

Prospective Field-Collected Dried Blood Spot DNA & RNA PCR for Infant Diagnosis

and Viral load Monitoring of HIV-1 Infection in Thailand

Young NL1-2, Chokephaibulkit K3, Chotpitayasunondh T4, Chaowanachan T1, Teeratkul A1, Jetsawang B1,

Neeyapun K1, Simonds RJ 1-2

1 HIV/AIDS Collaboration, Nonthaburi, Thailand2 CDC, Atlanta, GA, USA

3 Siriraj Hospital, Mahidol Univ., Bangkok, Thailand4 Queen Sirirkit NICH, MOPH, Bangkok, Thailand

: Poster Presentation at 6th International Congress on AIDS in Asia and the Pacific, October 5-10, 2001; Melbourne, Australia

: Oral Presentation in XIV International AIDS Conference, July 5-12, 2002; Barcelona, Spain

Specificity and sensitivity of DBS compared to venipuncture whole blood samples for

diagnosis of HIV-1 infection

Specificity and sensitivity of DBS compared to venipuncture whole blood samples for

diagnosis of HIV-1 infectionWhole bloodWhole blood DBSDBS

PositivePositive NegativeNegative PositivePositive NegativeNegative

AmplicorAmplicor DNA DNA PCRPCR aa

56/5656/56 106/106106/106 56/5656/56 106/106106/106

Biomerieux RNA Biomerieux RNA NASBANASBAbb

56/5656/56 106/106106/106 56/5656/56 106/106106/106

InIn--house realhouse real--time time PCRPCRcc

Not doneNot done Not doneNot done 54/5654/56 25/2525/25

AssayAssay

Sensitivity = 96-100%, specificity = 100%a whole blood samples assayed was white blood cell pelletb whole blood samples assayed was plasmac In the real-time PCR assay, only 25 negative samples were tested

Entry Points for TestingEntry Points for Testing

It is best to screen at well baby clinic that include:– immunization– growth monitoring– feeding center– Anticipatory guidance– Educational activities

Integrated management of childhood illness:– Integrate preventive and curative intervention to

improve practices both in health facilities and at home; aim against 5 common diseases: ARI, diarrhea, measles, malaria, and malnutrition

EDUCATIONAL CLASS NURSE STATION

PLAY AREA

PHYSICAL EXAMINATION

ROOM

BLOOD COLLECTING ROOMVACCINATION ROOMVACCINATION ROOM

Informed Consent for Testing in Children / Minors

Informed Consent for Testing in Children / Minors

Depend on local lawDepend on local lawIn Thailand, under 18 years old require In Thailand, under 18 years old require parental (or legal guardian, or caregiverparental (or legal guardian, or caregiver’’s) s) consent, unless marriedconsent, unless marriedPrincipal of Interest BalancingPrincipal of Interest Balancing-- In In symptomaticssymptomatics: to help with treatment: to help with treatment-- In In asymptomaticsasymptomatics, esp. young children: to , esp. young children: to

decide for ARV and PCP prophylaxis decide for ARV and PCP prophylaxis

Recommendation for Resource-Limited Settings

Recommendation for Resource-Limited Settings

Need to develop an easier, cheaper, and more practical test for early diagnosis. - Boosted p24 Ag assay?

-- Commercial by Commercial by PerkinPerkin ElmerElmer-- Very promising, cheaper (Very promising, cheaper ($5$5--10), but need more study 10), but need more study

and standardizationand standardization-- Sensitivity 100% if VL Sensitivity 100% if VL >>30,000, 46% if VL <30,00030,000, 46% if VL <30,000

ICM 2005;43:506-8, , specificity >98% specificity >98% PIDJ 2004;23:173-5.

Corporate HIV diagnosis into the “one stop service” for routine well child careDevelop a specific national policy and guidelines Secure adequate supplies and resources

Boosted p24 Ag Correlate Well With HIV-RNABoosted p24 Ag Correlate Well With HIV-RNA

Sutthent R. J Clin Micro 2003;41(3):1016-1022. Brinkhof M WG. JAIDS 2006; 41:557-62.

Boosted p24 Ag may be an alternative marker for treatment monitoring

Boosted p24 Ag may be an alternative marker for treatment monitoring