Diagnosis Laboratorium Dan Pemantauan Infeksi HIV 2017 Handout PDF

Diagnosis laboratorium & pemantauan
infeksi HIV
Diagnosis Laboratorium dan Pemantauan

Infeksi Human Immunodeficiency Virus (HIV)
July Kumalawati
Departemen Patologi Klinik
Fakultas Kedokteran Universitas Indonesia
RSUPN dr. Cipto Mangunkusumo
Jakarta
Simposium Pelantikan Pengurus Cabang

July Kumalawati
Balikpapan 2 April 2017
Human Immunodeficiency Virus (HIV)
• Retrovirus
• 2 ss-RNA
• reverse
transcriptase
• Struktur :
envelope
capsid
core
July Kumalawati
July Kumalawati 1
infeksi HIV
Antigen HIV
p17
p24
gp41
gp160
gp120

July Kumalawati
Susunan gen HIV

tat rev
gag vif nef

tat
LTR pol env LTR

rev
p100 gp160
protease RT endonuclease
p55
p17 p24 p15 p66 gp120 gp41

p51
July Kumalawati
July Kumalawati 2
infeksi HIV
Filogeni HIV

July Kumalawati
Cara penularan HIV

Darah dan produk darah
Tusukan benda tajam tercemar darah dan

cairan tubuh
Hubungan seksual
Melalui plasenta dari ibu ke janin

July Kumalawati Simposium Pelantikan Pengurus Cabang
July Kumalawati 3
infeksi HIV
Replikasi HIV
HIV
Provirus
RNA RNA
reverse
transcriptase
DNA
Protein
budding
July Kumalawati
Serokonversi pada infeksi HIV
anti-gp41, gp120, gp160
anti-p24
p24 p24
RNA HIV CD4
minggu tahun
infeksi
July Kumalawati
July Kumalawati 4
infeksi HIV
Perjalanan penyakit infeksi HIV

Stadium 1 Stadium 2 (ringan) Stadium 3 (sedang) Stadium 4 (berat)
• Unexplained moderate • Unexplained severe weight loss (>10% • HIV wasting syndrome
• Asymp- weight loss (<10% of of presumed or measured body

•
•
•
Pneumocystis jiroveci pneumonia (PCP)
Recurrent severe bacterial pneumonia
Chronic herpes simplex infection
tomatic presumed or measured weight) (orolabial, genital or anorectal, of more
than one month’s duration or visceral at
• Unexplained chronic diarrhoea for any site)
• PGL body longer than one month
• Oesophageal candidiasis (or candidiasis
of the trachea, bronchi or lungs)
• weight) • Unexplained persistent fever (above
•
•
Extrapulmonary TB (EPTB)
Kaposi sarcoma
• Recurrent respiratory tract 37.5°C, intermittent or constant, for

• Cytomegalovirus (CMV) infection
(retinitis or infection of other organs)
• Toxoplasmosis of the central nervous
infections (sinusitis, longer than one month) system (CNS)
• HIV encephalopathy
tonsillitis, otitis media and • Persistent oral candidiasis • Extrapulmonary cryptococcosis
including meningitis
• pharyngitis) • Oral hairy leukoplakia (OHL) • Disseminated non-tuberculous
mycobacterial infection
• Herpes zoster • Pulmonary TB • Progressive multifocal

leukoencephalopathy (PML)
• Severe bacterial infections (such as • Penicilliosis
• Angular cheilitis pneumonia, empyema, pyomyositis,
•
•
Chronic cryptosporidiosis
Chronic isosporiasis
• Recurrent oral ulcerations bone or joint
• Disseminated mycosis (extrapulmonary
histoplasmosis, coccidiodomycosis)
• Pruritic papular erruptions • infection, meningitis, bacteraemia)

• Recurrent septicaemia (including due to
non-typhoidal Salmonella)
• Lymphoma (cerebral or B-cell, non-
(PPE) • Acute necrotizing ulcerative Hodgkin)
• Invasive cervical carcinoma
• Seborrhoeic dermatitis stomatitis, gingivitis or periodontitis • Atypical disseminated leishmaniasis
• Symptomatic HIV-associated
• Fungal nail infections • Unexplained anaemia (<8 g/dl ), nephropathy or HIV-associated
cardiomyopathy
neutropenia (<0.5 x 109/litre) or
chronic thrombocytopenia (<50 x
109/litre)

July Kumalawati
Perjalanan penyakit infeksi HIV

Infeksi Awal Intermediate Lanjut
primer (CD4+ >500/µL) (500>CD4+ >200/µL) (CD4+ <200/µL)
Jumlah sel CD4+
Laten secara
Infeksi
klinis
HIV
primer (tanpa gejala) AIDS-related complex
July Kumalawati
July Kumalawati 5
infeksi HIV
Pemeriksaan laboratorium HIV
Biakan virus
Deteksi antigen : p24
Deteksi materi genetik : DNA provirus / RNA
Deteksi antibodi : penyaring & konfirmasi

July Kumalawati
Biakan virus
• metoda rujukan
• mahal
• perlu tenaga terlatih & teliti
• BERBAHAYA : pemaparan terhadap
HIV dalam jumlah besar

July Kumalawati 6
infeksi HIV
Deteksi antigen p24

anti-gp41, gp120, gp160
anti-p24
p24 p24
Kadar
minggu tahun
infeksi
July Kumalawati
Deteksi antigen p24
• Diagnosis dini pada neonatus

• Diagnosis dini pada penderita
seronegatif dengan riwayat
pemaparan terhadap HIV

July Kumalawati 7
infeksi HIV
Deteksi antibodi
• Penyaring : ELISA, aglutinasi, dot-blot

immunobinding assay
• Konfirmasi : Western blot,

Lineimmunoassay, imunofluoresensi,
Radioimmunoprecipitation assay

July Kumalawati
Pemakaian strategi pemeriksaan WHO

Tujuan tes Prevalensi Strategi
Darah semua I
donor + prevalensi
transplan-
tasi
Surveilans > 10 % I
≤ 10 % II
Diagnosis simtomatik > 30 % I
≤ 30 % II
asimtomatik > 10 % II
≤ 10 % III
July Kumalawati
July Kumalawati 8
infeksi HIV
Alur pemeriksaan anti-HIV untuk penyaring

darah donor & transplantasi (Strategi I)
A1
A1 positif A1 negatif
Anggap Anggap
sebagai sebagai
“positif” “negatif”
Jangan dipakai !!
July Kumalawati
Alur pemeriksaan anti-HIV untuk surveilans

(Strategi II)
A1
A2 Lapor
sebagai
“Non-reaktif”

July Kumalawati
July Kumalawati 9
infeksi HIV
Alur pemeriksaan anti-HIV untuk surveilans
A2
A1 pos A2 A1 pos
pos A2 neg
Ulangi A1&A2
A1 pos A1 pos A1 neg
A2 pos A2 neg A2 neg
Lapor
Lapor Lapor sebagai
sebagai sebagai “Non-reaktif”
“reaktif” “Indeterm”
July Kumalawati
Alur pemeriksaan anti-HIV untuk diagnosis

(Strategi III)
A1
A2 Lapor
sebagai
“Non-reaktif”

July Kumalawati
July Kumalawati 10
infeksi HIV
A2
A1 pos A2 A1 pos
pos A2 neg
A3 Ulangi A1&A2
A1 pos A1 pos A1 pos A1 neg
A2 pos A2 pos A2 neg A2 neg
A3 pos
Lapor
Lapor sebagai
sebagai “Non-reaktif”
“reaktif”
July Kumalawati
A2
A1 pos A2 A1 pos
pos A2 neg
A3 Ulangi A1&A2
A1 pos A1 pos A1 pos A1 pos A1 neg
A2 pos A2 neg A2 pos A2 neg A2 neg
A3 neg A3 pos
Lapor
sebagai
Lapor
“Non-reaktif”
sebagai
“Indeterm” Simposium Pelantikan Pengurus Cabang
July Kumalawati
July Kumalawati 11
infeksi HIV
A2
A1 pos A2 A1 pos
pos A2 neg
A3 Ulangi A1&A2
A1 pos A1 pos A1 pos A1 neg
A2 neg A2 pos A2 neg A2 neg
A3 neg
Lapor
sebagai
“Non-reaktif”
July Kumalawati
A1 pos
A2 neg
A3 neg
Risiko Risiko
tinggi rendah
Lapor Lapor
sebagai sebagai
“Indeterm” “Non-reaktif”

July Kumalawati
July Kumalawati 12
infeksi HIV
Lapor Lapor
sebagai sebagai
“reaktif” “Indeterm”
Tes Konfirmasi
Positif Indeterm Negatif

July Kumalawati
Catatan penting :
• Untuk individu yang “baru” didiagnosis perlu
dilakukan pemeriksaan ulang dengan bahan baru,
sebelum dikonfirmasi
• Untuk hasil yang “indeterminate”, perlu diulang
dengan bahan baru yang diambil sedikitnya 14 hari
sesudah yang pertama
• Bila hasil tetap “indeterminate” dengan bahan
baru, lakukan pemantauan ulang pada 3, 6 atau 12
bulan. Bila setelah 1 tahun hasil tetap
“indeterminate”, dianggap tidak terinfeksi HIV

July Kumalawati 13
infeksi HIV
Pemilihan reagensia
Penyaring darah & produk darah serta
transplantasi (strategi I) :
Sensitivitas tertinggi, sebaiknya ≥ 99 %
Surveilans (strategi I) :
Spesifisitas ≥ 95 %
Diagnosis (strategi III) :
Pertama : sensitivitas tertinggi (≥ 99 %)
Berikutnya (ke-2 & 3) : spesifisitas lebih tinggi dari
yang pertama
Asal antigen atau prinsip tes berbeda
Diskordan <5%
July Kumalawati
Pemeriksaan konfirmasi
Western blot CDC : 2 di antara p24,

gp41 atau gp120/160
LIA
RIPA WHO : 2 pita envelope
USFDA : p24, p31 & gp41

atau gp120/160
Imunofluoresensi
July Kumalawati
July Kumalawati 14
infeksi HIV
Western Blot

July Kumalawati
Deteksi materi genetik
• DNA provirus
• RNA Viral load
Elektroforesis
Hibridisasi dengan DNA probe
July Kumalawati
July Kumalawati 15
infeksi HIV
PCR

July Kumalawati
NASBA

July Kumalawati
July Kumalawati 16
infeksi HIV
bDNA

July Kumalawati
Simposium Pelantikan Pengurus

Vermeulen MCabang
et al.TRANSFUSION 2009;49:1115-1125.
July Kumalawati
July Kumalawati 17
infeksi HIV
Pemantauan Pengobatan infeksi HIV
• Viral load
(RNA HIV)
• Jumlah
CD4+

July Kumalawati
Balikpapan 2 April 2017 http://aidsinfo.nih.gov/guidelines (2016)
Pemantauan Pengobatan infeksi HIV
WHO. Policy brief: consolidated guidelines on the use of antiretroviral

July Kumalawati Simposiumdrugs
Pelantikan Pengurus
for treating andCabang
preventing HIV infection: what’s new. 2015
July Kumalawati 18
infeksi HIV
Pemantauan Efek Samping

Anti-Retroviral
http://aidsinfo.nih.gov/guidelines (2016)
July Kumalawati
Interferences in immunoassay
• Analyte-independent • Analyte-dependent
interferences: interferences:
– haemolysis – cross-reactivity
– lipaemia – heterophile antibodies,
– effects of anticoagulant human anti-animal
– sample storage antibodies, autoanalyte
antibodies
– rheumatoid factors
– other proteins
(complement, lysozyme
and paraprotein)

July Kumalawati
July Kumalawati 19
infeksi HIV
Nature of Interferences
• Interferences that alter the measurable analyte

concentration in the sample
– Analyte-binding proteins
– Pre-analytical factors, e.g., anticoagulants, sample
storage
– Autoanalyte antibodies
• Interferences that alter antibody binding
– Heterophile antibodies
– Human anti-animal antibodies
– High-dose hook effect

July Kumalawati
Analyte-independent interferences
• Immunoassays are generally unaffected by sample
haemolysis and icterus
• Lipaemia can interfere in some immunoassays especially

those by nephelometry and turbidimetry
• Binding of cations, e.g., Mg2+ or Ca2+, to analyte can

change antigen conformation . Differences in results for
samples collected in lithium heparin, EDTA, and sodium
fluoride/potassium oxalate or tubes without
anticoagulant

July Kumalawati 20
infeksi HIV
• Other non-specific, exogenous interferences:

– aberrant assay reagent or equipment interaction with
patient sample
– dissimilar reactions for the standard matrix and
patient sample:
• sample-induced changes in pH and ionic strength of the
reaction mixture
• pre-analytical variables affecting by physical masking of the
antibody label e.g., lipaemia

July Kumalawati
• Sample carryover due to inadequate assay washing
• Failure to detect a sample clot
• Samples or assay reagents contaminated with

substances e.g., enzyme inhibitors, fluorophores
from ophthalmic examination, or radioimaging
isotopes

July Kumalawati 21
infeksi HIV
Analyte-dependent interferences
• Cross-reacting species also result in over- or
underestimation of sample analyte concentration if
an immunoassay reagent contains antibodies
directed toward molecules other than the antigen of
interest
• Analyte-binding globulins can alter the measurable

analyte concentration in the sample either by their
removal from or blocking of the analyte

July Kumalawati
• Heterophile antibodies, human anti-animal
antibodies, autoanalyte antibodies, rheumatoid
factors and other proteins

July Kumalawati 22
infeksi HIV
• High dose Hook effect

July Kumalawati
Properties of interfering substances
• Unique to an individual
• Interfering antibody concentration can change over
time within the same individual
• Low affinity poly-specific antibodies can be present
in high concentrations or high affinity in low
concentrations
• Can produce falsely high (false-positive) or falsely
low (false-negative) results

July Kumalawati 23
infeksi HIV
Properties of interfering substances
• May interfere within one or more manufacturers'

immunoassay systems but not necessarily in all
assays
• May interfere in a number of immunoassays for
different analytes
• The inclusion of one or more interference blocking
agents in manufacturers' immunoassay reagents may
be insufficient to overcome the interference

July Kumalawati
Measures to overcome interferences
• Use of an alternate immunoassay that preferably

uses antibody from a different species
• Measurement before and after addition of a blocking
reagent, or a series of concentrations of the blocker,
or a combination of blockers from different species
• Dilutions of the sample using the manufacturer's
diluent containing non-immune globulin
• Sample pre-treatment

July Kumalawati 24
infeksi HIV

July Kumalawati
July Kumalawati 25

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Diagnosis laboratorium & pemantauan

Diagnosis Laboratorium dan Pemantauan

Simposium Pelantikan Pengurus Cabang

Human Immunodeficiency Virus (HIV)

Simposium Pelantikan Pengurus Cabang

Susunan gen HIV

gag vif nef

LTR pol env LTR

p17 p24 p15 p66 gp120 gp41

Simposium Pelantikan Pengurus Cabang

Cara penularan HIV

Tusukan benda tajam tercemar darah dan

Melalui plasenta dari ibu ke janin

Serokonversi pada infeksi HIV

anti-gp41, gp120, gp160

RNA HIV CD4

Perjalanan penyakit infeksi HIV

• Asymp- weight loss (<10% of of presumed or measured body

• Recurrent respiratory tract 37.5°C, intermittent or constant, for

• Herpes zoster • Pulmonary TB • Progressive multifocal

• Pruritic papular erruptions • infection, meningitis, bacteraemia)

Simposium Pelantikan Pengurus Cabang

Perjalanan penyakit infeksi HIV

Pemeriksaan laboratorium HIV

Deteksi antigen : p24

Deteksi materi genetik : DNA provirus / RNA

Deteksi antibodi : penyaring & konfirmasi

July Kumalawati Simposium Pelantikan Pengurus Cabang

Deteksi antigen p24

Deteksi antigen p24

• Diagnosis dini pada neonatus

July Kumalawati Simposium Pelantikan Pengurus Cabang

• Penyaring : ELISA, aglutinasi, dot-blot

• Konfirmasi : Western blot,

Simposium Pelantikan Pengurus Cabang

Pemakaian strategi pemeriksaan WHO

Alur pemeriksaan anti-HIV untuk penyaring

Alur pemeriksaan anti-HIV untuk surveilans

Simposium Pelantikan Pengurus Cabang

Alur pemeriksaan anti-HIV untuk surveilans

Alur pemeriksaan anti-HIV untuk diagnosis

Simposium Pelantikan Pengurus Cabang

Alur pemeriksaan anti-HIV untuk diagnosis

Alur pemeriksaan anti-HIV untuk diagnosis

Alur pemeriksaan anti-HIV untuk diagnosis

Alur pemeriksaan anti-HIV untuk diagnosis

Simposium Pelantikan Pengurus Cabang

Alur pemeriksaan anti-HIV untuk diagnosis

Positif Indeterm Negatif

Alur pemeriksaan anti-HIV untuk diagnosis

July Kumalawati Simposium Pelantikan Pengurus Cabang

Western blot CDC : 2 di antara p24,

USFDA : p24, p31 & gp41

Simposium Pelantikan Pengurus Cabang

Deteksi materi genetik

Simposium Pelantikan Pengurus Cabang

Simposium Pelantikan Pengurus Cabang

Simposium Pelantikan Pengurus Cabang

Simposium Pelantikan Pengurus

Pemantauan Pengobatan infeksi HIV

Simposium Pelantikan Pengurus Cabang

Pemantauan Pengobatan infeksi HIV

WHO. Policy brief: consolidated guidelines on the use of antiretroviral