Diagnostic ofMicrobiology

Aims

• To provide accurate information about: the presence or absence of microorganisms, or characterized biomarker of microorganism in a specimen that may be involved in patient’s disease process

Important requirements

• Adequate collection, transport, and processing of clinical specimens

• Cooperation among medical team members :• Clinical practitioners• Nurses• Laboratory practitioners

• Ongoing communication and education among all members of the team

Diagnostic Microbiology:

• Culture Assays for Bacteria and Fungi

• Culture Assays for virus

• Serologic Assays for Microorganism

• Molecular diagnostics (qualitative or quantitative)

• Genotyping (characterization of certain important gene of microorganism)

Spesimen

Mikroskopik

DitolakDiterima dengan catatan

Kultur pada agar

Diterima

Uji biokimiawi

Isolat

Penentuan Genus, Spesies dan Serotipe

Uji serologi

Uji kepekaan terhadap antibiotikaIdentitas bakteri

Hari ke 1

Hari ke 3

Hari ke 2

Kultur pada medium cair

BLOOD CULTURE FOR BACTERIA AND FUNGI

Factors for successful recovery of microbes from blood

• Possible types of bacteremia

• Specimen collection methods

• Blood volumes

• Number of specimens

• Timing of blood cultures

• Interpretation of results

Bacteremia

Blood Specimen Collection Technique

• Perform blood sample collection before administering antibiotics if clinically possible

• Label blood culture bottle and all tubes :– Patient’s

– Hospital & Doctor

– Specimen type

– Time of collection etc.

Specimens that have sat too long prior to processing must be rejected

• Select venipuncture site. Avoid the femoral vein area.

Blood volume• Adults : <30 CFU/ml, more blood that is cultured,

the greater the chance of isolating the organism• Children : <5 CFU/ml, infants : >1000 CFU/ml

Effect of Volume

*Reprinted from Infectious Disease Clinics of North America, Vol 16, M.L. Towns and L. B. Reller, Diagnostics methods: Current best practices and guidelines for isolation of bacteria and fungi in infective endocarditis, p. 363-376(2002) with permission from Elsevier.

Handling Positive Blood Cultures

• Growth indication (macroscopic) :• Hemolysis of RBC, gas bubbles, turbidity,

small aggregates of bacterial or fungal growth in the broth, on the surface, or along the walls of the bottles

• Gram-stained smear

• Subculture

Gram staining : report

• The presence of host cells and debris

• The Gram reactions, morphologies, and arrangement of bacterial cells present. Reporting the absence of bacteria and host cells can be equally as important

• Optionally, the relative amounts of bacterial cells (rare, few, moderate, many) may be provided

Subculture

• 5% sheep blood agar

• Mc Conkey / Endo agar

• Chocolate agar

• Supplemented anaerobic blood agar

Identification of microorganisms grown in culture

• Preliminary identification• Gram reaction and cell morphology• Growth requirement and condition

• Further identification• Ability to produce enzymes that can be detected by

simple tests• Ability to metabolize sugars oxidatively or

fermentatively• Ability to use a range of substrates for growth• Some species are identified on the basis of their

antigens by reacting cell suspensions with specific antisera

Fungi

• Identified from colonial characteristics and cell morphology

• Biochemical tests (substrates assimilation)

• Grow more slowly than bacteria and final identification may take up to 2 weeks

KULTUR DARAH

GRAMLAPORKAN/ HUBUNGI DOKTER

GRAM

AEROB

AD MAC AC

GRAM

ANAEROB

IDENTIFIKASI

KONVENSIONALAPI 20 E

API 20 NEAPI STREP

API 20 A (ANAEROB)API CAUX (JAMUR)

UJI KEPEKAAN

Antibiotic susceptibility

• Methods that are directly measure the activity of one or more antimicrobial agents against a bacterial isolates

• Methods that are directly detect the presence of a specific resistance mechanism in a bacterial isolate

• Special methods that are measure complex antimicrobial-organism interactions

Candida sp. on Sabouraud Agar

Blood Agar

Mueller Hinton Agar

DISK DIFFUSION TEST

• CLSI 2011• Mueller Hinton Agar (Ø 100 mm)• Standard kekeruhan bakteri ~ 0.5

MacFarland

MIC=2mg/L

2mg/L

1mg/L

0.5mg/L

0.25mg/L

4mg/L

8mg/L

amount ofantibiotic

cloudiness meansbacteria can grow atthat concentration of

antibiotic

no zone around disc =resistant

clear zonearound disc =

sensitive

bacterium

minimum inhibitory concentration (MIC) test

E-Test

CULTURE ASSAYS FOR VIRUS

Culturing Virus in Laboratory

3 types of media have been developed:

• Media consisting of whole organisms (bacteria, plants, or animals)

• Embryonated (fertilized) eggs

• Cell culture

Culturing viruses in bacteria• Some bacteria are easily

grown and maintained in vitro

• Phages can be grown in bacteria maintained in either liquid cultures or on agar plates

Method:• Bacteria and phages are

mixed with warm (liquid) nutrient agar and poured in a thin layer across the surface of an agar plate

• During incubation: bacteria infected lyses and release new phages that infect nearby bacteria, while uninfected bacteria grow normally

• After incubation: uniform bacterial lawn interrupted by clear zone called plaques

Culturing Viruses in Plants and Animals

• Plant and animal viruses can be grown in laboratory plants and animals

• Plant: tobacco ((tobacco mozaic viruses)

• Animals: rats, mice, guinea pigs, rabbits, pigs, primates

Culturing Viruses in Embryonated eggs

Chicken eggs:• Useful culture medium

(inexpensive, the largest of cells, free of contaminating microbes, contain a nourishing yolk)

• Viruses are injected into embryonated eggs at the sites that are best suited for the virus’s maintenance and replication

Culturing Viruses in Cell (Tissue) Culture

Cell culture: consists of cells isolated from an organism and grown on the surface of a medium or in broth• Diploid cell culture

Created from embryonic animal, plant or human cells that have been isolated and provided appropriate growth conditions

• Continuous cell culturesDerived from tumor cells, longer lasting. HeLa cells: derived from a woman named Henrietta

Lacks who died of cervical cancer, 1951

Plaque assay

http://www.nanohedron.com

SEROLOGIC ASSAYS FOR MICROORGANISM

Serologic diagnosis : why ?• Some microorganisms cannot be cultivated

on artificial media• Isolation of obligate intracellular organisms

requires animal inoculation or the use of cell culture

• Toxin detection • Cultivable organisms

• Labile in transport condition• Long incubation period• So fastidious as to render isolation unreliable

Serologic Methods

• Agglutination assays

• Precipitation assays

• Complement fixation test

• Neutralization test

• Microscope-assisted labeled-reagent technique

• Immunoassays with labeled reagents (EIA, RIA, FIA)

Antigen detection• Microbial-specific structural components (antigens) are

identified in specimens obtained from an infected host • Can be used to identify microorganisms once they have

been recovered in culture• Methods depends on the fact that some microbial

components are chemically unique and form areas on the molecule known as antigenic determinants

• The antigens can be recognized or bond by specifically with antibody molecules to form stable products

Antibody detection

• IgM antibodies • detected earlier in the infection (7-10 days)• Usually indicative of active, as opposed to past, infection

• IgG antibodies• Previous infections or immunization• Serodiagnosis of an infectious diseases requires

measurement of IgG concentration on both acute-phase and convalescent-phase serum specimens

• Chronic infections, epidemiology

False-Negative Serologic test results

Negative result for a patient who really is infected• May not have an intact immune system, and therefore

may not be able to respond to an antigenic stimulus• Congenital or acquired immunodeficiency diseases• Receiving either immunosuppressive therapy after organ

transplantation or cancer chemotherapy

• Neonates may not always respond to an infectious agent because their immune system are not fully mature

• For some infections (e.g. legionaires’ disease) antibody titers may not rise until months after acute infection

False-Positive Serologic test results

Positive result for a patient who is not infected by the specific agent for which the test is design

• Production of cross-reacting antibodySome antigen associated with different microorganisms are

closely related and a host may respond by producing antibody not only to the invading organism but also to antigenically closely related organism

• Reactivation of a latent organism due to infection by a different organism

• Receiving intravenous immunoglobulin

Agglutination

Slide Agglutination test

Hemagglutination and Hemagglutination Inhibition

HemaglutinasiAglutinasi dengan sel darah merah

Dasar hemaglutinasi bermacam-macam:a. reaksi antibodi dengan antigen pada permukaan sel darah merah: penentuan golongan darah

b.hemaglutinasi oleh virus atau riketsia:• pada permukaan sel darah merah terdapat reseptor spesifik

terhadap virus atau riketsia• antibodi spesifik terhadap bagian virus yang berinteraksi

dengan reseptor pada sel darah merah dapat menghambat hemaglutinas (hambatan hemaglutinasi: haemagglutinatio inhibition/HI)

HEMAGLUTINASI

AGLUTINASI SEL DARAH MERAH

SDM SDM

SDM SDM

ANTIGEN

ANTIBODI

CONTOH: PENENTUAN GOL. DARAH

DAPAT TERJADI :

1. ANTIBODI TERHADAP ANTIGEN PADA PERMUKAAAN SEL DARAH MERAH

HEMAGLUTINASI

2. VIRUS / RICKETTSIA

MEMPUNYAI HEMAGLUTININ, DAPAT MENGGUMPALKANSEL DARAH MERAH.

SDM SDM

SDM SDM

VIRUS

REAKSI INI DAPAT DIHAMBAT OLEH ANTIBODI REAKSI HAMBATAN HEMAGLUTINASI

VIRUS + ANTIBODI

+ SDMSDMHAMBATANHEMAGLUTINASI

SEL DARAH MERAH

ANTIBODI

SDM

Hemaglutinasi

Dasar hemaglutinasi bermacam-macam (lanjutan):•Sel darah merah berfungsi sebagai pembawa antigen (passive haemagglutination) :•sel-sel darah merah yang telah dilapisi oleh antigen tertentu dapat digunakan untuk mengetahui adanya antibodi terhadap antigen tersebut di dalam serum penderita

3. SEL DARAH MERAH SEBAGAI PEMBAWA ANTIGEN

SEL DARAH MERAH DAPAT DILAPISI DENGAN ANTIGENDIPERMUKAANNYA.

ANTIGEN ANTIBODI YANG SESUAI HEMAGLUTINASI

KEUNTUNGAN : MEMUDAHKAN MELIHAT HASIL REAKSI (PARTIKEL BESAR).

DISEBUT JUGA : HEMAGLUTINASI PASIF

Contoh : Reaksi TPHA (Treponema Pallidum Haemaglutination Test)

SDM

SDM SDM

SDM SDM

SDM SDM

SDM

+

PengenceranAntigen

1/20

1/40

1/80

1/160

1/320

1/640

1/1280

1/2560

Reaksi Hemaglutinasi (HA)

Titer Antigen : 1/160 (1 HAU )Untuk Reaksi HI digunakan Antigen 4 – 8 HAU

AntigenKontroleritrosit

PengenceranSerum pasien

1/20

1/40

1/80

1/160

1/320

1/640

1/1280

1/2560

SerumFase akut

SerumKonvalesens

Kontrol eritrosit

Kontrol antigen

Hambatan Hemaglutinasi (HI)

Bila jarak pengambilan serum fase akut dan serum konvalesens 8 hari, makaInterpretasi hasil pemeriksaan ini adalah: Definitif infeksi dengue, sekunder

!

Interpretation of dengue HI antibody response

!TABEL INI TIDAK USAH DIHAFAL, CUKUP DILATIHPENGGUNAANNYA, AGAR DAPAT CEPAT MENGINTERPRETASI DALAM UJIAN

Hasil reaksi Hemaglutinasi

1 2 3 4 5 6 7 8 9 10 11 12

1/2

1/8

1/4

1/32

1/16

1/64

1/128

1/256

Titer virus : 64 (1 unit)

Untuk reaksi hambatan-hemaglutinasi virus yang digunakan4 – 8 unit 8 x 1/64 = 8/64 = 1/8

Hasil reaksi hambatan hemaglutinasi

KESIMPULAN :

Titer antibodi serum I = 40 serum II = 640 Kenaikan titer > 4x

infeksi

1/20

1/80

1/40

1/320

1/160

1/640

1/1280

1/2560

I II K1 K2

PengenceranSerum pasien

1/20

1/40

1/80

1/160

1/320

1/640

1/1280

1/2560

SerumFase akut

Kontrol eritrosit

Kontrol antigen

Serum yang diperiksa hanyalah serum fase akut, maka hasil pemeriksaan ini: Tidak bisa diinterpretasi

!

Example:

Detection of Dengue Antibody.

Reaksi pengikatan komplemen(complemen fixation test)

• Komplemen dapat melekat pada kompleks antigen-antibodi

• Pada antigen yang tidak berupa sel, pengikatan komplemen tidak dapat terlihat

• Pembuktian pengikatan komplemen:• diperlukan indikator berupa campuran:

• suspensi sel darah merah kambing• larutan antibodi terhadap sel darah merah

kambing !

Ag Antibodi CAntiSDM

REAKSI PENGIKATAN KOMPLEMEN

SISTIM HEMOLISA

SDM

Reaksi pengikatan komplemen(complemen fixation test)

• Tahap 1:• antigen + antibodi (salah satunya diketahui)• tambahkan komplemen:• komplemen akan terikat bila antibodi sesuai dengan

antigen dan membentuk kompleks• komplemen tidak terikat bila kompleks antigen-antibodi

tidak terbentuk

• Tahap 2:• tambahkan sel darah merah dan antibodinya (sensitized

cells):• indikator adanya komplemen bebas di dalam larutan

!

SERUM - SERUM +

KOMPLEMEN

SISTIM HEMOLISASDM + ANTI SDM

PK

HEMOLISA(CFT negatif)

ANTIGEN

TIDAK HEMOLISA (CFT positif)

REAKSI PENGIKATAN KOMPLEMEN(COMPLEMENT FIXATION TEST = CFT)

Reaksi pengikatan komplemen(complemen fixation test)

Pembacaan: •Reaksi positif:

• tidak ada hemolisis karena komplemen telah terikat pada kompleks antigen-antibodi yang sesuai terikat bila antibodi sesuai dengan antigen

•Reaksi negatif:

• terjadi hemolisis karena komplemen tidak terikat antigen tidak sesuai dengan antibodi (tidak terbentuk kompleks)

Contoh aplikasi:- reaksi Wassermann untuk diagnosis sifilis- pemeriksaan auto-antibody terhadap antigen sel tubuh

!

PengenceranSerum pasien

1/20

1/40

1/80

1/160

1/320

1/640

1/1280

1/2560

SerumFase akut

SerumKonvalesens

Kontrol eritrosit Kontrol lisis

UJI FIKSASI KOMPLEMEN

Keterangan : = tidak lisis = lisis

• Seperti halnya pemeriksaan serologi lainnya, penilaian kenaikan titer serum fase akut dan fase konvalesens yang dianggap positif ialah kenaikan titer sebanyak 4 kali

• Kontrol eritrosit diperlukan untuk memastikan bahwa eritrosit tidak mengalami autolisis (lisis dengan sendirinya, tanpa adanya reaksi lisis oleh komplemen).

• Kontrol lisis diperlukan untuk menilai fungsi antibodi terhadap eritrosit dan komplemen dalam melisis eritrosit.

• Bila tidak ada kontrol eritrosit dan kontrol lisis, dapat terjadi kesulitan dalam penilaian hasil uji fiksasi komplemen, khususnya bila semua sumur pada pelat uji fiksasi komplemen menunjukkan gambaran yang sama (lisis semua atau tidak lisis semua)

- Deteksi antibodi (IgM atau IgG): IgG: - Disebut positif terinfeksi bila terjadi peningkatan titer antibodi sebanyak 4 kali IgM: - Interpretasi sama dengan IgM blot

- Deteksi antigen - Hasil dinyatakan positif bila nilainya diatas nilai

ambang (cut-off value)

ELISA

Example:

Detection Dengue NS1 Antigen

Hasil reaksi ELISA

1 2 3 4 5 6 7 8 9 10 11 12

A

C

B

E

D

F

G

H

Nilai OD

A. Neg Control 0.104

B. Pos Control 1.234

C. Calibrator 0.802

D. Calibrator 0.800

E. Calibrator 0.804

F. Sample A 0.949

G. Sample B 0.070

H. Sample C 0.098

Perhitungan:Rata-rata Calibrator = 0.802Calibration factor = 0.62Cutt-off value = 0.802 x 0.62 = 0.497Panbio units = Index value x 10

Index value = Sample absorbance Cut-off value

Sample A = 0.949/0.497 x 10 = 19.1Sample B = 0.070/0.497 x 10 = 1.4Sample C = 0.098/0.497 X 10 = 1.97

OD:1230

OD:1191

OD:1067

OD:821

OD:751

OD:335

OD:233

OD:128

OD:1100

OD:335

OD:1067

OD:1050

Serum II

Serum I Serum I

Serum IIP

enge

ncer

an a

ntib

odi s

tand

ar

Tidak diencerkan A

B

C

D

E

F

G

H

1/2

1/4

1/8

1/16

1/32

1/64

1/128

Pasien I :Serum II diambil7 hari setelahPengambilan serum I.

Perubahan titerAntibodi dariSerum I keSerum II ialah:Kenaikan 8X(1/4:1/32)

Arti perubahan titer ialah:Pasien sedangterinfeksi akut

Pasien II :Serum II diambil7 hari setelahpengambilan Serum I

Perubahan titerAntibodi dariSerum I keSerum II ialah :1 X (tidak adaperubahan)

Arti kenaikan Titer ini ialah:Pasien tidakdalam infeksi akut

Nilai Standar

1 2 3 4 5 6 7 8

Immunofluorescence Assay (IFA)

FITC (Fluorescein Isothiocyanate)

Antigen CMV

Monoklonal antibodi CMV

Anti mouse IgG/FITC

Examining using by fluorescence microscope

Positive result: green-yellow fluorescence

Immunoperoxide Assay

POD

Substrat(DAB + H2O2)

+

Positive result : brown color

Antigen CMV

Monoclonal antibody CMV

Anti mouse IgG/POD

Widal Agglutination Test

Principle of the Test• The test depends on the ability of antibody in the

patient’s serum to agglutinate the stained bacterial antigens. When this occurs the aggregates become clearly visible to the naked eye.

Antigen Products Available• Antigen Salmonella O, Group A, B, C, D• Antigen Salmonella H, Group A, B, C, D• The reagents used in this practical laboratory contain

0.1% sodium azide as a preservative which may be toxic if ingested and the antigens are preserved with 0.5% Formalin

Widal Agglutination Test

1:40 1:80 1:160 1:320 1:40 1:80 1:160 1:320Titer

S. paratyphi A (Grup A)

S. paratyphi B (Grup B)

S. paratyphi C (Grup C)

S. typhi (Grup D)

KontrolSerum +/-

(-) (-)(+) (+)

Dengue Blot for detection of IgG or IgM

• Qualitative presumptive detection of IgM and IgG antibodies to dengue virus in human serum, plasma and whole blood can be used for the presumptive differentiation between primary and secondary infection

• This test should only be used for patients with signs and symptoms that are consistent with dengue virus infection

• Positive results are presumptive and must be confirmed by virus isolation, paired serum analysis, antigen detection by immunohistochemistry or viral nucleic acid detection for confirmation of dengue virus infection

• Specimen used in this test is serum. The serum should be separated as soon as possible and refrigerated at 2-80C or stored frozen at -200C or colder if not tested within 2 days.

Primary Dengue Infection:

IgM : visible band

IgG : No band

Control: visible band

Secondary Infection

IgM : visible band

IgG : visible band

Control: visible band

!

Secondary Dengue Infection:IgM : visible bandIgG : visible bandControl: visible band

Negatif:IgM : No bandIgG : No bandControl: visible band

!

Suspected secondary Dengue Infection :IgM : No bandIgG : visible bandControl: visible band

This result may be due to infection by other members of flavivirus:- Japanese encephalitis- Chikungunja- Ross River Virus- Yellow Fever virus

!

Interpretation

• Primary infection: serum IgM antibodies can be detected from dengue patients as early as 3-5 days after the onset of fever, generally persisting for 30-90 days

• Secondary infection: characterized by high IgG levels that may or may not be accompanied by elevated IgM levels

• The sensitivity of this assay has been set: • primary dengue: IgM is positive while IgG is negative • secondary infections: a positive IgG result with or without a

positive IgM result. • Serological cross-reactivity across the flavivirus group is common.

Molecular Assays for Microorganism

Molecular Assays

• Hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection the paired hybrid

• Nucleic acid probe typically is labelled with enzymes, antigenic substrates, chemiluminescent molecules, or radioistopes to facilitate detection of the hybridization product

Nucleic Acid Amplification : PCR

• Amplification of a particular portion of DNA, contains diagnostically useful information

• PCR reaction mixture:– Target DNA for amplification– Master mix (oligonucleotide DNA primers, 4

nucleotide triphosphate, thermostable DNA polumerase, Mg chloride, buffers)

• Three phases: denaturation; primer annealing; primer extension

Polymerase Chain Reaction (PCR)

Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) untuk Diagnosis Infeksi Virus Dengue

Tujuan : - Deteksi RNA virus dengue - Penentuan tipe virus

Sampel : Serum/ plasma pada masa akut (hari demam 1 – 4)Metoda : - Isolasi RNA - RT-PCR : RT-PCR dilakukan berdasarkan metoda Lanciotti, yang terdiri dari 2

tahap, yaitu tahap pertama, RT diikuti PCR menggunakan primer universal, dilanjutkan dengan semi nested PCR menggunakan primer yang spesifik tipe, seperti dapat dilihat pada gambar 1 dan tabel 1.

- Elektroforesis pada gel agarosa 2%, Hasil yang diharapkan : Kontrol positif : (+) Kontrol negatif; isolasi RNA (-); kontrol negatif reagen : (-) DEN-1 = 482 bp DEN-3 = 290 bp DEN-2 = 119 bp DEN-4 = 392 bp

Primer Sekuens Nukleotida Posisi

Genom

Ukuran

Produk

(bp)

D1 5’-TCAATATGCTGAAACGCGCGAGAAACCG-3’ 134-161 511

D2 5’-TTGCACCAACAGTCAATGTCTTCAGGTTC-3’ 616-644 511

TS1 5’-CGTCTCAGTGATCCGGGGG-3’ 568-586 482

TS2 5’-CGCCACAAGGGCCATGAACAG-3’ 232-252 119

TS3 5’-TAACATCATCATGAGACAGAGC-3’ 400-421 290

TS4 5’-CTCTGTTGTCTTAAACAAGAGA-3’ 506-527 392

Hasil elektroforesis produk RT-PCR Dengue(M) marker Phi X 174 DNA/Hae III; (1) DEN-1 = 482 bp; (2) DEN-3 = 290 bp; (3) DEN-2 = 119 bp; (4) DEN-4 = 392 bp; (5) Kontrol isolasi RNA; (6) Kontrol negatif PCR; (7) Kontrol positif PCR.

M 1 2 3 4 5 6 7

Reverse Transcription (RT)-PCR

Reverse Transcription (RT)-PCR

Result of RT-PCR Assay for Detection of HIV-1

HIV-1 (118 bp)

k+ k- S M

Result of Duplex PCR Assay for Detection of Haemophilus influenzae and Moraxella catarrhalis

bp: base pair. S: sample. k-: Negative control. M: DNA Ladder.k+: Positive control. Hi: Haemophilus influenzae Mc: Moraxella catarrhalis.

350450400

100

150

200250

300

bp S k- M k+

500

Mc (237 bp)

Hi (525 bp)

Result of Multiplex PCR Assay for Detection of Candida Glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. Albicans

bp: base pair. M: DNA Ladder. k+: Positive control. k-: Negative control. Cg: Candida Glabrata.Cp: C. parapsilosis. Ct: C. Tropicalis. Ck: C. krusei. Ca: C. Albicans.

225250300

100125150175200

bp M k+ k-

500 Cg (483 bp)

Cp (229 bp)

Ck (182 bp)Ct or Ca1 (218 bp)

Ca2 (110 bp)

Real time PCR/RT-PCR Amplify RNA target by RT-PCR or DNA target by PCR Use a fluorescence/dye-labeled oligonucleutide Could be applied as Viral Load Assay (RT- PCR) or Viral Burden Assay (PCR)

Viral Load HIV

• Memeriksa jumlah RNA virus dalam 1 mililiter plasma (Deteksi RNA virus kuantitatif)

• Metoda: - RT-PCR - NASBA - bDNA assay

1. Menetapkan diagnosis

2. Memutuskan dimulai atau dihentikannya terapi

3. Mengganti komposisi obat-obat anti retrovirus

Viral Load

Kemampuan mengukur jumlah partikel virus(VIRAL LOAD)

Kinetika replikasi virus dalam penderitadapat diikuti

- Penanganan penyakit- Prognosis- Strategi pengobatan

Dimensi Baru:

“State of the Art” Penanganan infeksi HIV

• Penurunan nilai “Viral Load”

• Peningkatan jumlah sel T CD4+

DERAJAT REPLIKASI VIRUS

VIRAL LOAD

OBAT-OBATAN

KOMBINASIANTI VIRUS

INFEKSI OPORTUNISTIK

INFEKSI HIV

RESPONIMUN

FAKTOR-FAKTORPEJAMU

VIRUS RESISTEN OBAT

Viral Load Assays

HIV-1 Genotyping

HIV-1 Genotyping

The HIV-1 Genotyping System is used for identifying mutations in the pol gene of the human immunodeficiency virus, type one (HIV-1).

The entire Protease gene and approximately two thirds of the Reverse Transcriptase (RT) gene in the pol open reading frame are amplified (approximately 1.8 kilobases (kb).

The amplicon is subsequently used as a sequencing template to generate approximately 1.2kb of sequence data.

Genotypic analysis of the region of HIV-1 facilitates the study of the relationship between mutations and viral resistance to anti-retroviral drugs, specifically the protease and RT inhibitors.

The sequencing data are then used to provide supplemental information regarding the potential HIV-1 susceptibility to current anti-retroviral drugs.

Physicians use the data in conjunction with patient clinical data to make treatment decisions.

HIV-1 Genotyping

Polymerase Chain Reaction (PCR)

Sequencing Reaction

HIV-1 Genotyping:Contoh hasil sekuensing DNA

Result of HIV-1 Genotyping

Result of HIV-1 Genotyping

4. WESTERN BLOT (HIV)

Principles of Virology, Flint S.J. et al 2000

Ilustrasi alat untuk SDS PAGE

Load HIV

GEL of SDS PAGE

TRANSFER TO NITROCELLULOSE

NITROCELLULOSE

4. WESTERN BLOT

Serum pasien +

p55

p17

p24

p31

gp41

p51

p66

gp120gp160

Serumcontrol

HIV-2

Western Blot HIV

Interpretation band present

Indeterminate p24, p55, gp160

Indeterminate p24, p55

Indeterminate p24, gp41, p55, p66, gp120, gp160

Positive p17, p24, p31, gp41, p51, p55, p66,gp120 gp160