Diagnostic of Microbiology

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 patients 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)

Hari ke 1

Spesimen

Diterima

Diterima dengan catatan

Ditolak

Mikroskopik

Kultur pada agar

Kultur pada medium cair

Hari ke 2

Isolat

Penentuan Genus, Spesies dan Serotipe

Uji biokimiawi

Uji serologi

Hari ke 3

Identitas bakteri

Uji kepekaan terhadap antibiotika

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 :
Patients 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
100 90 80 70 60 % Relative 50 Yield 40 30 20 10 0 5 10 15 20 25 30 35
ml
*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. 363376(2002) with permission from Elsevier.

40 45

50

55 60

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

LAPORKAN/ HUBUNGI DOKTER

GRAM

AEROB

AD

MAC

AC

GRAM

ANAEROB

GRAM

IDENTIFIKASI

KONVENSIONAL API 20 E API 20 NE API 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

Blood Agar

Mueller Hinton Agar

Candida sp. on Sabouraud Agar

DISK DIFFUSION TEST

Disk Diffusion Test

CLSI 2011 Mueller Hinton Agar ( 100 mm) Standard kekeruhan bakteri ~ 0.5 MacFarland
Ukur Diameter Zona Hambat
WHONET Software Hasil Uji Kepekaan S, I,R

Dilusion Test
no zone around disc = resistant

minimum inhibitory concentration (MIC) test

clear zone around disc = sensitive bacterium

8mg/ L

4mg/ L

2mg/ L

1mg/ L

0.5m g/L

0.25 mg/L

cloudiness means bacteria can grow at amount of that concentration of antibiotic antibiotic MIC=2mg/L

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 Method: grown and maintained in Bacteria and phages are vitro mixed with warm (liquid) Phages can be grown in nutrient agar and poured in a bacteria maintained in either thin layer across the surface of liquid cultures or on agar an agar plate plates 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 viruss 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 cultures

Derived 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 antibody
Some 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

Hemaglutinasi
Aglutinasi 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 DAPAT TERJADI :

1. ANTIBODI TERHADAP ANTIGEN PADA PERMUKAAAN SEL DARAH MERAH

SDM

SDM

ANTIGEN

ANTIBODI

SDM

SDM

CONTOH: PENENTUAN GOL. DARAH

HEMAGLUTINASI

2. VIRUS / RICKETTSIA

MEMPUNYAI HEMAGLUTININ, DAPAT MENGGUMPALKAN SEL DARAH MERAH.

SDM SDM VIRUS SDM SDM ANTIBODI REAKSI INI DAPAT DIHAMBAT OLEH ANTIBODI REAKSI HAMBATAN HEMAGLUTINASI

SDM

SDM

SDM

HAMBATAN HEMAGLUTINASI

VIRUS + ANTIBODI

SEL DARAH MERAH

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 ANTIGEN DIPERMUKAANNYA.

SDM

SDM

SDM

SDM

+
SDM SDM SDM SDM

ANTIGEN

ANTIBODI YANG SESUAI

HEMAGLUTINASI

KEUNTUNGAN : MEMUDAHKAN MELIHAT HASIL REAKSI (PARTIKEL BESAR).

DISEBUT JUGA : HEMAGLUTINASI PASIF Contoh : Reaksi TPHA (Treponema Pallidum Haemaglutination Test)

Reaksi Hemaglutinasi (HA)

Pengenceran Antigen Antigen Kontrol eritrosit

1/20 1/40 1/80

1/160
1/320 1/640 1/1280 1/2560
Titer Antigen : 1/160 (1 HAU ) Untuk Reaksi HI digunakan Antigen 4 8 HAU

Hambatan Hemaglutinasi (HI)

Pengenceran Serum pasien Serum Fase akut Serum Konvalesens Kontrol eritrosit Kontrol antigen

1/20

1/40
1/80 1/160 1/320 1/640 1/1280 1/2560

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

Interpretation of dengue HI antibody response

TABEL INI TIDAK USAH DIHAFAL, CUKUP DILATIH PENGGUNAANNYA, AGAR DAPAT CEPAT MENGINTERPRETASI DALAM UJIAN

Hasil reaksi Hemaglutinasi

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

1/8
1/16 1/32 1/64 1/128 1/256

Titer virus : 64 (1 unit) Untuk reaksi hambatan-hemaglutinasi virus yang digunakan 4 8 unit 8 x 1/64 = 8/64 = 1/8

Hasil reaksi hambatan hemaglutinasi

I II K1 K2

1/20 1/40 1/80 1/160 1/320 1/640 1/1280 1/2560

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

Pengenceran Serum pasien

Serum Fase akut

Kontrol eritrosit

Kontrol antigen

1/20 1/40 1/80 1/160

1/320
1/640 1/1280 1/2560

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

REAKSI PENGIKATAN KOMPLEMEN

Ag

Antibodi

Anti SDM

SDM

SISTIM HEMOLISA

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

REAKSI PENGIKATAN KOMPLEMEN (COMPLEMENT FIXATION TEST = CFT)

SISTIM HEMOLISA SDM + ANTI SDM KOMPLEMEN ANTIGEN SERUM SERUM +

HEMOLISA (CFT negatif)

TIDAK HEMOLISA (CFT positif)

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

UJI FIKSASI KOMPLEMEN

Pengenceran Serum pasien Serum Fase akut Serum Konvalesens Kontrol eritrosit Kontrol lisis

1/20 1/40 1/80 1/160 1/320 1/640

1/1280
1/2560

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)

ELISA
- 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)

Example:
Detection Dengue NS1 Antigen

Hasil reaksi ELISA

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

A. Neg Control 0.104 B. Pos Control 1.234 C. Calibrator D. Calibrator E. Calibrator F. Sample A G. Sample B H. Sample C 0.802 0.800 0.804 0.949 0.070 0.098

A B C D E F G H

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

Index value = Sample absorbance Cut-off value Sample A = 0.949/0.497 x 10 = 19.1 Sample B = 0.070/0.497 x 10 = 1.4 Sample C = 0.098/0.497 X 10 = 1.97

1
Tidak diencerkan A

4
Serum II

8
Serum II

OD:1230

OD:1100

OD:1067

Pengenceran antibodi standar

1/2 1/4 1/8

B
OD:1191 OD:335 Serum I OD:1050 Serum I

C
OD:1067

D
OD:821

Pasien I : Serum II diambil 7 hari setelah Pengambilan serum I. Perubahan titer Antibodi dari Serum I ke Serum II ialah: Kenaikan 8X (1/4:1/32)

Pasien II : Serum II diambil 7 hari setelah pengambilan Serum I Perubahan titer Antibodi dari Serum I ke Serum II ialah : 1 X (tidak ada perubahan)

1/16 E
OD:751

1/32 F
OD:335

1/64 G
OD:233

1/128 H

OD:128

Nilai Standar

Arti perubahan titer ialah: Pasien sedang terinfeksi akut

Arti kenaikan Titer ini ialah: Pasien tidak dalam infeksi akut

Immunofluorescence Assay (IFA)

FITC (Fluorescein Isothiocyanate) Anti mouse IgG/FITC Monoklonal antibodi CMV Antigen CMV

Examining using by fluorescence microscope Positive result: green-yellow fluorescence

Immunoperoxide Assay
+
Substrat (DAB + H2O2)

POD Anti mouse IgG/POD Monoclonal antibody CMV Antigen CMV

Positive result : brown color

Widal Agglutination Test

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

Titer
S. paratyphi A (Grup A) S. paratyphi B (Grup B) S. paratyphi C (Grup C) S. typhi (Grup D) Kontrol Serum +/-

1:40

1:80

1:160

1:320

1:40

1:80

1:160

1:320

(-)

(+)

(-)

(+)

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.

Secondary Infection IgM IgG : visible band : visible band

Control: visible band

Primary Dengue Infection: IgM IgG : visible band : No band

Control: visible band

!
Secondary Dengue Infection: IgM : visible band IgG : visible band Control: visible band

Negatif: IgM : No band IgG : No band Control: visible band

Suspected secondary Dengue Infection : IgM : No band IgG : visible band Control: 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 134-161 616-644 568-586 232-252 400-421 506-527 Ukuran Produk (bp) 511 511 482 119 290 392

D1 D2 TS1 TS2 TS3 TS4

5-TCAATATGCTGAAACGCGCGAGAAACCG-3 5-TTGCACCAACAGTCAATGTCTTCAGGTTC-3 5-CGTCTCAGTGATCCGGGGG-3 5-CGCCACAAGGGCCATGAACAG-3 5-TAACATCATCATGAGACAGAGC-3 5-CTCTGTTGTCTTAAACAAGAGA-3

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.

Reverse Transcription (RT)-PCR

Reverse Transcription (RT)-PCR

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

k+ kS M

HIV-1 (118 bp)

Result of Duplex PCR Assay for Detection of Haemophilus influenzae and Moraxella catarrhalis
bp
S kM k+

500 450 400 350 300 250 200 150 100

Hi (525 bp)

Mc (237 bp)

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

Result of Multiplex PCR Assay for Detection of Candida Glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. Albicans bp
M k+ k-

500

Cg (483 bp)

300 250 225 200 175 150 125 100

Cp (229 bp) Ct or Ca1 (218 bp) Ck (182 bp)

Ca2 (110 bp)

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.

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

Viral Load

1. Menetapkan diagnosis
2. Memutuskan dimulai atau dihentikannya terapi 3. Mengganti komposisi obat-obat anti retrovirus

Kemampuan mengukur jumlah partikel virus

(VIRAL LOAD)

Kinetika replikasi virus dalam penderita dapat diikuti

Dimensi Baru:
- Penanganan penyakit - Prognosis - Strategi pengobatan

State of the Art Penanganan infeksi HIV

Penurunan nilai Viral

Load

Peningkatan jumlah sel T CD4+

INFEKSI OPORTUNISTIK KOMBINASI ANTI VIRUS OBAT-OBATAN

INFEKSI HIV

RESPON IMUN VIRUS RESISTEN OBAT

DERAJAT REPLIKASI VIRUS

FAKTOR-FAKTOR PEJAMU

VIRAL LOAD

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 +

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

Interpretation

band present

Indeterminate

p24, p55

Positive
Serum control HIV-2

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