Diagnosis Penyakit Viral

34
DIAGNOSIS PENYAKIT VIRAL Sy. Miftahul El Jannah

description

jjjjjj

Transcript of Diagnosis Penyakit Viral

Page 1: Diagnosis Penyakit Viral

DIAGNOSIS PENYAKIT VIRAL

Sy. Miftahul El Jannah

Page 2: Diagnosis Penyakit Viral

Viral laboratory studies are performed to:

(1)confirm the diagnosis by identifying the viral

agent of infection

(2)determine appropriate antiviral therapy

(3)define the course of the disease

(4)monitor the disease epidemiologically

(5)educate physicians and patients.

Page 3: Diagnosis Penyakit Viral

Hubungan tingkat penyakit dengan adanya virus dalam bahan pemeriksaan dan timbulnya antibodi spesifik

Tahap/masa sakit Ditemukan virus dlm pemeriksaan

Antibodi

Inkubasi Jarang Tidak

Prodromal Kadang-kadang Tidak

Awal Sering Kadang-kadang

Fase akut Sering Sering

Penyembuhan Jarang Biasanya

Konvalesensi Sangat jarang Biasanya

Page 4: Diagnosis Penyakit Viral

Prosedur Laboratorium dalam diagnosis penyakit virus

1. Isolasi dan identifikasi penyebab

2. Pengukuran antibodi yang terbentuk selama infeksi [serologi]

3. Pemeriksaan histologik jaringan yang terkena infeksi harus dilakukan pada semua kasus infeksi virus yang fatal & binatang yang dicurigai menderita infeksi virus rabies

Page 5: Diagnosis Penyakit Viral

Prosedur Laboratorium dalam diagnosis penyakit virus

4. Menemukan antigen virus dalam lesi dengan menggunakan antibodi yang ditandai [flouresens atau peroksida] pada cairan sekret nasofaring, sputum, kulit, kerokan konjunktiva, biopsi otak, dan jaringan otopsi

5. Hibridisasi titik dari asam nukleat virus [viral genome] dengan pemeriksaan asam nukleat yang dilabel radioaktif

6. Pemeriksaan mikroskop elektron dari cairan vesikel atau ekstrak jaringan

Page 6: Diagnosis Penyakit Viral

Common Pathogenic VirusesSpecimens for

CultureComments

Respiratory Tract

Adenovirus; influenza virus; enterovirus

(picornavirus); rhinovirus; paramyxovirus;

rubella virus; HSV

Nasal washing, throat

swab, nasal swab,

sputum

Enterovirus is also shed in stool

Gastrointestinal Tract

Reovirus; rotavirus; adenovirus; Norwalk

virus, calicivirus

Stool, rectal swab Samples are analyzed by electron

microscopy and antigen detection

(ELISA); viruses are not cultured

Maculopapular Rash

Adenovirus; enterovirus (picornavirus) Throat swab, rectal

swab

-

Rubella virus; measles virus Urine -

Vesicular Rash

Coxsackievirus; echovirus; HSV; VZV Vesicle fluid,

scraping, or swab,

enterovirus in stool

Initial diagnosis of HSV and VZV can be

obtained from vesicle scraping (Tzanck

smear)

Specimens for Viral Diagnosis

Page 7: Diagnosis Penyakit Viral

Central Nervous System (Aseptic Meningitis, Encephalitis)

Enterovirus (picornavirus) Stool PCR

Arboviruses (e.g., togaviruses, bunyavirus) Rarely cultured Diagnosis is by serologic tests

Rabies virus Tissue, saliva, brain

biopsy

Diagnosis is by immunofluorescence

analysis for antigen

HSV; CMV; mumps virus; measles virus Cerebrospinal fluid PCR, virus isolation, and antigen are

assayed

Urinary Tract

Adenovirus; CMV Urine CMV may be shed without apparent

disease

Blood

HIV; human T-cell leukemia virus; hepatitis

B, C, and D viruses

Blood Serologic antigen or antibody detection

(ELISA), PCR, and RT-PCR are

performed

Common Pathogenic VirusesSpecimens for

CultureComments

Page 8: Diagnosis Penyakit Viral

Isolasi virus Dilakukan bila:

1. Terjadi epidemi baru

2. Hasil tes serologik tumpah tindih sulit membedakan antara dua

spesies virus

3. Dilakukan konfirmasi diagnosis sementara dari pengamatan langsung

dengan mikroskop [mis: menemukan virus herpes dalam cairan vesikel]

4. Penyakit klinis yang sama dapat disebabkan oleh banyak mikroorganisme

Page 9: Diagnosis Penyakit Viral

Teknik Isolasi virus

• Semua bahan terbungkus aman pengiriman dibekukan di bawah -200C

• Bahan ditandai dengan jelas, diberi keterangan yang relevan

• Penyingkiran mikroorganisme lain [bakteri, jamur] dari bahan/spesimen

Page 10: Diagnosis Penyakit Viral

Mengawetkan virus

Pembekuan

• Botol sampel bermulut

lebar [termos] diisi CO2

padat [dry ice] ½ penuh

• Suhu penyimpanan

mendekati -760C

Liofilisasi

• Pembekuan cepat pada

suhu rendah bejana

mengandung alkohol dan

dry ice

Page 11: Diagnosis Penyakit Viral

Persiapan jaringan

Jaringan dicuci dalam perbenihan atau air steril

dipotong-potong digiling/blender

pasta homogen konsentrasi 10-20%

Page 12: Diagnosis Penyakit Viral

Penyingkiran kuman

Spesimen yang mengandung kuman [ swab tenggorok, feses, urin, jaringan terinfeksi, serangga]

1. Zat bakterisidala. Antibiotik dipakai berkombinasi dengan

pemusingan differensialb. Eter bagi virus yang tahan, 10-15%

2. Metode mekanika. Saringan milliporeb. Pemusingan differensial BJ

Page 13: Diagnosis Penyakit Viral

Pembiakan dalam biakan sel• Virus bermutiplikasi di dalam sel efek biologik [cytopathologic

effects/CPEs]

• Tebentuknya sel raksasa [Giant cells] dengan inti banyak virus

campak, mumps, parainfluenza

• Sel bulat besar,berkelompok spt buah anggur virus adeno

• Membentuk daerah-daerah fokal berbentuk bulat dan dendritik

virus rhino

• Sel membulat dan rata virus herpes simpleks

Page 14: Diagnosis Penyakit Viral

Syncytium formation by measles virus. Multinucleated giant cell (arrow) visible in a histologic section of lung biopsy tissue from a measles virus-induced giant cell pneumonia in an

immunocompromised child. (From Hart C, Broadhead RL: A color atlas of pediatric infectious diseases, London, 1992, Wolfe.)

Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)© 2005 Elsevier

Page 15: Diagnosis Penyakit Viral

HSV-induced CPE. A biopsy specimen of an HSV-infected liver shows an eosinophilic Cowdry type A intranuclear inclusion body (A) surrounded by a halo and a ring of marginated

chromatin at the nuclear membrane. An infected cell (B) exhibits a smaller condensed nucleus (pyknotic). CPE, Cytopathologic effect; HSV, herpes simplex virus. (Courtesy Dr. J.I. Pugh, St. Albans; from Emond RT, Rowland HAK: A color atlas of infectious diseases, ed 3,

London, 1995, Mosby.)Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)

© 2005 Elsevier

Page 16: Diagnosis Penyakit Viral

Negri bodies caused by rabies. A, A section of brain from a patient with rabies shows Negri bodies (arrow). B, Higher magnification from another biopsy specimen. (A from Hart C, Broadhead RL: A color atlas of pediatric infectious diseases, London, 1992,

Wolfe.)Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)

© 2005 Elsevier

Page 17: Diagnosis Penyakit Viral

Negri bodies caused by rabies. A, A section of brain from a patient with rabies shows Negri bodies (arrow). B, Higher magnification from another biopsy specimen. (A from Hart C,

Broadhead RL: A color atlas of pediatric infectious diseases, London, 1992, Wolfe.)Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)

© 2005 Elsevier

Page 18: Diagnosis Penyakit Viral

CPE of HSV infection. A, Uninfected Vero cells, an African green monkey kidney cell line. B, HSV-1-infected Vero cells showing rounded cells, multinucleated cells, and loss of the

monolayer. Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)© 2005 Elsevier

Page 19: Diagnosis Penyakit Viral

CPE of HSV infection. A, Uninfected Vero cells, an African green monkey kidney cell line. B, HSV-1-infected Vero cells showing rounded cells, multinucleated cells, and loss of the

monolayer. Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)© 2005 Elsevier

Page 20: Diagnosis Penyakit Viral

Pembiakan dalam biakan sel

• Tidak menimbulkan perubahan langsung

rubella

• Virus influenza dan orthomyxo

menyebabkan melekatnya eritrosit pada

permukaan sel [hemadsorpsi]

Page 21: Diagnosis Penyakit Viral

Hemadsorption of erythrocytes to cells infected with influenza viruses, mumps virus, parainfluenza viruses, or togaviruses. These viruses express a hemagglutinin on

their surfaces, which bind erythrocytes of selected animal species.

Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)© 2005 Elsevier

Page 22: Diagnosis Penyakit Viral

A virus can be grown in embryonated eggs,

Page 23: Diagnosis Penyakit Viral

Immunoelectron microscopy is a means of increasing the sensitivity and specificity of EM and is particularly

useful in the following situations:

1. The number of virus particles present is small.

2. Many different viruses have different morphology e.g. herpesviruses and

picornaviruses. IEM may identify the virus

3. In an outbreak situation where the pathogens responsible has been

identified so that it may be useful to go back to look at the negative

specimens again with IEM.

Page 24: Diagnosis Penyakit Viral

Immunoelectron microscopy

• There are 2 types of IEM

1. simple IEM, where the specimen is incubated with

specific antibody before staining in the hope that the

antibody will agglutinate the specimen

2. solid phase IEM (SPIEM), where the copy grid is

coated with specific antibody which is used to

capture virus particles from the specimen.

Page 25: Diagnosis Penyakit Viral

Rotavirus Adenovirus

AstovirusNorwalk like-virus

Page 26: Diagnosis Penyakit Viral

Uji serologi

• Complement Fixation Test/uji fiksasi komplemen– Menemukan Ag atau Ab yg hanya bereaksi dengan

komplemen– Ab+Ag mengikat komplemen– Komplemen tidak diikat bebas dalam larutan +

SDM hemolis

Page 27: Diagnosis Penyakit Viral
Page 28: Diagnosis Penyakit Viral

Advantages of CFT1.Ability to screen against a large number of viral and bacterial infections at the same time. 2.Cheap

Disadvantages of CFT1.Not sensitive - cannot be used for immunity screening 2.Time consuming and labor intensive 3.Often non-specific e.g. cross-reactivity between HSV and VZV

Page 29: Diagnosis Penyakit Viral

Hemaglutinasi [HE]

• Menemukan antibodi atas dasar agglutinasi SDM

• Ag dapat digunakan SDM atau Ag yang mensensitisasi SDM

• The advantages of HAI tests are that they are relatively easy and inexpensive to perform. The disadvantages are that HAI tests are not as sensitive as EIAs or RIAs,

Page 30: Diagnosis Penyakit Viral

Neutralization, hemagglutination, and hemagglutination inhibition assays. In the assay shown, tenfold dilutions of serum were incubated with virus. Aliquots of the mixture were then added to

cell cultures or erythrocytes. In the absence of antibody, the virus infected the monolayer (indicated by CPE) and caused hemagglutination (i.e., formed a gel-like suspension of

erythrocytes). In the presence of the antibody, infection was blocked (neutralization), and hemagglutination was inhibited, allowing the erythrocytes to pellet. The titer of antibody in the

serum was 100. pfu, Plaque-forming units.Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)© 2005 Elsevier

Page 31: Diagnosis Penyakit Viral

ELISA [Enzyme liked immunosorbent Assay]

• Digunakan untuk menemukan Ab• Ag diikatkan pada benda padat + Ab yang dicari + Ag yang

ditambah diberi tanda enzim [peroksidase • + substrat kromogenik, bila bereaksi dgn enzim perubahan

warna

• 7fosfatasr

Page 32: Diagnosis Penyakit Viral

Enzyme immunoassays for quantitation of antibody or antigen. A, Antibody detection. 1, Viral antigen, obtained from infected cells, virions, or genetic engineering, is affixed to a surface. 2, Patient serum is added and allowed to bind to the antigen. Unbound antibody is washed away. 3, Enzyme-conjugated antihuman antibody is added,

and unbound antibody is washed away. 4, Substrate is added and converted (5) into chromophore, precipitate, or light. B, Antigen capture and detection. 1, Antiviral antibody is affixed to a surface. 2, A specimen that contains antigen is added, and unbound antigen is washed away. 3, A second antiviral antibody is added to detect the

captured antigen. 4, Enzyme-conjugated anti-antibody is added, washed, and followed by substrate (5), which is converted (6) into chromophore, precipitate, or light.Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)

© 2005 Elsevier

Page 33: Diagnosis Penyakit Viral

Microplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivityMicroplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivityMicroplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivityMicroplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivityMicroplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivity

Microplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivity

Page 34: Diagnosis Penyakit Viral

Immunofluorescence and enzyme immunoassays for antigen localization in cells. Antigen can be detected by direct assay with antiviral antibody modified

covalently with a fluorescent or enzyme probe, or by indirect assay using antiviral antibody and chemically modified anti-immunoglobulin. The enzyme

converts substrate to a precipitate, chromophore, or light.Downloaded from: StudentConsult (on 27 October 2009 01:56 AM)

© 2005 Elsevier