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DIAGNOSIS PENYAKIT VIRAL
Sy. Miftahul El Jannah
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.
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
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
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
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
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
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
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
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
Persiapan jaringan
Jaringan dicuci dalam perbenihan atau air steril
dipotong-potong digiling/blender
pasta homogen konsentrasi 10-20%
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
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
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
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
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
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
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
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
Pembiakan dalam biakan sel
• Tidak menimbulkan perubahan langsung
rubella
• Virus influenza dan orthomyxo
menyebabkan melekatnya eritrosit pada
permukaan sel [hemadsorpsi]
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
A virus can be grown in embryonated eggs,
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.
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.
Rotavirus Adenovirus
AstovirusNorwalk like-virus
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
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
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,
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
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
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
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
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
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