pemeriksaan dbd

Source:  pemeriksaan dbd    Tag:  dengue virus genome

Pemeriksaan laboratorium penyakit infeksi tropis
Pada saat kita melakukan pemeriksaan lab. Kita semua harus tau,hafal,paham mengenai potegenisitas dari setiap penyakit.
1.       DBD ( demam berdarah dengue)
Sebaiknya ambil darah 2x,  yaitu saat fase akut dan fase konvalescens (diidentikan dengan fase penyembuhan yaitu, demam turun dan timbul petekie di seluruh/sebagian tubuh) (2-3 minggu kemudian). Tes rumple leed positif,  selalu ingat mengenai tanda hematocrit dan hb. Tes tourniquet pada hari demam 1 à 50%,  demam hari 2 à 70%, pada demam hari 3 à 90% negative palsu pada tes tourniquet dapat terjadi pada pasien obesitas, terlalu kurus, teknik jelek, saat shock. Trombosit biasanya menjadi sangat rendah saat hari ke 5.
Lakukan tes limfosit plasma biru / LPB pada hari ke 4-5 sensifitasnya 70-80% LPB per 100 leukosit. Akan menjadi positif bila lebih dari 8%
Mengetahui profil serologi itu penting.

 
Pada infeksi dengue primer igg diketahui optimal sekitar 14 hari setelah infeksi, igm 3-5 hari post infeksi pada dengue sekunder igg 2-3 hari setelah infeksi , igm 3-5 hari setelah infeksi
 

 
Immunological Response to Dengue Infection
The acquired immune response following a dengue infection consists of the production of IgM and IgG antibodies primarily directed against the virus envelope proteins. The immune response varies depending on whether the individual has a primary (first dengue or other flavivirus infection) versus a secondary (had dengue or other flavivirus infection in past) dengue infection. In general, diagnosis of dengue is dependent on the phase of the infection. The general timeline of a primary infection from virus isolation or identification, to IgM detection followed by IgG detection is as follows:

  A primary dengue infection is characterized by a slow and low titer antibody response . IgM antibody is the first immunoglobulin  isotype to appear. Anti-dengue IgG is detectable at low titer at the end of the first week of illness, and slowly increases.---- In contrast, during a secondary infection, antibody titers rise extremely rapidly and antibody reacts broadly with many flaviviruses. High levels of IgG are detectable even in the acute phase and they rise dramatically over the proceeding two weeks. The kinetics of the IgM response is more variable. IgM levels are significantly lower in secondary dengue infections and thus some anti-dengue IgM false-negative reactions are observed during secondary infections. According to the Pan American Health Organization (PAHO) guidelines 80% of all dengue cases have detectable IgM antibody by day five of illness, and 93-99% of cases have detectable IgM by day six to ten of illness, which may then remain detectable for over 90 days.
MAC-ELISA has become an important tool for routine dengue diagnosis, MAC-ELISA has a sensitivity and specificity of approximately 90% and 98%, respectively but only when used five or more days after onset of fever (i.e., in convalescent phase). Different formats such as capture ELISA, capture ultramicroELISA, dot-ELISA, AuBioDOT IgM capture and dipsticks have been developed. Serums, blood on filter paper, and saliva (but not urine) are useful for IgM detection if samples are taken in convalescent phase of illness (Vasquez et al., 2006). A variety of different commercial kits is available with variable sensitivity and specificity. Dengue diagnosis becomes even more challenging because dengue IgM antibodies also cross-react to some extent with other flaviviruses such asJEV, SLE, WNV and YFV.
II. Testing Algorithms for Dengue:
  1. a. PCR
    DEN Virus (DENV) can be detected in the blood (serum) from patients for approximately the first 5 days of symptoms. Currently, several PCR tests are employed to detect the viral genome in serum. In addition, virus can be isolated and sequenced for additional characterization. Real time RT–PCR assays have been developed and automated; but none of these tests are yet commercially available. Because antibodies are detected later, RT–PCR has become a primary tool to detect virus early in the course of illness. Current tests are between 80-90% sensitive, and more that 95% specific. A positive PCR result is a definite proof of current infection and it usually confirms the infecting serotype as well. However, a negative result is interpreted as "indeterminate". Patients receiving negative results before 5 days of illness are usually asked to submit a second serum sample for serological confirmation after the 5th day of illness (bellow).
  2. b. MAC ELISA
    IgM antibody capture ELISA (MAC-ELISA) format is most commonly employed in diagnostic laboratories and commercial available diagnostic kits. The assay is based on capturing human IgM antibodies on a microtiter plate using anti-human-IgM antibody followed by the addition of dengue virus specific antigen (DENV1-4). The antigens used for this assay are derived from the envelope protein of the virus. One of the limitation of this testing is the cross reactivity between other circulating flaviviruses. This limitation must be considered when working in regions where multiple flaviviruses co-circulate. IgM detection is not useful for dengue serotype determination due to cross-reactivity of the antibody.
  3. c. IgG ELISA
    The IgG ELISA used for the detection of a past dengue infection utilizes the same viral antigens as the MAC ELISA. This assay correlates with the hemagglutination assay (HI) previously used. In general IgG ELISA lacks specificity within the flavivirus serocomplex groups. Primary versus secondary dengue infection can be determined using a simple algorithm. Samples with a negative IgG in the acute phase and a positive IgG in the convalescent phase of the infection are primary dengue infections. Samples with a positive IgG in the acute phase and a 4 fold rise in IgG titer in the convalescent phase (with at least a 7 day interval between the two samples) is a secondary dengue infection.
  4. d. NS1 ELISA
    The non-structural protein 1 (NS1)
    of the dengue viral genome has been shown to be useful as a tool for the diagnosis of acute dengue infections. Dengue NS1 antigen has been detected in the serum of DENV infected patients as early as 1 day post onset of symptoms (DPO), and up to 18 DPO. The NS1 ELISA based antigen assay is commercially available for DENV and many investigators have evaluated this assay for sensitivity and specificity. The NS1 assay may also be useful for differential diagnostics between flaviviruses because of the specificity of the assay.
  5. e. PRNT
    Plaque Reduction and Neutralization Test (PRNT) and the microneutralization PRNT can be used when a serological specific diagnostic is required, as this assay is the most specific serological tool for the determination of dengue antibodies The PRNT test is used to determine the infecting serotype in convalescent sera. This assay measures the titer of the neutralizing antibodies in the serum of the infected individual and determines the level of protective antibodies this individual has towards the infecting virus. The assay is a biological assay based on the principle of interaction of virus and antibody resulting in inactivation of virus such that it is no longer able to infect and replicate in cell culture. Some of the variability of this assay is differences in interpretation of the results because of the cell lines and virus seeds used as well as the dilution of the sera. (
    http://www.cdc.gov/dengue/clinicallab/laboratory.html)
 

disarikan dari kuliah penegakan diagnosis infeksi tropis (dr Diah pramudianti SpPK)