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PREFACE

First of all, I would like to thank God for the blessing and health so I could finish this paper on time. I also want to thank my supervisor, Prof. Dr. Widyasari K, Sp. MK. for his guidance and help on this paper. Thanks to my family that has supported me during this writing. And thanks to my friends for their helps. Without their helps and supports, I wouldn¶t be able to finish this paper. This paper is far from perfect. There are a lot of mistakes in the writings, whether the grammar or the theory. I hope after reading this paper, readers could give me some advice and critics. Hopefully, with the critics and advice, I will be able to develop myself. I apologize for all the mistakes I¶ve made in this paper. I hope this paper could be useful to all the readers. Thank you.

Jakarta, January 2011

Aji Patriajati

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Evaluating these tests to see how well they perform in different countries and in different health care settings is an important process that helps to guide health care policy on whether these assays are likely to be useful in making a diagnosis. Dengue is a very important public health problem in many developing countries. Recently.Abstract Dengue is a viral infection of humans that is transmitted by mosquitoes. the results indicate these NS1 tests deserve inclusion in the diagnostic approach to dengue. Our hospital-based results. when best to use them. 3 . new tests to help diagnose patients with dengue have been developed. indicates that these tests are most sensitive when used during the first 3 days of illness and are most likely to be positive if the patient has primary dengue. Our results also show that a positive NS1 test result is a reflection of the amount of virus in the blood. so that patients with high amounts of virus in the blood are more likely to be NS1 positive. using two different types of NS1 tests for diagnosing dengue. and if so. Collectively.

There are an estimated 50 to 100 million cases of dengue infection each year. and the geographic range has extended to involve most tropical countries. in severe case hypovolaemic shock (DSS) may develop. and patient management relies on good supportive care. DHF is a vasculopathy characterized by capillary leakage and haematological dysregulation. primarily dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). a proportion result in clinically apparent disease that varies in severity from mild undifferentiated fever through to more severe syndromes. including about half a million cases of dengue hemorrhagic fever. is an increasing cause of morbidity and mortality throughout the tropical world. 4 . which are arboviruses belonging to the Flaviviridae family. There are no licensed vaccines or specific antiviral therapies for dengue. Dengue is caused by infection with one of four serotypes of dengue virus (DENV1-4). The number of cases of both dengue fever and dengue hemorrhagic fever has increased dramatically for the past few decades. Background Dengue is a major public health problem in many parts of the tropical developing world [1. I.CHAPTER I Introduction I.2. Nowadays scientist develop Detection of the dengue NS1 antigen during the symptomatic phase of illness represents an important advance in the diagnosis of dengue fever.2]. 1. Although most DENV infections are asymptomatic. Problems Dengue hemorrhagic fever.

5. Methods of Writing. The writing of paper is carried out by a library research and also via internet. Objectives The objectives of writing this paper are to describe : Comparison Non-Structural Protein NS1 sero-types specific Ig-G with IgM dengue Blot I. Limitation of the Problems 1. Early and accuratediagnosis can assist in patient management by directing clinical attention to the appearance of major warning signs of severe or even life threatening complications 2.I. 4. 5 . Expanded use of accurate dengue diagnostics provides important data on the epidemiology and health burden of dengue I. Accurate dengue diagnosis prevents unnecessary and possibly expensive antibiotic usage 3. 3. Prompt diagnosis of index cases can facilitate vector control activities in the community so as to mitigate further transmission 4.

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2 Genome Organization of Dengue Virus Dengue Virus (DV) belongs to the family Flaviviridae. The Dengue virus is a member of the virus family Flaviviridae and is transmitted to people through the bite of the mosquitoes Aedes aegypti and Aedes albopictus.CHAPTER II Dengue Virus Dengue is caused by one of four closely related virus serotypes of the genus Flavivirus. 100 amino acids). DEN-2. Infection in humans by one serotype produces life-long immunity against reinfection by that same serotype. and envelope (E. and the complete genome sequence is known for isolates of all four serotypes of dengue virus. membrane (M. The flavivirus genome is approximately 11kb (kilobases) in length. In addition to the plus-sense RNA genome of 10. each serotype is sufficiently different that there is no cross-protection and epidemics caused by multiple serotypes (hyperendemicity) can occur. The virion is approximately 50nm in diameter. 75 amino acids). etc) can be distinguished by serological methods. II. Dengue viruses share many characteristics with other flaviviruses. The genome is 7 . having a single-stranded RNA genome surrounded by an icosahedral nucleocapsid and covered by a lipid envelope. The four serotypes of dengue virus (designated DEN-1. with a diameter of approximately 500 Å. there are three structural proteins that occur in stoichiometric amounts in the particle: core (C. 1 Morphology of the Virus Electron micrographs showed that dengue virions are characterized by a relatively smooth surface. but only temporary and partial protection against the other serotypes. II.700 nucleotides. and an electron-dense core surrounded by a lipid bilayer. 495 amino acids). family Flaviviridae.

Schematic of the single stranded RNA genome with highly structured RNA elements in the 5' and 3' NTRs. a membrane-associated protein (M).NS3-NS4A-NS4B-NS5-3¶.000 and is the most conserved flavivirus protein. B. the viral proteinase functions in the cytosol. It appears that vesicle packets are sites of RNA replication that is probably catalyzed by a multi-protein 8 . The order of proteins encoded is 5¶-C-prM(M)-E-NS1NS2ANS2B. a glycoprotein is detected in high titers in patients with secondary dengue infections but its function is unknown. DV genomic organization and functions of viral proteins. NS4 region codes for two small hydrophobic proteins involved in the membrane bound RNA replication complex establishment. Virions are internalized by receptor-mediated endocytosis resulting in release of the viral genome from the nucleocapsid in a low pH dependent manner. fusion and interactions with virus receptors are associated with the envelope protein. is known to code for 2 proteins (NS2A and NS2B). NS1. NS5 codes for a protein with a molecular weight of 105.3 Dengue virus Replication cycle Dengue virus (DV) particles bind to cells via interactions between the surface glycoprotein and one or several poorly defined cellular receptor(s). In addition. viral proteins induce rearrangments of intracellular membranes forming distinct structures that have designated vesicle packets and convoluted membranes. Soon after infection. The domains responsible for neutralization. Putative membrane topology of DV proteins and proteinases involved in polyprotein cleavage. For some proteins their function in the viral life cycle is not yet established. encoding the nucleocapsid or core protein C. II.composed of three structural protein genes. NS3. they are marked with a µ?¶ C. This protein is assumed to be the virus encoded RNA dependent RNA polymerase. NS2 region. which are assumed to play a role in polyprotein processing. A. an envelope protein (E) and seven non-structural (NS) protein genes. particles may enter cells via Fcreceptor upon opsonization. NS6 and NS7 function yet to be found.

clinically. within humans. and dengue shock syndrome. Within the mosquito. cellular membranes and presumably also cellular proteins. or with hemorrhagic fever (prototype: yellow fever). Naked genomic RNA is infectious if introduced into the 9 . Although caused by the same viruses. However. with fever-arthralgia-rash (prototype: dengue fever). Human infection with both mosquitoborne flaviviruses is initiated by deposition of virus through the skin via the saliva of an infected arthropod. The mosquito then bites a susceptible person and transmits the virus to him or her. Virus particles are thought to assemble by budding into the ER and are transported through the host secretory pathway. as well as to every other susceptible person the mosquito bites for the rest of its lifetime. Viremia is concurrent with clinical illness.complex composed of viral proteins. Flaviviruses vary widely in their pathogenic potential and mechanisms for producing human disease. it can range from three to 14 days also. Severe leukopenia is often present. The viremia begins slightly before the onset of symptoms. Dengue viruses of all four serotypes cause three distinct syndromes: classic dengue fever. in regional lymph nodes. and then throughout the reticuloendothelial system. So the illness persists several days after the viremia has ended. The symptoms begin to appear in an average of four to seven days after the mosquito bite this is the intrinsic incubation period. The virus then replicates in the second person and produces symptoms. Louis encephalitis). after the onset of symptoms. it is useful to consider them in three major categories: those associated primarily with the encephalitis syndrome (prototype: St. Virus is present in the serum and in association with circulating monocytes. the virus replicates during an extrinsic incubation period of eight to twelve days. and epidemiologically distinct. The mechanism by which flaviviruses enter the cells probably involves an interaction between the E protein and cellular receptors. DV RNA is replicated via a negative strand intermediate that serves as a template for the production of excess amounts of positive strand progeny. dengue and dengue hemorrhagic fever are pathogenetically. While the intrinsic incubation period averages from four to seven days. Symptoms caused by dengue infection may last for three to 10 days. with an average of five days. dengue hemorrhagic fever. Virus replicates locally and in regional lymph nodes and results in viremia. Dengue viruses appear to replicate in macrophages at the site of the mosquito bite. followed by a postattachment fusion event that occurs in acidic intracytoplasmic vacuoles.

The body releases cytokines that cause the endothelial tissue to become permeable which results in hemorrhagic fever and fluid loss from the blood vessels.g. made from genomic RNA. This makes the viral infection much more acute. 10 .4 Antibody Responce Dengue infection will result in lifelong immunity to that serotype. The immune response attracts numerous macrophages. serves as a template to generate genomic RNA. another type of dengue virus infects the individual. The immune response produces specific antibodies to that subtype¶s surface proteins that prevents the virus from binding to macrophage cells (the target cell that dengue viruses infect) and gaining entry. Complementary (negative-sense) RNA. However. but only temporary immunity to other serotypes. no evidence of budding has been seen in flavivirus infected cells. This situation is referred to as Antibody-Dependent Enhancement (ADE) of a viral infection. which the virus proceeds to infect because it has not been inactivated. Unlike alpha viruses. NS-1 and RNAdependent RNA polymerase) are encoded in the 3'two-thirds. The 4 subtypes of dengue virus have 60-80% homology between each other. it serves as mRNA for all proteins. After a person is infected with dengue. Replication occurs in the cytoplasm. and nonstructural proteins (e. The immune system is tricked because the 4 subtypes have very similar surface antigens. II. the virus will activate the immune system to attack the first subtype. and the mechanisms of virion assembly and release remain obscure. Structural proteins are encoded at the 5' end of the genome. they develop an immune response to that dengue subtype.cytoplasm. The genomic RNA is capped but not polyadenylated. Virions appear within cytoplasmic vacuoles and appear to exit the cell as vacuoles fuse with the plasma membrane. Virions are formed in perinuclear regions of the cytoplasm in association with Golgi or smooth membranes. The antibodies bind to the surface proteins but do not inactivate the virus. The major difference for humans lies in subtle differences in the surface proteins of the different dengue subtypes..

In addition.. Thus. Currently virologic diagnostic methodsare based on virus isolation or detection of viral RNA in acute serum. Kyle and Harris. and mainly restricted to reference laboratories. Four serotypes of DENV serotypes cause the disease in humans (DENV-1 to DENV-4). such as encephalitis and hepatitis (Deen et al. Halstead. IgG titre significantly higher in secondary infection. and classic dengue fever (DF) to the more severe and sometimes fatal forms (DHF) and dengue shock syndrome (Deen et al. 3. presenting with similar symptoms and signs. undifferentiated fever. 2. IgG are detectable at approximately 14 days after onset of symptoms and are maintained for life. 2008). Serologic tests. producing a broad spectrum of illnesses. 1. One of the most challenging problems associated with management of the infected patient is to achieve a rapid and specific diagnosis of DENV infection during the acute phase. a specific and early diagnosis is determinant to provide an adequate supportive and timely clinical treatment. IgM titer can be slower to rise in secondary infection. which ranges from asymptomatic infection. which rely on the detection of DENV-specific immunoglobulin M (IgM) and 11 . resulting in 250. particularly in those countries where dengue coexists with other acute tropical febrile illnesses. however. IgG appears approximately 2 days after symptoms appear.000 to 500. 2002. The World Health Organization estimates that there may be 50 to 100 million cases of dengue virus (DENV) infections worldwide every year. 2006). Secondary Infection Approximately 5% patients do not produce detectable levels of specific IgM.. IgM antibodies detectable for up to 6 months. 3. IgM antibodies appear approximately 5 days after onset of symptoms and rise for the next 1-3 weeks.Primary Infection 1. expensive. 2006). other nonclassic clinical forms has been described. 2. both methodologies are time consuming. 2007.000 cases of dengue hemorrhagic fever (DHF) and approximately 25 000 deaths annually (Guzman and Kouri.

together with the Flavivirus cross-reactivity. 2000. which can be detected before seroconversion and.. Dussart et al. Libraty et al. whereas detection of both IgM and IgG antibodies is suggestive of secondary or later infection. NS1 antigen constitutes a suitable DENV biomarker. Several studies have shown that the DENV nonstructural 1 (NS1) antigen. This late and persistent IgM response.. 2002. the presence of IgM antibodies alone suggests primary infection. represents a new approach for the diagnosis of acute dengue infection (Alcon et al. a highly conserved glycoprotein. Nevertheless. Young et al. produced in both membrane-associated and secreted forms. During the acute phase.immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA).. Xu et al. are more commonly used for dengue diagnosis. 2006. Because of this.. detectable levels of IgM antibodies appear approximately 4 to 6 days after the fever onset and remains in serum for 90 days afterward. 2007). restricts the efficacy of ELISA tests for the diagnosis of dengue infections (WHO. is abundant in the serum of patients in the early stages of DENV infection. 2000) 12 . therefore.. 2002. 2006.

it is expensive and it takes at least 6±10 days for the virus to replicate in tissue cell culture or laboratory mosquitoes. At present. the three basic methods used by most laboratories for the diagnosis of dengue virus infection are viral isolation and identi¿ cation. Though virus isolation and characterisation are considered as the gold standard of laboratory diagnosis for acute dengue virus infection. and detection of dengue virus-speci¿c IgM antibodies by the IgMcapture enzyme-linked immunosorbent assay (MAC-ELISA) and/or the rapid dengue immunochromatographic test (DIT). Moreover. This evaluation clearly shows that the PLATELIATM DENGUE NS1 AG test kit gives an overall higher sensitivity rate than the current three established diagnostic test methods for laboratory diagnosis of acute dengue infection. depends on the time taken for an infected person¶s immunological response to produce IgM antibodies against dengue virus antigens. Detection of viral genomic sequence by RT-PCR is also an expensive method and is not widely available in most hospital diagnostic laboratories. as in most cases. and should not be interpreted as a diagnosis of acute infection without a paired second serum sample. Early laboratory diagnosis of acute dengue virus infection still remains a problem. both DIT (often considered as the rapid test for diagnosis of dengue infection) and MAC-ELISA do not provide early diagnosis of acute dengue infection. The third method. the Platelia NS1 antigen-capture ELISA gave a higher 13 . it is important to establish a diagnosis of acute dengue virus infection during the ¿rst few days after manifestation of clinical symptoms. and early public health control of dengue outbreaks. Thus.Chapter III Comparison NS1 with IgM ELISA Capture In order to provide timely information for the management of the patients. assay of anti-dengue speci¿ c IgM. a single serological detection of IgM is merely indicative of a recent dengue virus infection.the ¿ rst detectable IgM only appears on Days 4±5 of the illness. detection of viral genomic sequence by a nucleic acid ampli¿cation technology assay (RT-PCR). Compared to dengue virus isolation and molecular detection of viral RNA.

1 Positive detection rate of each dengue test method with respect to the sample age. The sensitivity rate of IgM assay for early diagnosis of dengue was poor in the ¿rst three days of the illness. 14 . the test could also be considered concurrently with an assay of dengue speci¿c IgM. Fig. However. the NS1 antigen-capture ELISA gave a signi¿ cantly higher detection rate in acute primary dengue than in acute secondary dengue. In this evaluation. notwithstanding the presence of dengue speci¿ c IgM was merely indicative of recent dengue infection. especially those with fever lasting ¿ve days or less. 1 shows the positive detection rate of Platelia NS1 antigen-capture ELISA.positive detection within the ¿ rst four days of illness. Despite the lower detection rate for serum samples from patients with acute secondary dengue. the NS1 antigen-capture ELISA has the added advantage of continuing to give good detection rates up to seven days of the illness. Thus. the Platelia NS1 antigen-capture ELISA still gave a higher detection rate than the other dengue diagnostic methods used in this laboratory. even at the ¿ fth post-fever day. and not con¿ rmative of acute dengue infection. For those patients with a history of fever for more than six days and are suspected to have acute dengue infection. which on the whole. Fig. gave a higher detection rate than the other test methods at the various sample ages. the Platelia NS1 antigen-capture ELISA should be considered as the test of choice for patients suspected of acute dengue illness. The ¿ nding of this evaluation shows that no dengue speci¿ c IgM was detected within the ¿rst two days of the fever and only 50% of patients had detectable dengue IgM in their sera.

as demonstrated by other studies. Further work is ongoing to evaluate the speci¿ city of the Platelia NS1 antigen-capture ELISA kit and the possibility of cross-reactivity with NS1 antigens of other À aviviruses.The Platelia NS1 antigen-capture ELISA test has the prospect of wide usage for early diagnosis of acute dengue virus infection in dengue endemic countries. since it uses the same instruments as that of the dengue IgM-capture ELISA (MAC ELISA) test.(18-20) 15 . The possibility of a correlation between a high level of circulating dengue NS1 antigen with the occurrence of dengue haemorrhagic fever. which is normally carried out in the hospital diagnostic laboratories. This study was limited by the lack of negative controls to evaluate the speci¿ city of the test kit. is also included in the ongoing evaluation work.

The usefulness of anti-dengue speci¿c IgM assays depends on the time taken for the immune response to produce IgM antibodies against dengue virus antigens. Recently. as in most cases the ¿rst detectable IgM only appears on days 4±5 of the illness. especially in hospital diagnostic laboratories in developing countries. A single serological detection of IgM is merely indicative of a recent exposure to dengue virus and should not be interpreted as a diagnosis of acute infection without a paired second serum sample for con¿rmation. Although virus isolation and characterization are considered the ³gold standard´ for laboratory diagnosis of acute dengue virus infection. and detection of dengue virus-speci¿c IgM antibodies by IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA) and/or rapid dengue immunochromatography test device for detection of dengue speci¿c IgM. This NS1 antigen-capture ELISA has the prospect of wide usage for early diagnosis of acute dengue virus infection since it uses the same instruments as for the 16 .Chapter IV Conclusion Early laboratory diagnosis of acute dengue virus infection still remains a major problem in many parts of the world especially in regions where dengue is hyper-endemic but resources are limited. Thus both rapid dengue immunochromatography test device for detection of dengue speci¿c IgM (often considered as the rapid test for the diagnosis of acute dengue infection) and MAC-ELISA do not provide early diagnosis of acute dengue infection. Three basic methods used commonly by most laboratories in resource rich countries for the diagnosis of acute dengue virus infection are viral isolation and identi¿cation. a highly sensitive and speci¿c commercial dengue NS1 antigen-capture ELISA kit has been evaluated and found to be better in comparison to virus isolation and RT-PCR for early laboratory con¿rmation of acute dengue virus infection based on a single serum sample (Kumarasamy et al. Reverse transcriptase-polymerase chain reaction (RT-PCR) is also an expensive method and is not available widely. molecular detection of viral genomic sequence by a nucleic acid ampli¿cation assay.. 2007). it is expensive and at least 6±10 days are required for the virus to replicate in tissue culture cells or laboratory mosquitoes (adult or larvae).

6%). it is still limited by the need for sophisticated instrumentation and higher technical skill which is normally only available in large hospital diagnostic laboratories. 17 .5%) and positive predictive value (99. However.dengue IgM-capture ELISA (MAC-ELISA). With its high speci¿city (99. The ¿nding of this study shows that the rapid dengue NS1 antigen immunochromatography test device meets the intended purpose. this rapid immunochromatography test device is highly recommended for use in a de¿ned population group with clinical features suggestive of acute dengue virus infection. but not as a routine screening test for an asymptomatic population. A simple yet highly sensitive and speci¿c rapid dengue test that does not require instrumentation will be highly desirable for wide application to con¿rm acute dengue even in an outpatient clinic setting or for application in the ¿eld.

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