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 wouldnt 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 Ive made in this paper. I hope this paper could be useful to all the readers. Thank you.

Jakarta, January 2011

Aji Patriajati

CONTENTS

Preface Contents Abstract CHAPTER

.. .. .. I I. 1 I. 2 I. 3 I. 4 I. 5 I. 6 Introduction Background Problems ...... ...... ......

1 2 3 4 4 4 5 5 5 6 7 7 7 8 10 13 16 18

Limitation of the Problems.. Objectives Methods of Writing Frame of Writing Dengue Virus Morphology Genome Organization Replication cycle Antibody Responce ...... ...... ...... ...... ...... .................. ...................................................... ..

CHAPTER

II II. 1 II. 2 II. 3 II. 4

CHAPTER CHAPTER

III IV

Comparison NS1 with IgM ELISA Capture ...... Conclusion .........

BIBLIOGRAFI .....

Abstract

Dengue is a viral infection of humans that is transmitted by mosquitoes. Dengue is a very important public health problem in many developing countries. Recently, new tests to help diagnose patients with dengue have been developed. 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, and if so, when best to use them. Our hospital-based results, using two different types of NS1 tests for diagnosing dengue, 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. Collectively, the results indicate these NS1 tests deserve inclusion in the diagnostic approach to dengue.

CHAPTER I
Introduction
I. 1. Background Dengue is a major public health problem in many parts of the tropical developing world [1,2]. Dengue is caused by infection with one of four serotypes of dengue virus (DENV1-4), which are arboviruses belonging to the Flaviviridae family. Although most DENV infections are asymptomatic, a proportion result in clinically apparent disease that varies in severity from mild undifferentiated fever through to more severe syndromes, primarily dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF is a vasculopathy characterized by capillary leakage and haematological dysregulation; in severe case hypovolaemic shock (DSS) may develop. There are no licensed vaccines or specific antiviral therapies for dengue, and patient management relies on good supportive care. 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.

I.2.

Problems Dengue hemorrhagic fever, is an increasing cause of morbidity and mortality throughout the

tropical world. There are an estimated 50 to 100 million cases of dengue infection each year, including about half a million cases of dengue hemorrhagic fever. The number of cases of both dengue fever and dengue hemorrhagic fever has increased dramatically for the past few decades, and the geographic range has extended to involve most tropical countries.

I. 3.

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. Accurate dengue diagnosis prevents unnecessary and possibly expensive antibiotic usage 3. Prompt diagnosis of index cases can facilitate vector control activities in the community so as to mitigate further transmission 4. Expanded use of accurate dengue diagnostics provides important data on the epidemiology and health burden of dengue

I. 4.

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. 5.

Methods of Writing. The writing of paper is carried out by a library research and also via internet.

I. 6.

Frame of Writing

Preface Contents Abstract CHAPTER

.. .. .. I I. 1 I. 2 I. 3 I. 4 I. 5 I. 6 Introduction Background Problems ...... ...... ......

1 2 3 4 4 4 5 5 5 5 6 6 7 7 7 9 14 16

Limitation of the Problems.. Objectives Methods of Writing Frame of Writing Dengue Virus Morphology Genome Organization Replication cycle Antibody Responce ...... ...... ...... ...... ...... .................. ...................................................... .. ....

CHAPTER

II II. 1 II. 2 II. 3 II. 4

CHAPTER CHAPTER

III IV

Comparison NS1 with IgM dengue blot Conclusion

..........

BIBLIOGRAFI ..

CHAPTER II
Dengue Virus
Dengue is caused by one of four closely related virus serotypes of the genus Flavivirus, family Flaviviridae, each serotype is sufficiently different that there is no cross-protection and epidemics caused by multiple serotypes (hyperendemicity) can occur. 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.

II. 1 Morphology of the Virus Electron micrographs showed that dengue virions are characterized by a relatively smooth surface, with a diameter of approximately 500 , and an electron-dense core surrounded by a lipid bilayer. In addition to the plus-sense RNA genome of 10,700 nucleotides, there are three structural proteins that occur in stoichiometric amounts in the particle: core (C, 100 amino acids), membrane (M, 75 amino acids), and envelope (E, 495 amino acids).

II.2 Genome Organization of Dengue Virus Dengue Virus (DV) belongs to the family Flaviviridae. The four serotypes of dengue virus (designated DEN-1, DEN-2, etc) can be distinguished by serological methods. Infection in humans by one serotype produces life-long immunity against reinfection by that same serotype, but only temporary and partial protection against the other serotypes. Dengue viruses share many characteristics with other flaviviruses, having a single-stranded RNA genome surrounded by an icosahedral nucleocapsid and covered by a lipid envelope. The virion is approximately 50nm in diameter. The flavivirus genome is approximately 11kb (kilobases) in length, and the complete genome sequence is known for isolates of all four serotypes of dengue virus. The genome is 7

composed of three structural protein genes, encoding the nucleocapsid or core protein C, a membrane-associated protein (M), an envelope protein (E) and seven non-structural (NS) protein genes. The domains responsible for neutralization, fusion and interactions with virus receptors are associated with the envelope protein. The order of proteins encoded is 5-C-prM(M)-E-NS1NS2ANS2B- NS3-NS4A-NS4B-NS5-3. NS1, a glycoprotein is detected in high titers in patients with secondary dengue infections but its function is unknown. NS2 region, is known to code for 2 proteins (NS2A and NS2B), which are assumed to play a role in polyprotein processing. NS3, the viral proteinase functions in the cytosol. NS4 region codes for two small hydrophobic proteins involved in the membrane bound RNA replication complex establishment. NS5 codes for a protein with a molecular weight of 105,000 and is the most conserved flavivirus protein. This protein is assumed to be the virus encoded RNA dependent RNA polymerase. NS6 and NS7 function yet to be found.

A. Schematic of the single stranded RNA genome with highly structured RNA elements in the 5' and 3' NTRs. B. DV genomic organization and functions of viral proteins. For some proteins their function in the viral life cycle is not yet established; they are marked with a ? C. Putative membrane topology of DV proteins and proteinases involved in polyprotein cleavage.

II.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, particles may enter cells via Fcreceptor upon opsonization. Virions are internalized by receptor-mediated endocytosis resulting in release of the viral genome from the nucleocapsid in a low pH dependent manner. Soon after infection, viral proteins induce rearrangments of intracellular membranes forming distinct structures that have designated vesicle packets and convoluted membranes. It appears that vesicle packets are sites of RNA replication that is probably catalyzed by a multi-protein 8

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

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

II.4 Antibody Responce

Dengue infection will result in lifelong immunity to that serotype, but only temporary immunity to other serotypes.

10

Primary Infection 1. IgM antibodies appear approximately 5 days after onset of symptoms and rise for the next 1-3 weeks. 2. IgM antibodies detectable for up to 6 months. 3. IgG are detectable at approximately 14 days after onset of symptoms and are maintained for life.

Secondary Infection Approximately 5% patients do not produce detectable levels of specific IgM. 1. IgM titer can be slower to rise in secondary infection. 2. IgG appears approximately 2 days after symptoms appear. 3. IgG titre significantly higher in secondary infection. The World Health Organization estimates that there may be 50 to 100 million cases of dengue virus (DENV) infections worldwide every year, resulting in 250,000 to 500,000 cases of dengue hemorrhagic fever (DHF) and approximately 25 000 deaths annually (Guzman and Kouri, 2002; Halstead, 2007; Kyle and Harris, 2008). Four serotypes of DENV serotypes cause the disease in humans (DENV-1 to DENV-4), producing a broad spectrum of illnesses, which ranges from asymptomatic infection, undifferentiated fever, and classic dengue fever (DF) to the more severe and sometimes fatal forms (DHF) and dengue shock syndrome (Deen et al., 2006). In addition, other nonclassic clinical forms has been described, such as encephalitis and hepatitis (Deen et al., 2006). 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, particularly in those countries where dengue coexists with other acute tropical febrile illnesses, presenting with similar symptoms and signs. Thus, a specific and early diagnosis is determinant to provide an adequate supportive and timely clinical treatment. Currently virologic diagnostic methodsare based on virus isolation or detection of viral RNA in acute serum; however, both methodologies are time consuming, expensive, and mainly restricted to reference laboratories. Serologic tests, which rely on the detection of DENV-specific immunoglobulin M (IgM) and 11

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

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Chapter III
Comparison NS1 with IgM ELISA Capture
In order to provide timely information for the management of the patients, and early public health control of dengue outbreaks, it is important to establish a diagnosis of acute dengue virus infection during the rst few days after manifestation of clinical symptoms. Early laboratory diagnosis of acute dengue virus infection still remains a problem. At present, the three basic methods used by most laboratories for the diagnosis of dengue virus infection are viral isolation and identi cation, detection of viral genomic sequence by a nucleic acid amplication technology assay (RT-PCR), and detection of dengue virus-specic IgM antibodies by the IgMcapture enzyme-linked immunosorbent assay (MAC-ELISA) and/or the rapid dengue immunochromatographic test (DIT). Though virus isolation and characterisation are considered as the gold standard of laboratory diagnosis for acute dengue virus infection, it is expensive and it takes at least 610 days for the virus to replicate in tissue cell culture or laboratory mosquitoes. Detection of viral genomic sequence by RT-PCR is also an expensive method and is not widely available in most hospital diagnostic laboratories. The third method, assay of anti-dengue speci c IgM, depends on the time taken for an infected persons immunological response to produce IgM antibodies against dengue virus antigens. Thus, 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, as in most cases,the rst detectable IgM only appears on Days 45 of the illness. Moreover, a single serological detection of IgM is merely indicative of a recent dengue virus infection, and should not be interpreted as a diagnosis of acute infection without a paired second serum sample. 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. Compared to dengue virus isolation and molecular detection of viral RNA, the Platelia NS1 antigen-capture ELISA gave a higher 13

positive detection within the rst four days of illness. However, the NS1 antigen-capture ELISA has the added advantage of continuing to give good detection rates up to seven days of the illness. In this evaluation, the NS1 antigen-capture ELISA gave a signi cantly higher detection rate in acute primary dengue than in acute secondary dengue. Despite the lower detection rate for serum samples from patients with acute secondary dengue, the Platelia NS1 antigen-capture ELISA still gave a higher detection rate than the other dengue diagnostic methods used in this laboratory. Fig. 1 shows the positive detection rate of Platelia NS1 antigen-capture ELISA, which on the whole, gave a higher detection rate than the other test methods at the various sample ages. The sensitivity rate of IgM assay for early diagnosis of dengue was poor in the rst three days of the illness, notwithstanding the presence of dengue speci c IgM was merely indicative of recent dengue infection, and not con rmative of acute dengue infection. 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, even at the fth post-fever day. Thus, the Platelia NS1 antigen-capture ELISA should be considered as the test of choice for patients suspected of acute dengue illness, especially those with fever lasting ve days or less. For those patients with a history of fever for more than six days and are suspected to have acute dengue infection, the test could also be considered concurrently with an assay of dengue specic IgM.

Fig. 1 Positive detection rate of each dengue test method with respect to the sample age.

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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, 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. 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 aviviruses. The possibility of a correlation between a high level of circulating dengue NS1 antigen with the occurrence of dengue haemorrhagic fever, as demonstrated by other studies, is also included in the ongoing evaluation work.(18-20)

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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. Three basic methods used commonly by most laboratories in resource rich countries for the diagnosis of acute dengue virus infection are viral isolation and identication, molecular detection of viral genomic sequence by a nucleic acid amplication assay, and detection of dengue virus-specic IgM antibodies by IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA) and/or rapid dengue immunochromatography test device for detection of dengue specic IgM. Although virus isolation and characterization are considered the gold standard for laboratory diagnosis of acute dengue virus infection, it is expensive and at least 610 days are required for the virus to replicate in tissue culture cells or laboratory mosquitoes (adult or larvae). Reverse transcriptase-polymerase chain reaction (RT-PCR) is also an expensive method and is not available widely, especially in hospital diagnostic laboratories in developing countries. The usefulness of anti-dengue specic IgM assays depends on the time taken for the immune response to produce IgM antibodies against dengue virus antigens. Thus both rapid dengue immunochromatography test device for detection of dengue specic 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, as in most cases the rst detectable IgM only appears on days 45 of the illness. 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 conrmation. Recently, a highly sensitive and specic 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 conrmation of acute dengue virus infection based on a single serum sample (Kumarasamy et al., 2007). 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

dengue IgM-capture ELISA (MAC-ELISA). However, it is still limited by the need for sophisticated instrumentation and higher technical skill which is normally only available in large hospital diagnostic laboratories. A simple yet highly sensitive and specic rapid dengue test that does not require instrumentation will be highly desirable for wide application to conrm acute dengue even in an outpatient clinic setting or for application in the eld. The nding of this study shows that the rapid dengue NS1 antigen immunochromatography test device meets the intended purpose. With its high specicity (99.5%) and positive predictive value (99.6%), this rapid immunochromatography test device is highly recommended for use in a dened population group with clinical features suggestive of acute dengue virus infection, but not as a routine screening test for an asymptomatic population.

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Bibliografi
1. World Health Organization. Dengue haemorrhagic fever: diagnosis, treatment and control. Handbook of the World Health Organization. Geneva, 2000: 1-84. 2. Gubler DJ, Meltzer M. Impact of dengue/dengue hemorrhagic fever on the developing world. Adv Virus Res 1999; 53:35-70. 3. Gibbons RV, Vaughn DW. Dengue: an escalating problem. BMJ 2002; 324:1563-6. 4. Monath TP, Heinz FX. Flaviviruses. In: Fields BW, Knipe DM, Knipe PM, Howley PM, eds. Fields Virology. Vol 1. 3rd ed. Philadelphia: Lippincott-Raven Press, 1996: 961-1034. 5. Henchal EA, Putnak JR. The dengue viruses. Clin Microbiol Rev 1990; 3:376-96. 6. Schlesinger JJ, Brandriss MW, Putnak JR, Walsh EE. Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody. J Gen Virol 1990; 71:593-9. 7. Mackenzie JM, Jones MK, Young PR. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 1996; 220:232-40.

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