Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus

MM Parida; SR Santhosh; PK Dash; PV Lakshmana Rao


Future Virology. 2008;3(2):179-192. 

In This Article

Abstract and Introduction

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is a considerable public health concern in Southeast Asian and African countries. Despite the fact that CHIKV resurgence is associated with epidemics of unprecedented magnitude, only a few specific serological and molecular diagnostic tools are available. CHIKV diagnosis is essentially based on virus isolation, ELISA and reverse transcription (RT)-PCR assays. RT-PCR is the method of choice for the early detection and confirmation of virus in clinical samples. Further advancement in terms of rapid, reliable detection and quantification with improved sensitivity has been accomplished through development of both fully automated TaqMan® and SYBR® Green I-based real-time RT-PCR assays. In addition, another simple, rapid, novel and cost-effective isothermal gene amplification method known as RT loop-mediated isothermal amplification (RT-LAMP) has also been reported for the early detection and quantification of viral genomes in acute-phase patient serum samples. Of notable importance is the substantial reduction in time required for the confirmation of results by RT-LAMP assay (30 min) and monitoring of amplification by SYBR Green I dye-mediated naked-eye visualization. These findings demonstrate that the real-time RT-PCR and RT-LAMP assays have potential applications in clinical diagnostics owing to simultaneous detection and quantification of CHIKV in acute phase patient serum samples.

Chikungunya virus (CHIKV) is an important human pathogen that causes a syndrome characterized by fever, chills, headache and severe joint pain usually of the smaller joints, with or without swelling. The name is derived from the Makonde word meaning "that which bends up" in reference to the stooped posture developed as a result of the arthritic symptoms of the disease.[1] CHIKV, a member of the Alphavirus genus in the family Togaviridae, was first isolated from the serum of a febrile human in the Newala district (Tanzania) in 1953. CHIKV is classified serologically as a member of the Semliki Forest virus (SFV) antigenic complex closely related to O"nyong-nyong virus (ONNV) because of its cross reactivity.[2,3]

CHIKV is geographically distributed throughout Africa and Southeast Asia, and its transmission to humans is mainly via the Aedes mosquito species. CHIKV is most prevalent in urban areas and epidemics are sustained by the human-mosquito-human transmission cycle, since humans act as very efficient reservoirs for the virus.[1] Since 1953, CHIKV has caused numerous well-documented outbreaks and epidemics in both Africa and Southeast Asia, involving hundreds of thousands of people. A CHIKV outbreak of unprecedented magnitude swept the Indian Ocean territories principally involving Reunion Island, Comoros, Mauritius, Seychelles and Southwestern India in 2005-2006.[4,5,6,7] Recently, the virus has also crossed the tropics and its presence has been recorded for the first time in the Emilia Romagna Region of northeastern Italy; the Aedes albopictus being implicated as the mosquito vector.[8] The epidemic was a surprise owing to its unexpected emergence, its unprecedented magnitude, explosive spread and clinical severity of unprecedented levels.[9,10] The precise reasons for the re-emergence of CHIKV in the Indian subcontinent as well as other small countries in the southern Indian Ocean are an enigma. However, the plausible explanations include increased tourism, the introduction of virus into a naive population and viral mutation.[11]

The severity of the illness varies, tending to be less severe in children. Most patients recover after a few weeks but a small proportion of individuals may experience chronic joint pain for some years. Deaths related directly to infection with CHIKV have not been documented to date, but CHIKV may be a contributing factor in the death of some individuals with underlying health problems. Polyarthralgia, the typical clinical sign of CHIKV is very painful. Symptoms are generally self limiting and last 1-10 days. However, arthralgia may persist for months or years. In some patients, minor hemorrhagic signs, such as epistaxis or gingivorrhagia, have also been described. The symptoms are most often clinically indistinguishable from those observed in dengue fever. Indeed, the simultaneous isolation of both dengue and CHIKV from the sera of the same patients has previously been reported, indicating the presence of dual infections.[12] It is, therefore, very important to clinically distinguish dengue from CHIKV infection. In contrast to dengue, hemorrhagic manifestations are relatively rare in CHIKV infection and shock is not typically observed in CHIKV infection. Fever and polyarthralgia are the major symptoms that result in a positive clinical diagnosis. However, in 5% of cases the disease is asymptomatic.[13] Neurological complications, such as meningoencephalitis, hepatic cytolyses, severe lymphopenia, severe dermatological involvement, death and neonatal infections have also been reported in a small proportion of patients during the recent Indian, as well as the French Reunion Island outbreaks.[11,14]

Although not listed as a hemorrhagic fever virus, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms as discussed above. Thus, the differential diagnosis of these two infections is essential for clinical management and epidemiological study in the tropics. Despite the fact that CHIKV resurgence is associated with epidemics of unprecedented magnitude, only a few specific serological and molecular diagnostic tools are available. A definitive diagnosis of Chikungunya infection can be made only with the aid of laboratory support since clinically, symptoms resemble those of dengue fever. Laboratory diagnosis is therefore critical to establish the differential diagnosis and enable the initiation of a specific public health response.

Three main laboratory tests are used for diagnosing CHIKV infection: virus isolation, serological tests for demonstration of virus-specific antibodies and genomic detection by PCR-based methods. Virus isolation is the most definitive test and is considered to be the gold standard. Detection of most alphaviruses is currently dependent on virus isolation from the blood of viremic patients, infected tissues or blood-feeding arthropods. The isolation of CHIKV is comparatively more simple and effective owing to the highly cytopathic and fast-growing nature of the virus, which will grow to very high titers.[15] CHIKV replicates in various cell lines, including insect cells, for example, C6/36, nonhuman viz, Vero, chick embryo fibroblast-like cells, BHK21, L929 and Hep-2 cells, and human cell lines, for example, HeLa, MRC5 in which it will often induce a significant cytopathic effect. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, are susceptible to infection and permit viral production. By contrast, CHIKV does not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells.[15] However, the isolation process is time-consuming and the degree of success is dependent on a number of complicating factors, for example, time of collection, transportation, maintenance of cold chain, storage and processing of samples.

Serodiagnosis of CHIKV relies on the demonstration of a fourfold increase in Chikungunya IgG titer between the acute and convalescent phase sera. However, obtaining paired sera is usually impractical. Alternatively, the demonstration of IgM antibodies specific for CHIKV in acute-phase sera is used in instances where paired sera cannot be collected. The most commonly used test is the IgM capture (MAC)-ELISA.[16,17] Cross-reaction with other Alphavirus antibodies such as ONNV and SFV usually limits the application of MAC-ELISA as a confirmatory test. A positive virus culture supplemented with neutralization is taken as definitive proof for the presence of CHIKV. However, as already discussed, it is not always practical to adapt these approaches for routine and early detection. Therefore, a molecular approach based on reverse transcription (RT)-PCR technologies is useful for early confirmatory diagnosis prior to the appearance of IgM antibody, irrespective of the presence of viable virus ( Table 1 ).


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