**5. Diagnosis**

120 Current Topics in Tropical Medicine

2009). Moreover, since high concentrations of these pro-inflammatory factors were found in the joints of humans afflicted with RRV-induced polyarthritis, they probably have a causative role in chronic joint and muscle pains that plague patients (Lidbury et al., 2008). The finding that aberrant Type I interferon signalling in mice led to severe forms of CHIKF (Couderc et al., 2008) further highlighted the important role cytokines play in the

Chikungunya is not generally considered to be a life-threatening disease. Usually the clinical course is fairly mild, but fatal cases directly or indirectly linked to infection with CHIKV have been observed during the Indian-Ocean outbreak (Josseran et al., 2006). The main evidence of a mortality linked to Chikungunya fever epidemics has been obtained in La Reunion, Mauritius, and India by comparing expected and observed mortality data. In all cases, during the months when the epidemics were raging, the observed mortality significantly exceeded the expected one. In particular, in La Reunion the monthly crude death rates in February and March 2006 were 34.4%and 25.2% higher, respectively, than expected. These corresponded to 260 excess deaths (an increase of 18.4%) with a rough estimate of the case-fatality rate for Chikungunya fever of ≈1/1,000 cases. The case-fatality rate calculated on increased crude death rates in Mauritius and Ahmedabad, India, is substantially higher than that calculated in La Reunion: approximately 4.5% (15,760 confirmed or suspected cases and 743 excess deaths) and 4,9% (60,777confirmed or suspected cases and 2,944 excess deaths), respectively (Beesoon et al., 2008; Mavalankar et al., 2008). These differences may be attributed to many factors (greater disease severity, preexisting patient conditions, different patient management, or coincident excess deaths from other causes) but may also be due to a different efficacy of the surveillance systems for Chikungunya fever, that probably worked poorly in Mauritius and India, leading to underestimating the total number of cases (Fusco et al., 2010). The possible link between

Neurological complications such as meningo-encephalitis were reported in a few patients during the first Indian outbreak in 1973, and during the 2006 Indian outbreak (Chatterjee et al., 1965; Ravi, 2006). The possible mechanisms underlying these processes remain unknown. Studies performed on animal models showed that CHIKV-infected young mice had weakness and walking difficulties which could be due to necrosis and inflammation of skeletal muscles (Ziegler et al., 2008). CHIKV antigens and viral replication have been detected in human myogenic precursors such as satellite cells but not in muscle fibers (Ozden et al., 2007), suggesting that muscle satellite cells could be potential virus reservoirs. The pathologic symptoms of encephalitis owing to CHIKV infection as well as central nervous system (CNS) infections (Chatterjee & Sarkar, 1965) were expected, since *in vitro* experiments showed that the virus could infect and replicate for extended periods in mouse brain cells (Precious et al., 1974). More recently, it was found that mouse CNS tissues such as the choroid plexi could also be targets of CHIKV, lending more credence to the fact that CHIKV infections do affect CNS cells and tissues (Couderc et al., 2008). Work is currently underway by several research groups around the world to decipher this mechanism in CHIKV infections. Moreover, during the 2006 Indian-Ocean outbreak, rare cases of Guillain-Barré syndrome (GBS) associated with CHIKV infection have been described (Lebrun et al.,

CHIKV infection and multiorgan failure is still under investigation.

pathology of CHIKV infection.

**4.3 Other pathologies** 

2009; Wielanek et al., 2007).

Diagnosis of infection with CHIKV is based on molecular biology (RT-PCR) and serology methods. The first one is useful during the initial viraemic phase, at the onset of symptoms and normally for the following 5-10 days, when CHIKV RNA reaches very high levels (viral loads of 3.3 x 109 copies/ml) and can be detected (Carletti et al., 2007; Parola et al., 2006). Afterwards, the diagnosis is based on serological methods (ELISA, immunofluorescence, hemoagglutination inhibition (HI) and infectivity neutralization (Nt)).

IgM specific against CHIKV are detectable 2-3 days after the onset of symptoms by ELISA immunofluorescent assay and persists for several weeks, up to 3 months (Litzba et al, 2008; Sam & AbuBakar, 2006); rarely, IgM can be detected for longer periods, up to 1 year. IgG specific against CHIKV appear soon after IgM antibodies (2-3 days) and persists for years. Testing of a couple of sera collected in the acute and the convalescent phases of the disease is mandatory for the identification of recent infection using serology methods that cannot distinguish IgG Ab from IgM Ab (i.e. HI and Nt). It is also very useful to confirm results obtained with other methods, especially taking into account the although rare persistence of IgM antibodies. Viral isolation can be performed from serum of infected patient on insect or mammalian cell lines (i.e. C6/36 or Vero E6) during the early phase of the disease, when the viral load is very high and the immune response is still not detectable; however it is useful only for epidemiology or pathogenesis studies or for thorough molecular characterization (Fusco et al., 2010). The sensitivity and specificity of rapid bedside tests commercially available are poorly established, and the possibility of false-positive reactions resulting from cross-reactivity with dengue or other arboviruses such as o'nyong-nyong virus has to be considered (Blackburn et al., 1995). Serologically, chikungunya virus is most closely related to o'nyong-nyong virus and is a member of the Semliki Forest antigenic complex. Individual serological testing is not particularly useful, except when faced with atypical or severe forms, or in travellers returning from an epidemic zone (Pile et al., 1999).
