Molecular and serological techniques to detect co-circulation of DENV,ZIKV and CHIKV in suspected dengue-like syndrome patients

BackgroundArboviruses are important emerging viruses worldwide. The signs and symptoms of Zika virus (ZIKV) infection are similar to those presented by infections with dengue virus (DENV) and chikungunya virus (CHIKV). Furthermore, diagnosis of ZIKV infection is particularly challenging in dengue endemic regions and with co-circulation of DENV, CHIKV, and ZIKV, making diagnosis based solely on clinical and epidemiological data unreliable. As these three viral infections share similar clinical manifestations, differential diagnosis is crucial.ObjectivesIn this study, diagnoses of ZIKV, CHIKV and DENV infections were investigated in 30 patients with suspected dengue fever residing in the area of co-circulation of these three arboviruses.Study designThe study included whole blood and/or serum samples obtained from 30 patients with suspected dengue fever. All patients were tested for DENV infection as well as for CHIKV and ZIKV infections. Assays for detecting anti-DENV IgM and DENV RNA by semi-nested RT-PCR and ZIKV and CHIKV RNA by real-time RT-PCR were performed.ResultsDENV RNA was not detectable in any of the clinical samples, whereas ZIKV RNA was detectable in 17 samples (56.7%). Co-infection by ZIKV and CHIKV was documented in one case. Of the 17 ZIKV-positive individuals, 8 showed reactivity for anti-DENV IgM, which suggested recent DENV infection, cross-reactivity or co-infection.ConclusionOur findings confirm that accurate laboratory testing is of paramount importance for differential diagnosis in areas of simultaneous transmission of different arboviruses with similar clinical presentations.     

Chikungunya Virus Seroprevalence and Associated Factors among Hospital Attendees in Two States of Southwest Nigeria: A Preliminary Assessment

Chikungunya virus (CHIKV) is a re-emerging pathogen causing long-term polyarthritis and encephalitis. In conducting a preliminary investigation, we hypothesized that there is no serologic evidence of CHIKV infection among attendees of selected hospitals in Lagos and Osun States, Nigeria. Sera from 304 consecutively selected participants were screened for CHIKV IgG and IgM using ELISA. Findings were analyzed vis-à-vis participants’ demographic and clinical data. Over 90.0% of the participants had never heard of CHIKV despite the fact that a large proportion of them (88.8%) had secondary/tertiary education. Overall, 41.8% were positive for, at least, one antibody type (IgG or IgM), while about 16.0% of the participants had dual seropositivity (CHIKV IgG and IgM) with gender as associated factor (odds ratio [OR]: 2.8, p = 0.03). Prevalence rates were 31.8% and 38.4% for CHIKV IgG and IgM, respectively. Only hospital location (Osogbo) was associated with CHIKV IgG (OR: 2.2, p = 0.009), while gender alone was associated with CHIKV IgM (OR: 3.0, p = 0.001). Participants seropositive for CHIKV antibodies were mostly adults (18–59 yrs) belonging to the active work-force; five (22.7%) and three (20.0%) of the pregnant participants had CHIKV IgG and IgM, respectively. Detection of CHIKV IgM in some participants might make them potentially infectious to the newborn and mosquito vectors. Importantly, participants positive for either IgG or IgM had fever (72.8%, 67.2%) and general body pains (61.7%, 57.6%), respectively. This ELISA-based study revealed serologic evidence of CHIKV infection among hospital attendees in Lagos and Osun states with the group-specific prevalence rates being considerably high.

Abbreviations:Chikungunya virus (CHIKV); Chikungunya (CHIK); enzyme-linked immunosorbent assay (ELISA); immunoglobulin G or M (IgG/IgM); odds ratio (OR); non-structural proteins (nsP); hemagglutination inhibiting (HI); complement fixing (CF); neutralization test (NT); immunofluorescence assay (IFA); plaque reduction neutralization test (PRNT); confidence interval (CI); analysis of variance (ANOVA); body temperature (BT); Building Nigeria’s Response to Climate Change (BNRCC).     

Detection of chikungunya virus-specific IgM on laser-cut paper-based device using pseudo-particles as capture antigen

The incidence of arbovirus infections has increased dramatically in recent decades, affecting hundreds of millions of people each year. The Togaviridae family includes the chikungunya virus (CHIKV), which is typically transmitted by Aedes mosquitoes and causes a wide range of symptoms from flu-like fever to severe arthralgia. Although conventional diagnostic tests can provide early diagnosis of CHIKV infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop efficient, affordable, simple, rapid, and robust diagnostic tools that can be used in point-of-care settings. Early diagnosis is crucial to improve patient management and to reduce the risk of complications. A glass-fiber laser-cut microfluidic device (paper-based analytical device [PAD]) was designed and evaluated in a proof of principle context, for the analysis of 30 µL of patient serum. Biological raw materials used for the functionalization of the PAD were first screened by MAC-ELISA (IgM capture enzyme-linked immunosorbent assay) for CHIKV Immunoglobulin M (IgM) capture and then evaluated on the PAD using various human samples. Compared with viral lysate traditionally used for chikungunya (CHIK) serology, CHIKV pseudo-particles (PPs) have proven to be powerful antigens for specific IgM capture. The PAD was able to detect CHIKV IgM in human sera in less than 10 minutes. Results obtained in patient sera showed a sensitivity of 70.6% and a specificity of around 98%. The PAD showed few cross-reactions with other tropical viral diseases. The PAD could help health workers in the early diagnosis of tropical diseases such as CHIK, which require specific management protocols in at-risk populations.     

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection

BackgroundSince the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important.ObjectivesWe evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide.Study designA panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing.ResultsThe panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used.ConclusionThe external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance.     

Chikungunya fever: a re-emerging viral infection

Chikungunya (CHIK) fever is a re-emerging viral disease characterized by abrupt onset of fever with severe arthralgia followed by constitutional symptoms and rash lasting for 1-7 days. The disease is almost self-limiting and rarely fatal. Chikungunya virus (CHIKV) is a RNA virus belonging to family Togaviridae, genus Alphavirus. Molecular characterization has demonstrated two distinct lineages of strains which cause epidemics in Africa and Asia. These geographical genotypes exhibit differences in the transmission cycles. In contrast to Africa where sylvatic cycle is maintained between monkeys and wild mosquitoes, in Asia the cycle continues between humans and the Aedes aegypti mosquito. CHIKV is known to cause epidemics after a period of quiescence. The first recorded epidemic occurred in Tanzania in 1952-1953. In Asia, CHIK activity was documented since its isolation in Bangkok, Thailand in 1958. Virus transmission continued till 1964. After hiatus, the virus activity re-appeared in the mid-1970s and declined by 1976. In India, well-documented outbreaks occurred in 1963 and 1964 in Kolkata and southern India, respectively. Thereafter, a small outbreak of CHIK was reported from Sholapur district, Maharashtra in 1973. CHIKV emerged in the islands of South West Indian Ocean viz. French island of La Reunion, Mayotee, Mauritius and Seychelles which are reporting the outbreak since February, 2005. After quiescence of about three decades, CHIKV re-emerged in India in the states of Andhra Pradesh, Karnataka, Maharashtra, Madhya Pradesh and Tamil Nadu since December, 2005. Cases have also been reported from Rajasthan, Gujarat and Kerala. The outbreak is still continuing. National Institute of Communicable Diseases has conducted epidemiological, entomological and laboratory investigations for confirmation of the outbreak. These have been discussed in detail along with the major challenges that the country faced during the current outbreak.     

Expression of the capsid protein of Chikungunya virus in a baculovirus for serodiagnosis of Chikungunya disease

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIK fever, which typically manifests as a febrile illness. To develop a CHIKV-specific diagnostic test, CHIKV capsid protein was expressed using a baculovirus expression system. The seroreactvity of the recombinant CHIKV capsid protein was evaluated by ELISA and immuochromatographic assay (ICA), using 40 anti-CHIKV-positive and 20 anti-CHIKV-negative sera, an additional 20 normal sera samples from healthy Koreans, and 20 anti-Dengue virus sera samples. The sensitivity of the recombinant CHIKV capsid protein was 85% and 87.5% as measured by ELISA and ICA, respectively. The specificity of the recombinant CHIKV capsid protein was 100% both by ELISA and by ICA. No cross-reactivity of the capsid protein was seen with anti-Dengue virus sera samples. There was a significant correlation between the ELISA- and ICA-measured seroreactivities of the recombinant CHIKV capsid protein for anti-CHIKV IgM-positive sera samples. These results suggest that the recombinant CHIKV capsid protein could be used in a diagnostic test for identifying CHIKV disease.     

Molecular characterization of Chikungunya virus during an outbreak in South India

Introduction: Re-emergence of Chikungunya is a major public health problem in the southern states of India. Objectives: This study was undertaken to investigate an outbreak of Chikungunya, in June–August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. Materials and Methods: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. Results: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368–GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. Conclusion: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.     

Evaluation of arthropod-borne viruses and other infectious disease pathogens as the causes of febrile illnesses in the Khartoum Province of Sudan

The relative importance of arthropod-borne and other disease pathogens as the cause of an outbreak of febrile illnesses was assessed during August 1988, following severe flooding in Khartoum, Sudan. A total of 200 patients with acute febrile illness and 100 afebrile controls were enrolled in the study during October and November 1988, at the Omdurman Military Hospital, Khartoum, Sudan. Sera were tested for IgM and IgG antibodies to six arthropod-borne viruses by an enzyme-linked immunoabsorbent assay, and for similar antibodies to Lassa fever, Crimean-Congo hemorrhagic fever, and Ebola and Marburg viruses by an indirect fluorescence assay. Thick and thin blood smears were examined microscopically for malaria parasites, and fecal and blood specimens were tested for bacteria by standard culture methods. Among the acute and convalescent sera collected from 67 febrile patients, five cases were caused by sandfly fever Sicilian (SFS), six by sandfly fever Naples (SFN), and 12 by unidentified phleboviruses. Of 233 remaining unpaired, acute-phase sera collected from cases and controls, 49 (21%) had IgM antibodies to SFS or SFN, RVF, West Nile (WN), and Chikungunya (CHIK) viruses. Forty-three (22%) of 192 febrile cases and two of the 100 afebrile controls were positive for Plasmodium falciparum, and bacterial enteropathogens were associated with 25 (13%) cases and four controls. These data indicated that phleboviruses and to a lesser extent, WN, P. falciparum, and enterobacterial pathogens were causes of acute febrile illnesses following the 1988 flood in Khartoum, Sudan. © 1996 Wiley-Liss, Inc.     

Urinary detection of toscana virus nucleic acids in neuroinvasive infections

BackgroundToscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion.ObjectivesTo evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen.Study designA total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing.ResultsPhlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample.ConclusionsThese preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available.     

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection

Purpose

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens.

Materials and Methods

CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA.

Results

The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%.

Conclusion

The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.     

Chikungunya virus of Asian and Central/East African genotypes in Malaysia

BackgroundChikungunya virus (CHIKV) of the Central/East African genotype has caused large outbreaks worldwide in recent years. In Malaysia, limited CHIKV outbreaks of the endemic Asian and imported Central/East African genotypes were reported in 1998 and 2006. Since April 2008, an unprecedented nationwide outbreak has affected Malaysia.ObjectiveTo study the molecular epidemiology of the current Malaysian CHIKV outbreak, and to evaluate cross-neutralisation activity of serum from infected patients against isolates of Asian and Central/East African genotypes.Study designSerum samples were collected from 83 patients presenting in 2008, and tested with PCR for the E1 gene, virus isolation, and for IgM. Phylogenetic analysis was performed on partial E1 gene sequences of 837 bp length. Convalescent serum from the current outbreak and Bagan Panchor outbreak (Asian genotype, 2006) were tested for cross-neutralising activity against representative strains from each outbreak.ResultsCHIKV was confirmed in 34 patients (41.0%). The current outbreak strain has the A226V mutation in the E1 structural protein, and grouped with Central/East African isolates from recent global outbreaks. Serum cross-neutralisation activity against both Central/East African and Asian genotypes was observed at titres from 40 to 1280.ConclusionsThe CHIKV strain causing the largest Malaysian outbreak is of the Central/East African genotype. The presence of the A226V mutation, which enhances transmissibility of CHIKV by Aedes albopictus, may explain the extensive spread especially in rural areas. Serum cross-neutralisation of different genotypes may aid potential vaccines and limit the effect of future outbreaks.     

Recombinant CHIK virus E1 coat protein of 11 KDa with antigenic domains for the detection of Chikungunya

Chikungunya is an acute febrile illness caused by an alpha virus technically called as CHIK virus. A smaller size of CHIK virus E1 coat protein -11 kDa was expressed in prokaryotic expression system. The recombinant protein was purified and confirmed by western blot analysis. The positions of the antigenic domain in the protein were identified and the immunoreactivity of recombinant protein with anti-CHIK IgM antibodies was ascertained. The antigen showed an 88% sensitivity and 100% specificity by Indirect ELISA. No cross reactivity of the antigen was observed with anti-Dengue virus serum samples. The results strongly support that the recombinant CHIK coat protein could be used as a diagnostic antigen for the detection of Chikungunya by Indirect ELISA. The relevance of a smaller size recombinant antigen highlights its large scale application in serodiagnosis of CHIK virus since bacterial expression is more simple and cost effective than eukaryotic system.     

固相酶联免疫测定法检测NS1抗原在登革病毒感染早期诊断中的应用

目的探讨固相酶联免疫测定（ELISA）法检测NS1抗原在登革病毒感染早期诊断中的应用价值。方法选取登革病毒感染早期患者血清171份,非登革病毒感染发热患者血清11份,正常人血清10份,采用ELISA法检测全部192份血清的登革病毒NS1抗原和IgM抗体;采用逆转录-聚合酶链反应-限制性内切酶酶切片段长度多态性分析（RT-PCR-RFLP）技术对发病5 d内的125份血清进行扩增和鉴定分型;并采用C6/36细胞微量培养法对发病第1、2天的41份血清进行登革病毒分离培养。结果登革病毒感染患者发病2 d内、3～5 d以及6～10 d血清NS1抗原的检出率分别是92.7%（38/41）、83.3%（70/84）、10.9%（5/46）;IgM抗体的检出率分别是2.4%（1/41）、51.2%（43/84）、97.8%（45/46）;非登革病毒感染的发热患者及正常人血清中,有1例疟疾患者血清登革病毒IgM抗体呈阳性,NS1抗原无一例阳性。RT-PCR在登革病毒感染患者发病第1、2天和3～5天的检出率分别是85.4%（35/41）、83.3%（70/84）;登革病毒感染患者发病第1、2天血清的病毒分离培养阳性率分别是80.0%（16/20）、38.1%（8/21）,总分离率58.5%（24/41）;RT-PCR-RFLP分型鉴定技术及间接免疫荧光法（IFA）均证实2006年广州流行株为登革Ⅰ型病毒。结论ELISA法检测登革病毒NS1抗原操作技术成熟,且具有敏感性高、特异性好的特点,对登革病毒感染的早期诊断和疫情的早期控制具有重要意义,适合于基层医疗机构常规应用。     

Crimean-Congo haemorrhagic fever presenting as epididymo-orchitis

BackgroundCrimean-Congo haemorrhagic fever (CCHF) is a potentially fatal disease caused by a tick-borne virus.ObjectivesA 53-year-old man presented with fever and acute painful scrotal swelling simulating acute epididymo-orchitis.Study designBased on the clinical and epidemiological findings, CCHF virus infection and epididymo-orchitis were suspected. This symptom, rarely reported in viral haemorrhagic fevers, was observed in this case.ResultsThe diagnosis was confirmed by detection of the IgM antibody to CCHF virus and positive RT-PCR.ConclusionWe report the first case of imported CCHF presenting as epididymo-orchitis. This symptom is a rare complication of CCHF, and the clinician should consider this entity in the differential diagnosis of adults with epididymo-orchitis.     

Detection of Zika virus in saliva

BackgroundDuring the largest Zika virus (ZIKV) outbreak ever reported that occurred from October 2013 to March 2014 in French Polynesia, we observed that several patients presenting the symptoms of acute phase Zika fever were tested negative in blood by ZIKV real-time PCR (RT-PCR).ObjectivesAs we have previously detected ZIKV RNA in the saliva of a young child, we investigated the use of saliva as an alternative sample for routine ZIKV RNA detection.Study designOver a 6 month period, 1,067 samples collected from 855 patients presenting symptoms of Zika fever (saliva only, blood only or both samples) were tested using a specific ZIKV RT-PCR. A medical questionnaire was available for most of the patients.ResultsZIKV was more frequently detected in saliva compared to blood. For the 182 patients with both samples collected, tests were positive for 35 (19.2%) in saliva while negative in blood and tests were positive for 16 (8.8%) in blood while negative in saliva; the difference in mean days after symptoms onset and the percentage of the main symptoms of Zika fever for patients only positive in saliva or in blood was not significant.ConclusionThe use of saliva sample increased the rate of molecular detection of ZIKV at the acute phase of the disease but did not enlarge the window of detection of ZIKV RNA. Saliva was of particular interest when blood was difficult to collect (children and neonates especially).