Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever.

Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

Discriminating Tonate Virus from Dengue Virus Infection: A Matched Case-Control Study in French Guiana, 2003-2016.

Tonate virus (TONV) is an arbovirus discovered in 1973 in French Guiana (FG) belonging to the Venezuelan equine encephalitis virus complex, Alphavirus genus. Only few publications and cases have been reported in FG. The objectives of the present study were to describe the clinical picture of TONV and to compare its presentation with that of dengue virus (DENV). A retrospective study was performed in Cayenne hospital from 2003 to 2016 including all patients exclusively positive for TONV IgM and not for other alphaviruses. They were classified as high probability: typical clinical picture of arbovirus infection (i.e., fever, chills, headaches, muscle, and joint pains) and IgM seroconversion; medium probability: typical clinical picture + single positive IgM on a unique serum sample without control; and low probability: atypical clinical picture of infection and single positive IgM. Only patients with high and medium probability were included in the analysis and compared with a gender- and age-matched control group of DENV diagnosed by NS1 antigen (two controls per case). During the study period, 45 cases of TONV were included and compared with 90 cases of DENV. Twenty-eight (62.2%) were men; the median age was 34 years (IQ [22-49]). In the bivariate analysis, variables significantly associated with TONV versus DENV were the presence of cough (33.3% versus 10.3%) and anemia (32.5% versus 11.1%) and the absence of nausea (4.4% versus 32.2%), rash (2.2% versus 27.4%), fatigue (17.8% versus 41.0%), anorexia (6.7% versus 30.1%), muscle pain (42.2% versus 61.4%), headache (53.3% versus 70.8%), leukopenia (9.8% versus 44.4), and lymphopenia (42.5% versus 89.9%). There were no cases with severe neurological involvement, and there were no deaths. Tonate virus may be evoked as a cause of fever in patients living or returning from the Amazonian area. Positive TONV IgM does not prove the diagnosis and should not preclude from searching for alternative infectious diagnoses.

Kunjin flaviviral encephalomyelitis in an Arabian gelding in New South Wales, Australia.

Flaviviruses, including Kunjin virus, are arboviruses that cause encephalomyelitis in humans and horses. This case report describes an Arabian gelding exhibiting neurological signs of flavivirus encephalomyelitis, the diagnostic investigation and confirmation of an unreported case of Kunjin virus equine encephalomyelitis in Australia.

[Insect-borne diseases and insect bites in Finland]. / Hyönteisten levittämät taudit ja puremat Suomessa.

Clinically significant endemic, arthropod-borne infectious diseases in Finland include Lyme disease, tick-borne encephalitis, tularemia and Karelian fever. The diagnosis of early borreliosis or tularemia is clinical and the treatment is initiated already before the results of eventual blood tests. The diagnosis of late stage borreliosis, tick-borne encephalitis or Karelian fever is confirmed mainly with serologic tests. The clinical significance of endemic anaplasmosis, Rickettsia helvetica, Inkoo and Uukuniemi viruses as well as anthropod-borne bunyaviruses and bartonelloses is either minor or remains open.

Field evaluation of clinical features during chikungunya outbreak in Mayotte, 2005-2006.

BACKGROUND: To record and assess the clinical features of chikungunya fever (CHIKF), with a view to enable diagnosis based on clinical criteria rather than costly laboratory procedures in field conditions. METHODS: As part of a cross-sectional serologic survey conducted in Mayotte after a massive chikungunya outbreak in 2006, we collected data on clinical features of chikungunya infection and assessed the performance and accuracy of clinical case definition criteria combining different symptoms. RESULTS: Of 1154 participants included, 440 (38.1%) had chikungunya-specific IgM or IgG antibodies by enzyme-linked immunosorbent assay (ELISA). Of symptomatic participants, 318 (72.3%) had confirmed chikungunya, the dominant symptoms reported were incapacitating polyarthralgia (98.7%), myalgia (93.1%), backache (86%), fever of abrupt onset (85%) and headache (81.4%). There was a strong linear association between symptomatic infection and age (chi(2) for trend = 9.85, P < 0.001). Only 52% of persons with presumptive chikungunya sought medical advice, principally at public primary health care facilities. The association of fever and polyarthralgia had a sensitivity of 84% (95% CI: 79-87) and a specificity of 89% (95% CI: 86-91). This association allowed to classify correctly 87% (95% CI: 85-89) of individuals with serologically confirmed chikungunya. CONCLUSIONS: Our results suggest that the pair fever and incapacitating polyarthralgia is an accurate and reliable tool for identifying presumptive CHIKF cases in the field. These criteria provide a useful evidence base to support operational syndromic surveillance in laboratory-confirmed chikungunya epidemic settings.

IL-1beta, IL-6, and RANTES as biomarkers of Chikungunya severity.

BACKGROUND: Little is known about the immunopathogenesis of Chikungunya virus. Circulating levels of immune mediators and growth factors were analyzed from patients infected during the first Singaporean Chikungunya fever outbreak in early 2008 to establish biomarkers associated with infection and/or disease severity. METHODS AND FINDINGS: Adult patients with laboratory-confirmed Chikungunya fever infection, who were referred to the Communicable Disease Centre/Tan Tock Seng Hospital during the period from January to February 2008, were included in this retrospective study. Plasma fractions were analyzed using a multiplex-microbead immunoassay. Among the patients, the most common clinical features were fever (100%), arthralgia (90%), rash (50%) and conjunctivitis (40%). Profiles of 30 cytokines, chemokines, and growth factors were able to discriminate the clinical forms of Chikungunya from healthy controls, with patients classified as non-severe and severe disease. Levels of 8 plasma cytokines and 4 growth factors were significantly elevated. Statistical analysis showed that an increase in IL-1beta, IL-6 and a decrease in RANTES were associated with disease severity. CONCLUSIONS: This is the first comprehensive report on the production of cytokines, chemokines, and growth factors during acute Chikungunya virus infection. Using these biomarkers, we were able to distinguish between mild disease and more severe forms of Chikungunya fever, thus enabling the identification of patients with poor prognosis and monitoring of the disease.

Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections.

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of >/=2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

Historical, epidemiological and ecological aspects of arboviruses in Argentina: Togaviridae, Alphavirus

This is a review of the arboviruses in Argentina belonging to families Flaviridae, Bunyaviridae and Rhabdoviridae. Of the many viruses belonging to these families, the flavivirus St. Louis encephalitis (SLE), has been most intensively studied. SLE virus strains have been recovered from three sources: 2 strains from humans with an undifferentiated, febrile disease; 6 from mosquitoes; and 2 from rodents. The viruses recovered from rodents are attenuated and those from mosquitoes are virulent based on a neuroinvasiveness test in mice; the degree of virulence of the mosquito strains remain to be analyzed. Serological surveys indicate a wide distribution and endemicity of SLE virus in the temperate and subtropical areas (central and northern Argentina), but no data are available from the andean region or from the South. The virulent SLE virus strains appear to be transmitted between Culex (Cx.)spp. from which they were isolated, and wild birds, based on antobody prevalence. A urban cycle may involve Cx. quinquefasciatus (source of a viral isolate and a competent experimental vector) and abundant birds (house sparrows, doves, and/or chickens), chickens are experimentally competent host species. Despite similarities in the ecology of SLE between Argentina and North America, urban outbreaks of SLE have not been recognized. Possible explanations for this discrepancy include virus strain differences in virulenc, ecologic factors determining the rate of virus transmission, and the lack of disease recognition and specific laboratory diagnosis of human meningoencephalitis. The transmission cycle of attenuated SLE virus strains isolated from rodentshas not been studied. Ilheus virus has isolated only once from a human being. The available serological data are difficult to interpret due to cross-reactivity with other flaviviruses, and the ecologyand medical importance of this agent remain uncertain. Dengue has not been recognized in Argentina since 1916, although is vector, Aedes aegypti, was not erradicated until 1963. Dengue was previously present in coastal localities of Chaco. Corrientes and Misiones Provinces. Within the last few years, Argentina was reinfested by Ae. aegypti. Although no human cases have vet been reported, outbreaks of dengue in bordering countries (Brazil, Paraguay, Bolivia) since 1986, cleary signal that the country in once againat risk of importantion ans spread of the virus

An uncommon presentation for a common problem.

One of the axioms of general practice is that 'common things present commonly', both in the community incidence of disease and in the symptoms with which illnesses usually present. This was somewhat enigmatically put by a renowned neurologist at the Mayo Clinic: "When you hear hoof-beats, why think of zebras?". A most difficult, yet important feat for the GP is to keep on the alert for the uncommon symptom of a common cause, and the common symptom with an uncommon cause.

[Development and use of an immunoenzyme technique for the routine diagnosis of the porcine reproductive and respiratory syndrome]. / Développement et utilisation d'une épreuve immuno-enzymatique pour le diagnostic courant du syndrome dysgénésique et respiratoire du porc.

An indirect enzyme-linked immunosorbent assay was developed for rapid detection of serum antibodies against the virus responsible for the porcine reproductive and respiratory syndrome (PRRS). This test is more sensitive than the reference technique (immunoperoxidase test applied to cultures of alveolar macrophages), particularly for detecting animals at the stage of seroconversion. It is also very specific for PRRS virus, because all specific hyperimmune sera against other porcine viruses, and all serum samples taken from herds before the disease appeared in western France were negative. The test has been used for routine diagnosis of PRRS. The results obtained during nine months from over 21,000 samples have confirmed the value of the test for diagnosis and epidemiological surveillance of the disease.

Evaluation of vero cell lysate antigen for the ELISA of flaviviruses.

The vero cell lysate antigen for the enzyme-linked immunosorbent assay (ELISA) of flaviviruses was evaluated for sensitivity, specificity including cross-reactions, and background by comparing with the standard ELISA. Human sera, in serial dilutions, were taken from subjects 14, 35, and 210 days postvaccination with 17D antigen. Early after injection, high sensitivity (82.9%) was shown by the cell lysate antigen method. Late after infection, high sensitivity was achieved by the standard method (96.2% and 94%), with significant difference (P = 0.0001). However, sensitivity achieved by the cell lysate antigen method was also acceptable (91.7% & 88.9%). The cell lysate antigen method showed high specificity and low cross reactivity early after infection. At 35 days postvaccination, no difference in specificity was observed between the two methods, but higher cross-reactions were observed for the standard method. This pattern continued at 210 days postvaccination, with significantly higher cross-reactions with the standard ELISA. The optical density differences by the two methods did not show significant relationship with the serial dilutions of human sera. No difference was observed in early and late infections in the background values of the negative control (Western equine encephalitis) between the two methods. The ELISA by the cell lysate antigen, within the limits of the experiments done, was found to be a good replacement for the ELISA by the standard method.

Isolation of Getah virus from mosquitos collected on Hainan Island, China, and results of a serosurvey.

An isolate of Getah virus was obtained from Culex mosquitos collected in Mao'an Village, Baoting County, Hainan Province, China, in 1964. The virus (strain M-1) replicated in laboratory-bred Aedes aegypti and Cx. fatigans (= quinquefasciatus), and was transmitted by laboratory-bred Ae. albopictus to healthy newborn albino mice. Skeletal muscles of newborn albino mice experimentally infected with the virus showed degeneration, atrophy, necrosis, and inflammatory changes of muscle fibers. Antibody prevalence in humans and animals ranged from 10.3% by neutralization tests of samples from healthy people in 1979 to 26.4% by CF tests of samples from people with febrile illnesses in 1982. The high prevalence of antibody in pigs, horses, and goats (17.6% to 37.5%) indicated that infection with Getah or a closely related virus is relatively common in domestic animals.

Rapid affinity-purification and biotinylation of antibodies.

A simple and rapid method to affinity-purify and biotinylate antibodies was developed. The method utilizes separation of antigens by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose and binding of the antibodies to the specific antigen. The antibodies are biotinylated, while still bound to the antigen, thus avoiding the conjugation of the active antigen-binding sites of the antibodies. These antibodies have been successfully used in double-label immunofluorescence studies, but they should be likewise applicable in other immunological protocols.