Zika virus (ZIKV), a flavivirus, and chikungunya virus (CHIKV), an alphavirus, are infectious RNA arboviruses transmitted to humans by the bite of Aedes species mosquitoes. Both viruses have only recently emerged in the Western Hemisphere (1, 2), and along with dengue virus (DENV), another flavivirus, now circulate widely in Brazil. The acute illness caused by these viruses, characterized by fever, rash, myalgia, arthralgia, and conjunctivitis, is nonspecific, and differential diagnosis on the basis of clinical findings alone is challenging. Later infectious sequelae include chronic arthritis for CHIKV (2) and encephalitis, immune-mediated syndromes, and stroke for DENV (3). Recently, the association between ZIKV infection and severe fetal complications such as microcephaly in pregnant women has been established (4), and the virus has also been linked to neurological complications such as Guillain-Barré syndrome (5). Thus, broadbased assays are needed for differential diagnosis of vectorborne febrile illnesses and to identify potential coinfections. Here we report the utility of metagenomic next-generation sequencing (mNGS) as a screening tool to identify coinfections and the use of genome recovery and phylogenetic analyses directly from patient serum samples in the context of the ongoing ZIKV outbreak. We also show that the clinical presentation of arboviral coinfections seems to favor the virus present at a higher titer in acutely infected individuals. MATERIALS AND METHODSZIKV serum sample collection, ZIKV RT-PCR, and DENV antibody testing. Written consent from patients was obtained under a study protocol approved by the Brazil Ministry of Health (Certificado de Apresentação para Apreciação Ética 45483115.0.0000.0046, no. 1159.184, Brazil). Serum samples were obtained from 15 patients seen at Aliança Hospital in Salvador, Bahia, Brazil, from April 2015 to January 2016 who were given a presumptive diagnosis of an acute viral illness by emergency department physicians and were found to be positive by qualitative reverse transcription-PCR (RT-PCR) testing for ZIKV. Serum samples Bahia01 to Bahia15 were subjected to RNA extraction using the QIAamp viral RNA minikit (Qiagen), and RNA was reverse transcribed using the Superscript II reverse transcriptase kit (Invitrogen), followed by qualitative RT-PCR testing for ZIKV using primers targeting the NS5 gene (6). Serum samples were also tested for DENV infection using an enzyme-linked immunosorbent assay (ELISA) specific for the NS1 antigen and anti-DENV IgG/IgM according to the manufacturer's instructions (Dengue Duo Test; Bioeasy Diagnostica, Brazil).Metagenomic next-generation sequencing. A separate serum aliquot was extracted for total nucleic acid using the Qiagen viral RNA minikit (Qiagen), followed by DNase treatment using a cocktail of Turbo DNase (Thermo Fisher Scientific) and Baseline-Zero DNase (Epicentre Biotechnologies), followed by NGS library construction using the NexteraXT kit (Illumina) as previously described (7,8). Runs of single-end, 160-base pair...
Background: There is growing concern about individuals reported to suffer repeat COVID-19 disease episodes, these in a small number of cases characterised as de novo infections with distinct sequences, indicative of insufficient protective immunity even in the short term. Methods: Observational case series and case-control studies reporting 33 cases of recurrent, symptomatic, qRT-PCR positive COVID-19. Recurrent disease was defined as symptomatic recurrence after symptom-free clinical recovery, with release from isolation > 14 days from the beginning of symptoms confirmed by qRT-PCR. The case control study-design compared this group of patients with a control group of 62 patients randomly selected from the same COVID-19 database. Results: Of 33 recurrent COVID-19 patients, 26 were female and 30 were HCW. Mean time to recurrence was 50.5 days which was associated with being a HCW (OR 36.4 (p < 0.0 0 01)), and blood type A (OR 4.8 (p = 0.002)). SARS-CoV-2 antibodies were signifcantly lower in recurrent patients after initial COVID-19 (2.4 ± 0.610; p < 0.0 0 01) and after recurrence (6.4 ± 11.34; p = 0.007). Virus genome sequencing identified reinfection by a different isolate in one patient. Conclusions: This is the first detailed case series showing COVID-19 recurrence with qRT-PCR positivity. For one individual detection of phylogenetically distinct genomic sequences in the first and second episodes confirmed bona fide renfection, but in most cases the data do not formally distinguish between reinfection and re-emergence of a chronic infection reservoir. These episodes were significantly associated with reduced Ab response during initial disease and argue the need for ongoing vigilance without an assumption of protection after a first episode.
BackgroundSerologic detection of Zika virus (ZIKV) infections is challenging because of antigenic similarities among flaviviruses.ObjectiveTo evaluate the sensitivity and specificity of commercial ZIKV IgM and IgG enzyme-linked immunoassay (ELISA) kits.MethodsWe used sera from febrile patients with RT-PCR-confirmed ZIKV infection to determine sensitivity and sera from RT-PCR-confirmed dengue cases and blood donors, both of which were collected before ZIKV epidemics in Brazil (2009–2011 and 2013, respectively) to determine specificity.ResultsThe ZIKV IgM-ELISA positivity among RT-PCR ZIKV confirmed cases was 0.0% (0/14) and 12.5% (1/8) for acute- and convalescent-phase sera, respectively, while its specificity was 100.0% (58/58) and 98.3% (58/59) for acute- and convalescent-phase sera of dengue patients, and 100.0% (23/23) for blood donors. The ZIKV IgG-ELISA sensitivity was 100.0% (6/6) on convalescent-phase sera from RT-PCR confirmed ZIKV patients, while its specificity was 27.3% (15/55) on convalescent-phase sera from dengue patients and 45.0% (9/20) on blood donors’ sera. The ZIKV IgG-ELISA specificity among dengue confirmed cases was much greater among patients with primary dengue (92.3%; 12/13), compared to secondary dengue (7.1%; 3/42).ConclusionsIn a setting of endemic dengue transmission, the ZIKV IgM-ELISA had high specificity, but poor sensitivity. In contrast, the ZIKV IgG-ELISA showed low specificity, particularly for patients previously exposed to dengue infections. This suggests that this ZIKV IgM-ELISA is not useful in confirming a diagnosis of ZIKV infection in suspected patients, whereas the IgG-ELISA is more suitable for ZIKV diagnosis among travelers, who reside in areas free of flavivirus transmission, rather than for serosurveys in dengue-endemic areas.
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