Dr. Chang works as research assistant professor in National Center for Clinical Laboratories, Beijing Hospital, focusing on the detection of transfusiontransmitted infectious pathogens. Dr. Zhao is chief of the laboratory department of Wuhan Blood Center. His primary focus is detection of transfusion-transmitted infectious pathogens. References Chinese Center for Disease Control and Prevention;The Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. Vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19)-China, 2020.

Acute febrile illness is a common problem managed by clinicians and health systems globally, particularly in the Tropics. In many regions, malaria is a leading and potentially deadly cause of fever; however, myriad alternative etiologies exist. Identifying the cause of fever allows optimal management, but this depends on many factors including thorough knowledge of circulating infections. Arboviruses such as dengue (DENV) cause fever and may be underdiagnosed in sub-Saharan Africa where malaria is a major focus. We examined cases of fever in western Cameroon that tested negative for malaria and found 13.5% (13/96) were due to DENV, with 75% (9/12) of these being DENV serotype 2 infections. Two complete DENV2 genomes were obtained and clustered closely to recent isolates from Senegal and Burkina Faso. The seroprevalence of DENV in this region was 24.8% (96/387). Neutralizing antibodies to DENV2 were detected in all (15/15) seropositive samples tested. Chikungunya (CHIKV) is an arthritogenic alphavirus that is transmitted by Aedes mosquitoes, the same principal vector as DENV. The seroprevalence for CHIKV was 15.7% (67/427); however, CHIKV did not cause a single case of fever in the 96 subjects tested. Of note, being seropositive for one arbovirus was associated with being seropositive for the other (Χ2 = 16.8, p<0.001). Taken together, these data indicate that Aedes-transmitted arboviruses are endemic in western Cameroon and are likely a common but underappreciated cause of febrile illness. This work supports the need for additional study of arboviruses in sub-Saharan Africa and efforts to improve diagnostic capacity, surveillance systems, and arbovirus prevention strategies.

Background In 2018, Paraguay experienced a large dengue virus (DENV) outbreak. The primary objective of this study was to characterize dengue cases in the Central Department, where the majority of cases occur, and identify factors associated with DENV infection. Methods Patients were enrolled from January-May 2018 if they presented with a suspected arboviral illness. Acute-phase specimens (≤8 days after symptom onset) were tested using rRT-PCR, a rapid diagnostic test for DENV nonstructural protein 1 (NS1) and anti-DENV IgM and IgG, and ELISA for IgG against NS1 from Zika virus (ZIKV). Results A total of 231 patients were enrolled (95.2% adults) at two sites: emergency care and an outpatient clinical site. Patients included 119 (51.5%) dengue cases confirmed by rRT-PCR (n = 115, 96.6%) and/or the detection of NS1 and anti-DENV IgM (n = 4, 3.4%). DENV-1 was the predominant serotype (109/115, 94.8%). Epidemiologically, dengue cases and non-dengue cases were similar, though dengue cases were less likely to reside in a house/apartment or report a previous dengue case. Clinical and laboratory findings associated with dengue included red eyes, absence of sore throat, leucopenia and thrombocytopenia. At an emergency care site, 26% of dengue cases (26/100) required hospitalization. In univariate analysis, hospitalization was associated with increased viral load, anti-DENV IgG, and thrombocytopenia. Among dengue cases that tested positive for IgG against ZIKV NS1, the odds of DENV NS1 detection in the acute phase were decreased 10-fold (OR 0.1, 0.0–0.3). Conclusions Findings from a predominantly adult population demonstrate clinical and laboratory factors associated with DENV infections and the potential severity of dengue in this group. The combination of viral load and specific IgG antibodies warrant further study as a prognostic to identify patients at risk for severe disease.

Broad testing for respiratory viruses among persons under investigation (PUIs) for SARS-CoV-2 has been performed inconsistently, limiting our understanding of alternative viral infections and co-infections in these patients. RNA metagenomic next-generation sequencing (mNGS) offers an agnostic tool for the detection of both SARS-CoV-2 and other RNA respiratory viruses in PUIs. Herein, we used RNA mNGS to assess the frequencies of alternative viral infections in SARS-CoV-2 RT-PCR negative PUIs (n=30) and viral co-infections in SARS-CoV-2 RT-PCR positive PUIs (n=45). mNGS identified all viruses detected by routine clinical testing (Influenza A (N=3), Human metapneumovirus (N=2), Human coronavirus OC43 (N=2) and Human coronavirus HKU1(N=1)). mNGS also identified both co-infections (1, 2.2%) and alternative viral infections (4, 13.3%) that were not detected by routine clinical workup (Respiratory syncytial virus (N=3), Human metapneumovirus (N=1), Human coronavirus NL63 (N=1)). Among SARS-CoV-2 RT-PCR positive PUIs, lower cycle threshold (CT) values correlated with greater SARS-CoV-2 read recovery by mNGS (R2: 0.65, p-value: <0.001). Our results suggest that current broad-spectrum molecular testing algorithms identify most respiratory viral infections among SARS-CoV-2 PUIs, when available and implemented consistently.

To provide an accessible and inexpensive method to surveil for SARS-CoV-2 mutations, we developed a multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348 bp region of spike , and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log 10 GE/mL for the three initial targets (∼1-2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with Ct values < 30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 Ct values ≥ 30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in receptor binding domain, and it can be quickly modified to detect new mutations that emerge.