inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Clonal serotype 1c multidrug-resistant Shigella flexneri detected in multiple institutions by sentinel-site sequencing.

Shigella flexneri is a major diarrhoeal pathogen, and the emergence of multidrug-resistant S. flexneri is of public health concern. We report the detection of a clonal cluster of multidrug-resistant serotype 1c (7a) S. flexneri in Singapore in April 2022. Long-read whole-genome sequence analysis found five S. flexneri isolates to be clonal and harboring the extended-spectrum ß-lactamases bla CTX-M-15 and bla TEM-1. The isolates were phenotypically resistant to ceftriaxone and had intermediate susceptibility to ciprofloxacin. The S. flexneri clonal cluster was first detected in a tertiary hospital diagnostic laboratory (sentinel-site), to which the S. flexneri isolates were sent from other hospitals for routine serogrouping. Long-read whole-genome sequence analysis was performed in the sentinel-site near real-time in view of the unusually high number of S. flexneri isolates received within a short time frame. This study demonstrates that near real-time sentinel-site sequence-based surveillance of convenience samples can detect possible clonal outbreak clusters and may provide alerts useful for public health mitigations at the earliest possible opportunity.

Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

Experience of a Tertiary Hospital in Singapore with Management of a Dual Outbreak of COVID-19 and Dengue.

During the COVID-19 pandemic, distinguishing dengue from cases of COVID-19 in endemic areas can be difficult. In a tertiary hospital contending with COVID-19 during a dengue epidemic, a triage strategy of routine COVID-19 testing for febrile patients with viral prodromes was used. All febrile patients with viral prodromes and no epidemiologic risk for COVID-19 were first admitted to a designated ward for COVID-19 testing, where enhanced personal protective equipment was used by healthcare workers until COVID-19 was ruled out. From January to May 2020, 11,086 admissions were screened for COVID-19; 868 cases of COVID-19 were diagnosed in our institution, along with 380 cases of dengue. Only 8.5% (943/11,086) of suspected COVID-19 cases were concurrently tested for dengue serology due to a compatible overlapping clinical syndrome, and dengue was established as an alternative diagnosis in 2% (207/10,218) of suspected COVID-19 cases that tested negative. There were eight COVID-19 cases with likely false-positive dengue serology and one probable COVID-19/dengue coinfection. From April to May 2020, 251 admissions presenting as viral prodromes with no respiratory symptoms were screened; of those, 15 cases had COVID-19, and 2/15 had false-positive dengue IgM. Epidemiology investigations showed no healthcare-associated transmission. In a dengue epidemic season coinciding with a COVID-19 pandemic, dengue was established as an alternative diagnosis in a minority of COVID-19 suspects, likely due to early availability of basic diagnostics. Routine screening of patients with viral prodromes during a dual outbreak of COVID-19 and dengue enabled containment of COVID-19 cases masquerading as dengue with false-positive IgM.

Molecular determinants of plaque size as an indicator of dengue virus attenuation.

The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines.