Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration.

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations (hbaa+ Mac and timp4.3+ Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.

COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR.

The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume less reagents by increasing the capacity of testing and reducing the number of experiments performed, and are therefore especially suitable for under-developed countries with limited resources. Here, we propose a simple, reliable pooling strategy for COVID-19 testing using clinical nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. The strategy includes the pooling of 10 NP/OP swabs for extraction and subsequent testing via quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and may also be applied to the screening of other pathogens.

A novel strategy for community screening of SARS-CoV-2 (COVID-19): Sample pooling method.

The rapid global spread of the coronavirus disease (COVID-19) has inflicted significant health and socioeconomic burden on affected countries. As positive cases continued to rise in Malaysia, public health laboratories experienced an overwhelming demand for COVID-19 screening. The confirmation of positive cases of COVID-19 has solely been based on the detection of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) using real-time reverse transcription polymerase chain reaction (qRT-PCR). In efforts to increase the cost-effectiveness and efficiency of COVID-19 screening, we evaluated the feasibility of pooling clinical Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens during nucleic acid extraction without a reduction in sensitivity of qRT-PCR. Pools of 10 specimens were extracted and subsequently tested by qRT-PCR according to the WHO-Charité protocol. We demonstrated that the sample pooling method showed no loss of sensitivity. The effectiveness of the pooled testing strategy was evaluated on both retrospective and prospective samples, and the results showed a similar detection sensitivity compared to testing individual sample alone. This study demonstrates the feasibility of using a pooled testing strategy to increase testing capacity and conserve resources, especially when there is a high demand for disease testing.

Importance of Proactive Malaria Case Surveillance and Management in Malaysia.

Although Plasmodium vivax infections in Malaysia are usually imported, a significant autochthonous outbreak of vivax malaria was detected in a remote indigenous (Orang Asli) settlement located in northern peninsular Malaysia. Between November 2016 and April 2017, 164 cases of P. vivax infection were detected. Although 83.5% of the vivax cases were identified through passive case detection and contact screening during the first 7 weeks, subsequent mass blood screening (combination of rapid diagnostic tests, blood films, and polymerase chain reaction [PCR]) of the entire settlement (N = 3,757) revealed another 27 P. vivax infections, 19 of which were asymptomatic. The mapped data from this active case detection program was used to direct control efforts resulting in the successful control of the outbreak in this region. This report highlights the importance of proactive case surveillance and timely management of malaria control in Malaysia as it nears malaria elimination.

The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

BACKGROUND: The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. METHODS: The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. RESULTS: Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. CONCLUSION: This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.