Malaria Detection Accelerated: Combing a High-Throughput NanoZoomer Platform with a ParasiteMacro Algorithm.

Eradication of malaria, a mosquito-borne parasitic disease that hijacks human red blood cells, is a global priority. Microscopy remains the gold standard hallmark for diagnosis and estimation of parasitemia for malaria, to date. However, this approach is time-consuming and requires much expertise especially in malaria-endemic countries or in areas with low-density malaria infection. Thus, there is a need for accurate malaria diagnosis/parasitemia estimation with standardized, fast, and more reliable methods. To this end, we performed a proof-of-concept study using the automated imaging (NanoZoomer) platform to detect the malarial parasite in infected blood. The approach can be used as a steppingstone for malaria diagnosis and parasitemia estimation. Additionally, we created an algorithm (ParasiteMacro) compatible with free online imaging software (ImageJ) that can be used with low magnification objectives (e.g., 5×, 10×, and 20×) both in the NanoZoomer and routine microscope. The novel approach to estimate malarial parasitemia based on modern technologies compared to manual light microscopy demonstrated 100% sensitivity, 87% specificity, a 100% negative predictive value (NPV) and a 93% positive predictive value (PPV). The manual and automated malaria counts showed a good Pearson correlation for low- (R2 = 0.9377, r = 0.9683 and p < 0.0001) as well as high- parasitemia (R2 = 0.8170, r = 0.9044 and p < 0.0001) with low estimation errors. Our robust strategy that identifies and quantifies malaria can play a pivotal role in disease control strategies.

Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of Bacillus subtilis spores.

The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca2+-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs' spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores' rapid phase darkening, which is considered as due to rapid Ca2+DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40-80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.

Heat Activation and Inactivation of Bacterial Spores: Is There an Overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has the potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination and then inactivating the less-heat-resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single-spore levels. The heat treatments used were 40 to 80°C and for 0 to 300 min. The results were as follows. (i) Heat activation at 40 to 70°C promoted l-valine- and l-asparagine-glucose-fructose-potassium (AGFK)-induced germination in a time-dependent manner. (ii) The optimal heat activation temperatures for AGFK and l-valine germination via the GerB plus GerK or GerA germinant receptors were 65°C and 50 to 65°C, respectively. (iii) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. (iv) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. (v) For the spores that should withstand adverse environmental temperatures in nature, heat activation seemed functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. IMPORTANCE Bacterial spores are thermal-stress-resistant structures that can thus survive food preservation strategies and revive through the process of spore germination. The more heat resistant spores are, the more heterogeneous their germination upon the addition of germinants. Upon germination, spores can cause food spoilage and food intoxication. Here, we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Quantitative Insights Into ß-Lactamase Inhibitor's Contribution in the Treatment of Carbapenemase-Producing Organisms With ß-Lactams.

Objectives: Carbapenemase-producing organisms (CPOs) are associated with high mortality rates. The recent development of ß-lactamase inhibitors (BLIs) has made it possible to control CPO infections safely and effectively with ß-lactams (BLs). This study aims to explicate the quantitative relationship between BLI's ß-lactamase inhibition and CPO's BL susceptibility restoration, thereby providing the infectious disease society practical scientific grounds for regulating the use of BL/BLI in CPO infection treatment. Methods: A diverse collection of human CPO infection isolates was challenged by three structurally representative BLIs available in the clinic. The resultant ß-lactamase inhibition, BL susceptibility restoration, and their correlation were followed quantitatively for each isolate by coupling FIBA (fluorescence identification of ß-lactamase activity) and BL antibiotic susceptibility testing. Results: The ß-lactamase inhibition and BL susceptibility restoration are positively correlated among CPOs under the treatment of BLIs. Both of them are dependent on the target CPO's carbapenemase molecular identity. Of note, without sufficient ß-lactamase inhibition, CPO's BL susceptibility restoration is universally low across all tested carbapenemase molecular groups. However, a high degree of ß-lactamase inhibition would not necessarily lead to a substantial BL susceptibility restoration in CPO probably due to the existence of non-ß-lactamase BL resistance mechanisms. Conclusion: BL/BLI choice and dosing should be guided by quantitative tools that can evaluate the inhibition across the entire ß-lactamase background of the CPO upon the BLI administion. Furthermore, rapid molecular diagnostics for BL/BLI resistances, especially those sensitive to ß-lactamase independent BL resistance mechanisms, should be exploited to prevent ineffective BL/BLI treatment.