Amplicon Deep Sequencing Reveals Multiple Genetic Events Lead to Treatment Failure with Atovaquone-Proguanil in Plasmodium falciparum.

Atovaquone-proguanil (AP) is used as treatment for uncomplicated malaria, and as a chemoprophylactic agent against Plasmodium falciparum. Imported malaria remains one of the top causes of fever in Canadian returning travelers. Twelve sequential whole-blood samples before and after AP treatment failure were obtained from a patient diagnosed with P. falciparum malaria upon their return from Uganda and Sudan. Ultradeep sequencing was performed on the cytb, dhfr, and dhps markers of treatment resistance before and during the episode of recrudescence. Haplotyping profiles were generated using three different approaches: msp2-3D7 agarose and capillary electrophoresis, and cpmp using amplicon deep sequencing (ADS). A complexity of infection (COI) analysis was conducted. De novo cytb Y268C mutants strains were observed during an episode of recrudescence 17 days and 16 h after the initial malaria diagnosis and AP treatment initiation. No Y268C mutant reads were observed in any of the samples prior to the recrudescence. SNPs in the dhfr and dhps genes were observed upon initial presentation. The haplotyping profiles suggest multiple clones mutating under AP selection pressure (COI > 3). Significant differences in COI were observed by capillary electrophoresis and ADS compared to the agarose gel results. ADS using cpmp revealed the lowest haplotype variation across the longitudinal analysis. Our findings highlight the value of ultra-deep sequencing methods in the understanding of P. falciparum haplotype infection dynamics. Longitudinal samples should be analyzed in genotyping studies to increase the analytical sensitivity.

Ultrasensitive Reverse Transcriptase Loop-Mediated Isothermal Amplification (US-LAMP)-Based Detection of Malaria Infection from Dried Blood Spots.

Submicroscopic malaria diagnosis requires highly sensitive tools instead of the conventional microscopy and rapid diagnostic tests (RDTs). While polymerase chain reaction (PCR) is more sensitive than RDTs and microscopy, the required capital cost and technical expertise hinder implementation of PCR in low- and middle-income countries. This chapter describes an ultrasensitive reverse transcriptase loop-mediated isothermal amplification (US-LAMP) test for malaria with a high sensitivity and specificity, while also being practical to implement in low-complexity laboratory settings. The workflow combines a silica spin column-based total nucleic extraction from dried blood spots (DBS) with US-LAMP amplifying the Plasmodium (Pan-LAMP) target and subsequent identification Plasmodium falciparum (Pf-LAMP).

Correlation of Clinical Severity With Stool Microbiome Changes in Clostridioides difficile Infection.

CONTEXT.­: Clostridioides difficile infection (CDI) is the world-leading cause of infectious nosocomial diarrhea and pseudomembranous colitis. Antibiotics are the first line of treatment against CDI despite the high likelihood of antibiotic failure and/or recurrence. More data are needed to correlate clinical variables with 16S rRNA microbiome profiles in CDI-infected patients. OBJECTIVE.­: To determine the relationship(s) between a patient's clinical factors and the stool bacteriome of CDI-positive patients and CDI-negative patients with diarrheal symptoms. DESIGN.­: This study used stool samples and clinical data from 358 patients with nosocomial diarrhea, who were divided by their CDI diagnosis (CDI-negative: n = 180; CDI-positive; n = 178). The stool bacteriome was profiled by amplicon deep sequencing of the 16S rRNA gene, followed by correlating clinical data. RESULTS.­: The stool bacteriome was significantly different by severity assessment regardless of CDI status. Phyla and species varied significantly by CDI diagnosis. Severity, defined as a serum white blood cell count greater than 15 cells/µL and/or a creatinine level greater than 1.5 mg/dL, correlated significantly with dysbiosis of the stool bacteriome profile of CDI-positive patients compared to CDI-negative patients. Serum white blood cell count was significantly higher in patients with bacterial dysbiosis, and high levels of creatinine were associated with low bacteriome diversity. CONCLUSIONS.­: Clinical severity of CDI influences the stool microbiome of infected patients. To date, this study has the largest data set comparing 16S rRNA microbiome profiles and clinical variables between CDI-infected and noninfected individuals.

The Role of Subgenomic RNA in Discordant Results From Reverse Transcription-Polymerase Chain Reaction Tests for COVID-19.

CONTEXT.­: Reverse transcription-polymerase chain reaction (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when 1 RT-PCR target is positive for SARS-CoV-2 and 1 RT-PCR target is negative for SARS-CoV-2 within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. OBJECTIVE.­: To understand the role of subgenomic RNA transcripts in discordant results from RT-PCR tests for COVID-19. DESIGN.­: A panel of 6 droplet digital PCR assays was designed to quantify the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity. Eleven clinical nasopharyngeal swabs were also tested with this panel. RESULTS.­: In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. The same trends in the relative abundance of copies across different targets observed in infected cells were observed in clinical samples, although trends were more pronounced in infected cells. CONCLUSIONS.­: This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by these data.

A rapid near-patient detection system for SARS-CoV-2 using saliva.

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.