Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow.

Since the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient's samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results' display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.

Gut Dysbiosis in Chagas Disease. A Possible Link to the Pathogenesis.

Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi. Cardiomyopathy and damage to gastrointestinal tissue are the main disease manifestations. There are data suggesting that the immune response to T. cruzi depends on the intestinal microbiota. We hypothesized that Chagas disease is associated with an altered gut microbiome and that these changes are related to the disease phenotype. The stool microbiome from 104 individuals, 73 with Chagas disease (30 with the cardiac, 11 with the digestive, and 32 with the indeterminate form), and 31 healthy controls was characterized using 16S rRNA amplification and sequencing. The QIIME (Quantitative Insights Into Microbial Ecology) platform was used to analyze the data. Alpha and beta diversity indexes did not indicate differences between the groups. However, the relative abundance of Verrucomicrobia, represented primarily by the genus Akkermansia, was significantly lower in the Chagas disease groups, especially the cardiac group, compared to the controls. Furthermore, differences in the relative abundances of Alistipes, Bilophila, and Dialister were observed between the groups. We conclude that T. cruzi infection results in changes in the gut microbiome that may play a role in the myocardial and intestinal inflammation seen in Chagas disease.

ELISA Saliva for Trypanosoma cruzi Antibody Detection: An Alternative for Serological Surveys in Endemic Regions.

Chagas is a neglected disease endemic in Latin America. Vector transmission control had been aggressively performed. Recent entomological surveillance in Brazil has revealed natural infection rates ranging from 0.40% to 0.52%. Although serological surveys are complex to develop, they are important for disease control. In this study, we validated the use of saliva in ELISA commercial kits with a cohort of 100 patients with Chagas disease followed at Hospital das Clinicas in São Paulo, Brazil, and 50 healthy controls. Five ELISA kits for detecting antibodies against Trypanosoma cruzi were tested. The best discrimination between Chagas patients and controls was observed with the Wiener kit, which yielded a sensitivity of 97% and a specificity of 100%. Our findings reveal that the use of saliva may be an alternative to large-scale screening surveys in detecting T. cruzi antibodies; it is a noninvasive sample collection method potentially key to large-scale screening in children.

Changes in the intestinal microbiota of superobese patients after bariatric surgery.

OBJECTIVES: The gut microbiota is associated with obesity and weight loss after bariatric surgery and has been related to its changing pattern. Exactly how the bacterial population affects weight loss and the results of surgery remain controversial. This study aimed to evaluate the intestinal microbiota of superobese patients before and after gastric bypass surgery (RYGB). METHOD: DNA fragments for the microbiota obtained from stool samples collected from nine superobese patients before and after bariatric surgery were sequenced using Ion Torrent. RESULTS: We observed that with a mean follow-up of 15 months, patients achieved 55.9% excess weight loss (EWL). A significant population reduction in the Proteobacteria phylum (11 to 2%, p=0.0025) was observed after surgery, while no difference was seen in Firmicutes and Bacteroidetes. Further analyses performed with two specific individuals with divergent clinical outcomes showed a change in the pattern between them, with a significant increase in Firmicutes and a decrease in Bacteroidetes in the patient with less weight loss (%EWL 50.79 vs. 61.85). CONCLUSIONS: RYGB affects the microbiota of superobese patients, with a significant reduction in Proteobacteria in patients with different weight loss, showing that different bacteria may contribute to the process.

Changes in the intestinal microbiota of superobese patients after bariatric surgery

OBJECTIVES: The gut microbiota is associated with obesity and weight loss after bariatric surgery and has been related to its changing pattern. Exactly how the bacterial population affects weight loss and the results of surgery remain controversial. This study aimed to evaluate the intestinal microbiota of superobese patients before and after gastric bypass surgery (RYGB). METHOD: DNA fragments for the microbiota obtained from stool samples collected from nine superobese patients before and after bariatric surgery were sequenced using Ion Torrent. RESULTS: We observed that with a mean follow-up of 15 months, patients achieved 55.9% excess weight loss (EWL). A significant population reduction in the Proteobacteria phylum (11 to 2%, p=0.0025) was observed after surgery, while no difference was seen in Firmicutes and Bacteroidetes. Further analyses performed with two specific individuals with divergent clinical outcomes showed a change in the pattern between them, with a significant increase in Firmicutes and a decrease in Bacteroidetes in the patient with less weight loss (%EWL 50.79 vs. 61.85). CONCLUSIONS: RYGB affects the microbiota of superobese patients, with a significant reduction in Proteobacteria in patients with different weight loss, showing that different bacteria may contribute to the process.