Automated sample-to-answer system for rapid and accurate diagnosis of emerging infectious diseases

Automated sample-to-answer systems that promptly diagnose emerging infectious diseases, such as zoonotic diseases, are crucial to preventing the spread of infectious diseases and future global pandemics. However, automated, rapid, and sensitive diagnostic testing without professionals and sample capacity and type limitations remains unmet needs. Here, we developed an automated sample-to-answer diagnostic system for rapid and accurate detection of emerging infectious diseases from clinical specimens. This integrated system consists of a microfluidic platform for sample preparation and a bio-optical sensor for nucleic acid (NA) amplification/detection. The microfluidic platform concentrates pathogens and NAs in a large sample volume using adipic acid dihydrazide and a low-cost disposable chip. The bio-optical sensor allows label-free, isothermal one-step NA amplification/detection using a ball-lensed optical fiber-based silicon micro-ring resonator sensor. The system is integrated with software to automate testing and perform analysis rapidly and simply;it can distinguish infection status within 80 min. The detection limit of the system (0.96 × 101 PFU) is 10 times more sensitive than conventional methods (0.96 × 102 PFU). Furthermore, we validated the clinical utility of this automated system in various clinical specimens from emerging infectious diseases, including 20 plasma samples for Q fever and 13 (11 nasopharyngeal swabs and 2 saliva) samples for COVID-19. The system showed 100% sensitivity and specificity for detecting 33 samples of emerging infectious diseases, such as Q fever, other febrile diseases, COVID-19, human coronavirus OC43, influenza A, and respiratory syncytial virus A. Therefore, we envision that this automated sample-to-answer diagnostic system will show high potential for diagnosing emerging infectious diseases in various clinical applications. © 2023 Elsevier B.V.

Automated sample-to-answer system for rapid and accurate diagnosis of emerging infectious diseases

Automated sample-to-answer systems that promptly diagnose emerging infectious diseases, such as zoonotic diseases, are crucial to preventing the spread of infectious diseases and future global pandemics. However, automated, rapid, and sensitive diagnostic testing without professionals and sample capacity and type limitations remains unmet needs. Here, we developed an automated sample-to-answer diagnostic system for rapid and accurate detection of emerging infectious diseases from clinical specimens. This integrated system consists of a microfluidic platform for sample preparation and a bio-optical sensor for nucleic acid (NA) amplification/detection. The microfluidic platform concentrates pathogens and NAs in a large sample volume using adipic acid dihydrazide and a low-cost disposable chip. The bio-optical sensor allows label-free, isothermal one-step NA amplification/detection using a ball-lensed optical fiber-based silicon micro-ring resonator sensor. The system is integrated with software to automate testing and perform analysis rapidly and simply;it can distinguish infection status within 80min. The detection limit of the system (0.96 × 101 PFU) is 10 times more sensitive than conventional methods (0.96 × 102 PFU). Furthermore, we validated the clinical utility of this automated system in various clinical specimens from emerging infectious diseases, including 20 plasma samples for Q fever and 13 (11 nasopharyngeal swabs and 2 saliva) samples for COVID-19. The system showed 100% sensitivity and specificity for detecting 33 samples of emerging infectious diseases, such as Q fever, other febrile diseases, COVID-19, human coronavirus OC43, influenza A, and respiratory syncytial virus A. Therefore, we envision that this automated sample-to-answer diagnostic system will show high potential for diagnosing emerging infectious diseases in various clinical applications.

From Clinical Specimen to Whole Genome Sequencing of A(H3N2) Influenza Viruses: A Fast and Reliable High-Throughput Protocol.

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

Saliva specimen complements anal swab in assessing patients with COVID-19 for discharge from hospital.

Since December 2019, coronavirus disease 2019 (COVID-19) caused by SARS coronavirus 2 (SARS-CoV-2) has spread and threatens public health worldwide. The recurrence of SARS-CoV-2 RNA detection in patients after discharge from hospital signals a risk of transmission from such patients to the community and challenges the current discharge criteria of COVID-19 patients. A wide range of clinical specimens has been used to detect SARS-CoV-2. However, to date, a consensus has not been reached regarding the most appropriate specimens to use for viral RNA detection in assessing COVID-19 patients for discharge. An anal swab sample was proposed as the standard because of prolonged viral detection. In this retrospective longitudinal study of viral RNA detection in 60 confirmed COVID-19 patients, we used saliva, oropharyngeal/nasopharyngeal swab (O/N swab) and anal swab procedures from admission to discharge. The conversion times of saliva and anal swab were longer than that of O/N swab. The conversion time of hyper sensitive-CRP was the shortest and correlated with that of CT scanning and viral detection. Some patients were found to be RNA-positive in saliva while RNA-negative in anal swab while the reverse was true in some other patients, which indicated that false negatives were inevitable if only the anal swab is used for evaluating suitability for discharge. These results indicated that double-checking for viral RNA using multiple and diverse specimens was essential, and saliva could be a candidate to supplement anal swabs to reduce false-negative results and facilitate pandemic control.

Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis.

Testing is one of the commendable measures for curbing the spread of coronavirus disease (COVID-19). But, it should be done using the most appropriate specimen and an accurate diagnostic test such as real-time reverse transcription-polymerase chain reaction (qRT-PCR). Therefore, a systematic review was conducted to determine the positive detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different clinical specimens using qRT-PCR. A total of 8136 pooled clinical specimens were analyzed to detect SARS-CoV-2, the majority were nasopharyngeal swabs (69.6%). A lower respiratory tract (LRT) specimens had a positive rate (PR) of 71.3% (95% confidence interval [CI]: 60.3%-82.3%) while no virus was detected in the urinogenital specimens. Bronchoalveolar lavage fluid (BLF) specimen had the PR of 91.8% (95% CI: 79.9%-103.7%), followed by rectal swabs; 87.8% (95% CI: 78.6%-96.9%) then sputum; 68.1% (95% CI: 56.9%-79.4%). A low PR was observed in oropharyngeal swabs; 7.6% (95% CI: 5.7%-9.6%) and blood samples; 1.0% (95% CI: -0.1%-2.1%) whereas no SARS-CoV-2 was detected in urine samples. Feces had a PR of 32.8% (95% CI:1 5.8%-49.8%). Nasopharyngeal swab, a widely used specimen had a PR of 45.5% (95% CI: 31.2%-59.7%). In this study, SARS-CoV-2 was highly detected in LRT specimens while no virus was detected in urinogenital specimens. BLF had the highest PR followed by rectal swab then sputum. Nasopharyngeal swab which is widely used had moderate PR. Low PR was recorded in oropharyngeal swab and blood samples while no virus was found in urine samples. Last, the virus was detected in feces, suggesting SARS-CoV-2 transmission by the fecal route.

Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens.

COVID-19 pandemic caused by SARS-CoV-2 infection continue to cause the morbidity and mortality in many countries. Limitations of the gold standard qRT-PCR for diagnosis of this infection includes need for expensive equipment, specialized molecular laboratory, and experienced staff. Currently, CRISPR-based diagnostic method was approved by the U.S. FDA for rapid detection. Several studies developed SARS-CoV-2 detection based on CRISPR-Cas12a platform; however, the validations with RNA extracted from clinical specimens were limited. Therefore, this study evaluated the clinical performance of previously described CRISPR-Cas12a based diagnostic assays for SARS-CoV-2. According to the results, the CRISPR-Cas12a assays on N1 and S genes provided diagnostic accuracy (≥ 95 %) comparable to the qRT-PCR results. The assays with E, N2 and S genes yielded acceptable sensitivity of detection (≥ 95 %) whereas N1 and S genes provided outstanding specificity of detection (100 %). Preferably, multiple target genes should be detected by using CRISPR-Cas12a to ensure the most effective SARS-CoV-2 detection. Therefore, the N1 and S genes would be attractive target genes for SARS-CoV-2 detection based on CRISPR-Cas12a.