Development and field validation of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for the rapid detection of chikungunya virus in patient and mosquito samples.

OBJECTIVES: We aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform for the rapid detection of chikungunya virus (CHIKV) in both patient and mosquito samples from Brazil. METHODS: We optimized an RT-LAMP assay and then evaluated the specificity and sensitivity using visual detection. In comparison with the RT-qPCR reference method, we validated the utility of this assay as a molecular diagnostic test in a reference laboratory for arbovirus diagnostics using 100 serum samples collected from suspected CHIKV cases. RESULTS: Our RT-LAMP assay specifically detected CHIKV without cross-reactivity against other arboviruses. The limit of detection of our RT-LAMP was estimated in -1.18 PFU (confidence interval [CI] ranging from -2.08 to 0.45), resulting in a similar analytical sensitivity when directly compared with the reference standard RT-qPCR assay. Then, we demonstrate the ability of our RT-LAMP assay to detect the virus in different human specimens (serum, urine, and saliva), and crude lysate of Aedes aegypti mosquitoes in as little as 20-30 minutes and without a separate RNA isolation step. Lastly, we showed that our RT-LAMP assay could be lyophilized and reactivated by adding water, indicating potential for room-temperature storage. Our RT-LAMP had a clinical sensitivity of 100% (95% CI, 90.97-100.00%), clinical specificity of 96.72% (95% CI, 88.65-99.60%), and overall accuracy of 98.00% (95% CI, 92.96-99.76%). DISCUSSION: Taken together, these findings indicate that the RT-LAMP assay reported here solves important practical drawbacks to the deployment of molecular diagnostics in the field and can be used to improve testing capacity, particularly in low- and middle-income countries.

Clinical and laboratory diagnosis of monkeypox (mpox): Current status and future directions.

The emergence and rapid spread of the monkeypox virus (MPXV) to non-endemic countries has brought this once obscure pathogen to the forefront of global public health. Given the range of conditions that cause similar skin lesions, and because the clinical manifestation may often be atypical in the current mpox outbreak, it can be challenging to diagnose patients based on clinical signs and symptoms. With this perspective in mind, laboratory-based diagnosis assumes a critical role for the clinical management, along with the implementation of countermeasures. Here, we review the clinical features reported in mpox patients, the available laboratory tests for mpox diagnosis, and discuss the principles, advances, advantages, and drawbacks of each assay. We also highlight the diagnostic platforms with the potential to guide ongoing clinical response, particularly those that increase diagnostic capacity in low- and middle-income countries. With the outlook of this evolving research area, we hope to provide a resource to the community and inspire more research and the development of diagnostic alternatives with applications to this and future public health crises.

Recent insights into SARS-CoV-2 omicron variant.

The SARS-CoV-2 omicron variant (B.1.1.529) was first identified in Botswana and South Africa, and its emergence has been associated with a steep increase in the number of SARS-CoV-2 infections. The omicron variant has subsequently spread very rapidly across the world, resulting in the World Health Organization classification as a variant of concern on 26 November 2021. Since its emergence, great efforts have been made by research groups around the world that have rapidly responded to fill our gaps in knowledge for this novel variant. A growing body of data has demonstrated that the omicron variant shows high transmissibility, robust binding to human angiotensin-converting enzyme 2 receptor, attenuated viral replication, and causes less severe disease in COVID-19 patients. Further, the variant has high environmental stability, high resistance against most therapeutic antibodies, and partial escape neutralisation by antibodies from convalescent patients or vaccinated individuals. With the pandemic ongoing, there is a need for the distillation of literature from primary research into an accessible format for the community. In this review, we summarise the key discoveries related to the SARS-CoV-2 omicron variant, highlighting the gaps in knowledge that guide the field's ongoing and future work.

Portable sample processing for molecular assays: application to Zika virus diagnostics.

This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL-1 level from a 5 µL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

Collapse of the public health system and the emergence of new variants during the second wave of the COVID-19 pandemic in Brazil.

The worldwide spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the most severe public health crisis since the 1918 Spanish influenza pandemic. After the introduction of public health interventions aimed at reducing the number of COVID-19 cases, many countries across the world obtained success at containing the fast spread of SARS-CoV-2 during the first wave of the pandemic. However, the SARS-CoV-2 has resurged in many countries causing a even more devastating second wave. Brazil is one the most affected countries and currently is facing one of the worst public health crises in its history. Here, we discuss the unprecedented challenges faced by the Brazilian public health system in the midst of the second wave of the COVID-19 pandemic, particularly regarding the collapse of the Brazilian health system and the emergence of new variants of concern (VOCs). Finally, we suggest some insights using a one health approach that will help the country to face and overcome the current COVID-19 crisis.

Simultaneous Circulation of DENV, CHIKV, ZIKV and SARS-CoV-2 in Brazil: an Inconvenient Truth.

The emergence of SARS-CoV-2 has prompted the mobilization of a network of public health laboratories to diagnose COVID-19 patients, trace contacts and identify hot-spot areas for active community transmission at the expense of arbovirus diagnosis and control practices. In this article, we discuss the unprecedented challenges faced by the Brazilian public health system in dealing with the incursion of SARS-CoV-2 in the midst of ongoing triple arboviral epidemics caused by dengue, chikungunya, and Zika virus. Finally, we highlight the importance of the introduction of one health approach as an effective inter-disciplinary response and management to mitigate the catastrophic effect caused by these pathogens.

Epidemiological and clinical characteristics of the first 557 successive patients with COVID-19 in Pernambuco state, Northeast Brazil.

BACKGROUND: South America is the current epicenter of COVID-19 pandemic. Yet, the epidemiological and clinical features of the disease have not been described in Brazil, the third most affected country in the world. METHODS: In this retrospective study, we describe the demographics, epidemiology and clinical features of the first 557 consecutive patients positive for SARS-CoV-2 living in Pernambuco state, Northeast Brazil. RESULTS: The first COVID-19 cases occurred in the high income population. The age of infected patients ranged from 27 days to 97 years with a median of 47 years. The ratio of males to female in the SARS-CoV-2-infected group was 0.83:1. The most common symptom was cough (74.51%), followed by fever (66.79%), dyspnea (56.01%), sore throat (28.19%) and O2 saturation <95% (24.42%). 86.44% of the lethal cases were patients older than 51 years. The median time from illness onset to diagnosis was 4.0 days (range 0-39 days) Severe patients diagnosed after 14 days of symptoms onset had higher viral load than patients with mild disease. CONCLUSIONS: Our study provides important information about COVID-19 in the tropics and will assist physicians and health officials to face the current pandemics as SARS-CoV-2 continues to spread in the human population.

Insights into SARS-CoV-2, the Coronavirus Underlying COVID-19: Recent Genomic Data and the Development of Reverse Genetics Systems.

The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease - eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) - across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution.

Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review.

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil.

The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.