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Nucleic acid amplification tests (NAAT) are widely used for the detection of living organisms, recently applied in Lab-on-a-Chip (LoC) devices to make portable DNA analysis platforms. While portable LoC-NAAT can provide definitive test results on the spot, it requires specialized temperature control equipment. This work focuses on delivering a generalized low-cost, wireless smart thermostat for isothermal NAAT protocols in 2 cm × 3 cm LoC cartridges. We report on the design, prototyping, and evaluation results of our smart thermostat. The thermostat was evaluated by experimental and simulated thermal analysis using 3D printed LoC cartridges, in order to verify its applicability to various isothermal NAAT protocols. Furthermore, it was tested at the boundaries of its operating ambient temperature range as well as its battery life was evaluated. The prototype thermostat was proven functional in 20–30 °C ambient range, capable of maintaining the required reaction temperature of 12 isothermal NAAT protocols with 0.7 °C steady-state error in the worst case.

Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

Mycoplasma genitalium is one of the major causes of nongonococcal urethritis (NGU) worldwide but an uncommon sexually transmitted infection (STI) in the general population. The risk of sexual transmission is probably lower than for Chlamydia trachomatis. Infection in men is usually asymptomatic and it is likely that most men resolve infection without developing disease. The incubation period for NGU caused by Mycoplasma genitalium is probably longer than for NGU caused by C. trachomatis. The clinical characteristics of symptomatic NGU have not been shown to identify the pathogen specific etiology. Effective treatment of men and their sexual partner(s) is complicated as macrolide antimicrobial resistance is now common in many countries, conceivably due to the widespread use of azithromycin 1 g to treat STIs and the limited availability of diagnostic tests for M. genitalium. Improved outcomes in men with NGU and better antimicrobial stewardship are likely to arise from the introduction of diagnostic M. genitalium nucleic acid amplification testing including antimicrobial resistance testing in men with symptoms of NGU as well as in their current sexual partner(s). The cost effectiveness of these approaches needs further evaluation. The evidence that M. genitalium causes epididymo-orchitis, proctitis, and reactive arthritis and facilitates human immunodeficiency virus transmission in men is weak, although biologically plausible. In the absence of randomized controlled trials demonstrating cost effectiveness, screening of asymptomatic men cannot be recommended.

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis.

Nucleic acid amplification tests (NAATs) are powerful medical diagnostic tools for point-of-care (POC) and other field applications. However, traditional methods like quantitative PCR (qPCR) require complex, expensive equipment and trained operators, limiting their use to centralized labs. Isothermal alternatives, like Loop-mediated Isothermal Amplification (LAMP), are better adapted for POC devices. Lab-on-PCB systems have the potential to overcome the challenges faced by conventional microfabrication-based systems. This study presents a novel lab-on-PCB device for nucleic acid amplification and electrochemical detection using reverse transcription LAMP (RT-LAMP) of SARS-CoV-2. The system consists of two disposable PCB-based chips making it close to zero cost. One PCB is for heating and nucleic acid amplification, while the other is for electrochemical detection using Cyclic Voltammetry (CV) with a redox-active intercalator. The PCB slides are connected to a compact electronic device (< 10 USD) for controlling the heating and electroanalytical readout. Using this device, we achieved successful rapid (< 1.5 h) nucleic acid amplification and detection at a target concentration of 10 copies/reaction. This work represents a notable step toward developing integrated, portable NAAT devices for POC diagnostics.

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed.

Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present the use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction under molecular crowding conditions and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the high pH for alkaline lysis and crowding effects for ionic binding of the DNA under high salt conditions. In this study, we introduce Paper-based Abridged Solid-Phase Extraction with Alkaline Poly(ethylene) Glycol Lysis (PASAP). The anionic mixed cellulose ester (MCE) paper is used as solid phase and allows for fluid transport by wicking, eliminating the need for pipetting skills and the use of a magnet to retain beads. Following the release of DNA from the cells due to the lytic activity of the PASAP solution, the DNA binds to the anionic surface of the MCE paper, concentrating at the bottom while the sample matrix is transported towards the top by wicking. The paper was washed by dipping it in 40% isopropanol for 10 s. After air-drying for 30 s, the bottom section of the paper (3 mm × 4 mm) was snapped off using the cap of a PCR tube and immersed in the colourimetric loop-mediated isothermal amplification (cLAMP) solution for direct amplification and colourimetric detection. The total sample processing was completed in 15 min and ready for amplification. cLAMP enabled the detection of 10 2 CFU/mL of Escherichia coli ( E. coli ) from culture media and the detection of E. coli in milk < 10 3  CFU/mL (10 CFU) after incubation at 68 °C for 60 min, demonstrating applicability of the method to complex biological samples.

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

ABSTRACT Mpox (formerly—monkeypox) is the second viral pit break after COVID‐19. It is a zoonotic viral illness caused by an orthopox virus belonging to the same genus as various cowpox and vaccinia viruses. The mpox virus has two clades, clade IIb, responsible for the disease's global expansion in 2022. The virus can be isolated from rodents, squirrels, and dormice; however, the actual reservoir is unknown. Animal‐to‐human and human‐to‐human transmission can occur through noninvasive as well as invasive routes. The disease is more prevalent among homosexuals and injectable drug users, as long‐term contact is required for transmission. The disease typically presents with fever, myalgia, rash, and lymphadenopathy following an incubation period of 1–2 weeks. Skin lesions are considered preferred diagnostic specimen, with polymerase chain reaction (PCR) remaining the gold standard for confirming diagnosis. Symptomatic treatment antipyretics, antihistamines, and warm baths are given. Oral and intravenous antivirals like tecovirimat and cidofovir are used only in emergency settings, as the clinical trials on their efficacy are still in progress. Vaccinia intravenous immunoglobulins IVIG can be used in immunocompromised individuals. ACAM 2000, IMVAMUNE, and Dryvax are the available vaccines. Non‐pharmacological interventions like hand hygiene, social distancing, and personal protective equipment can significantly reduce viral transmission, whereas early diagnosis can limit the prevalence by providing timely public health interventions. This paper highlights key public health interventions and control strategies implemented during mpox outbreaks, emphasizing early detection, targeted prevention, and the role of vaccination and education in mitigating disease transmission.

Global TB control efforts have been severely hampered by the lack of diagnostic tests that are accurate, simple to use and can be applied at the point of clinical care. This has been further compounded by the widespread inability to test for drug resistance. The Xpert(®) MTB/RIF assay is a rapid molecular assay that can be used close to the point of care by operators with minimal technical expertise, enabling diagnosis of TB and simultaneous assessment of rifampicin resistance to be completed within 2 h. Moreover, this can be accomplished using unprocessed sputum samples as well as clinical specimens from extrapulmonary sites. We review in detail the development of this assay, its evaluation within the laboratory, its utility among adult and pediatric TB suspects, its use as a screening tool for HIV-associated TB and studies of its implementation at the district and sub-district levels in resource-limited settings. Following endorsement by the WHO in 2010, we consider the next steps in the implementation of the assay and its potential impact in high burden settings.