Nucleic Acid Amplification Techniques Research Articles

Endonuclease Q as a Robust Enhancer for Nucleic Acid Amplification

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Non-Microbiological Mycobacterial Detection Techniques for Quality Control of Biological Products: A Comprehensive Review.

Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients' health. For this reason, the European Pharmacopoeia mandates the specific testing of biological products for mycobacteria, a critical regulatory requirement aimed at ensuring the safety of these products before they are released to the market. The current pharmacopeial reference, i.e., microbial culture method, cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time consuming and requires a continuous supply of culture media, posing logistical challenges. Thus, to overcome these issues, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment for biological products and underlines their advantages and limitations. Nucleic acid amplification techniques or direct measurement of mycobacteria stand out as the most suitable alternatives for mycobacterial testing in biological products.

Towards real-time airborne pathogen sensing: Electrostatic capture and on-chip LAMP based detection of airborne viral pathogens

Considerable loss of life, economic slowdown, and public health risk associated with the transmission of airborne respiratory pathogens was underscored by the recent COVID-19 pandemic. Airborne transmission of zoonotic diseases such as the highly pathogenic avian influenza (HPAI) and porcine reproductive and respiratory syndrome virus (PRRSV) has caused major disruptions to domestic and global food security. Current ambient air pathogen monitoring systems involves the collection of air samples from indoor settings suspected of viral contamination, followed by subsequent processing of capture samples to determine the presence and species of airborne viral matter. Nucleic acid amplification techniques are considered the gold standard for pathogen diagnostics. Currently, the necessary extraction and purification of viral RNA from air collector systems prior to sample analysis is both time consuming and performed manually. A monitoring system with separate air sampling and biochemical detection procedures is prone to delay the response to emergent viral threats. In this paper, we present a pathogen monitoring system that overcomes these limitations related to extraction and purification of viral samples and lays the groundwork for a real-time monitor for airborne viral pathogens. We demonstrate a high flow electrostatic precipitator system, that uses small collection wells as counter electrodes for pathogen collection. Integrated reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is used for detection of captured viral matter within wells. On-chip heating of collection wells is enabled by integrated planar heaters and small volumes of reagent (30 μL) directly to the collection wells. We present the design of such a system and show experimental results that demonstrate the use of this device for detection of aerosolized SARS-CoV-2 virus like particles (VLPs), a model pathogen for SARV-CoV-2.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

From Wet Mount to Nucleic Acid Amplification Techniques: Current Diagnostic Methods and Future Perspectives Based on Patenting of New Assays, Stains, and Diagnostic Images for Trichomonas vaginalis Detection

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world. The estimated global prevalence in 2016 was 156 million adults aged 15–49. However, these data are underestimated, since the most used diagnostic method is the wet mount, which has low sensitivity, the information regarding the estimated duration of infection is limited and there is evidence of undiagnosed asymptomatic cases in both sexes. Currently 80% of cases—including both sexes—are asymptomatic, which makes the disease silent and chronic in course, leading to complications. The aim of this review was to discuss the diagnostic methods for T. vaginalis detection that are currently available and applicable in the clinical laboratory routine. Overall, nucleic acid amplification techniques are the best option for T. vaginalis detection, with higher sensitivity and specificity than other tests. Although these techniques present higher cost, their implementation should be supported to ensure correct trichomoniasis diagnosis and treatment beyond contributing to questions on epidemiology and control.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies.

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic.

Rapid Manufacturing of Glass‐Based Digital Nucleic Acid Amplification Chips by Ultrafast Bessel Pulses

Advanced nucleic acid amplification techniques require separating test samples into numerous units (digitalization), which enables higher sensitivity and accuracy than traditional methods. Reagent partition tools are commercially available, but are usually complex and expensive, hindering mass adoption. Herein, the fabrication of a large‐scale micro‐through‐hole array on glass substrates for an advanced digital nucleic acid amplification technique (NAAT) is demonstrated. To meet the requirement of hole quantities (over tens of thousands) for valid nucleic acid statistics, ultrafast Bessel pulses with continuous translation of the glass substrate are applied. A single‐shot Bessel pulse can lead to a single hole, and 100 holes can be produced per second (scalable to thousands per second). Experiments using the fabricated chips show that the performance meets the NAAT requirements. This work provides a highly efficient and low‐cost scheme for reagent partitioning that promotes the wide accessibility of advanced digital NAATs as well as a variety of other applications.

Sequence‐Guided Localization of DNA Hybridization Enables Highly Selective and Robust Genotyping

AbstractGenetic variations are always related to human diseases or susceptibility to therapies. Nucleic acid probes that precisely distinguish closely related sequences become an indispensable requisite both in research and clinical applications. Here, a Sequence‐guided DNA LOCalization for leaKless DNA detection (SeqLOCK) is introduced as a technique for DNA hybridization, where the intended targets carrying distinct “guiding sequences” act selectively on the probes. In silicon modeling, experimental results reveal considerable agreement (R2 = 0.9228) that SeqLOCK is capable of preserving high discrimination capacity at an extraordinarily wide range of target concentrations. Furthermore, SeqLOCK reveals high robustness to various solution conditions and can be directly adapted to nucleic acid amplification techniques (e.g., polymerase chain reaction) without the need for laborious pre‐treatments. Benefiting from the low hybridization leakage of SeqLOCK, three distinct variations with a clinically relevant mutation frequency under the background of genomic DNA can be discriminated simultaneously. This work establishes a reliable nucleic acid hybridization strategy that offers great potential for constructing robust and programmable systems for molecular sensing and computing.

Poly(ethylene glycol)-Engrafted Graphene Oxide for Gene Delivery and Nucleic Acid Amplification

Graphene oxide (GO) is an oxidized form of graphene accommodating various oxygen-containing functional groups such as hydroxyl, epoxy, and carboxyl groups on its surface. GO has been extensively utilized in various biomedical applications including the delivery of biomolecules and the development of biosensors owing to its beneficial properties such as high surface area, nucleic acid adsorption, and fluorescence quenching through fluorescence resonance energy transfer (FRET). However, despite these favorable properties, the direct utilization of GO in these applications is often limited by low dispersibility in a physiological medium, cytotoxicity, low biocompatibility, and a strong binding affinity of nucleic acids to GO surface. The large surface area of GO and the presence of various functional groups on its surface make it highly amenable to facile surface modifications, offering scope for GO surface functionalization to overcome these limitations. When polyethylene glycol (PEG), which is a biocompatible polymer, is conjugated to GO, the PEGylated GO enhances the biocompatibility and dispersibility, reduces cytotoxicity, and allows controlled drug delivery with controllable binding affinity towards nucleic acid. PEG-engrafted GO retains the beneficial properties of GO while effectively addressing its limitations, rendering it suitable for various biomedical applications. In this review, we present the recent advancements of PEGylated GO in gene/drug delivery and the facilitation of nucleic acid amplification techniques, which aid in the development of therapeutic and diagnostic tools, respectively.

Novel helix loop-mediated isothermal amplification (HAMP) assay for colorimetric detection of Staphylococcus aureus in milk.

Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus.

Loop-mediated Iso-thermal amplification (LAMP) used for COVID-19 detection

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique, which can be efficiently amplified at constant temperature by using a Bst DNA polymerase with high chain replacement activity. Compared with traditional nucleic acid amplification technology, this technique has the advantages of high sensitivity, strong specificity, simple operation and low detection cost. In the outbreak of COVID-19 in recent years, LAMP technology has been successfully used to develop rapid domestic COVID-19 nucleic acid detection kits with the above advantages, which can achieve rapid detection of COVID-19 in only 20min, greatly improving the convenience and immediacy of nucleic acid detection. In this paper, the principle and development of LAMP technology were summarized by using databases such as NCBI PubMed, Web of Science, CNKI, etc., focusing on the application of LAMP technology in novel coronavirus detection, and the future development direction of LAMP technology was analyzed by describing the latest LAMP technology.

The Era of Molecular Biology: Diagnostics for Nipah Virus

Abstract: Nipah virus (NiV) is an emerging pathogen that, unlike other priority pathogens identified by the WHO, is endemic to Southeast Asia. It is most commonly transmitted through exposure to saliva or excrement from the Pteropus fruit bat or direct contact with intermediate animal hosts, such as pigs. NiV infection causes severe febrile encephalitic disease and/or respiratory disease; treatment options are limited to supportive care. The serological and nucleic acid amplification techniques have been developed for NiV and are used in laboratory settings, including some early multiplex panels for differentiation of NiV infection. The rural and remote nature of NiV outbreak settings, there remains a need for rapid diagnostic tests that can be implemented at the point of care. Additionally, more reliable assays for surveillance of communities and livestock will be vital to achieving a better understanding of the fruit bat host and transmission risk to other intermediate hosts, enabling the implementation to outbreak prevention and the management of this NiV. An improved understanding of NiV viral diversity and infection kinetics or dynamics will be central to the development of new diagnostics to enable effective validation and external quality assessments

A Blood Drying Process for DNA Amplification.

The presence of numerous inhibitors in blood makes their use in nucleic acid amplification techniques difficult. Current methods for extracting and purifying pathogenic DNA from blood involve removal of inhibitors, resulting in low and inconsistent DNA recovery rates. To address this issue, a biphasic method is developed that simultaneously achieves inhibitor inactivation and DNA amplification without the need for a purification step. Inhibitors are physically trapped in the solid-phase dried blood matrix by blood drying, while amplification reagents can move into the solid nano-porous dried blood and initiate the amplification. It is demonstrated that the biphasic method has significant improvement in detection limits for bacteria such as Escherichia coli, Methicillin-resistant Staphylococcus aureus, Methicillin-Sensitive Staphylococcus aureus using loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Several factors, such as drying time, sample volume, and material properties are characterized to increase sensitivity and expand the application of the biphasic assay to blood diagnostics. With further automation, this biphasic technique has the potential to be used as a diagnostic platform for the detection of pathogens eliminating lengthy culture steps.

Sample-to-answer sensing technologies for nucleic acid preparation and detection in the field.

Efficient sample preparation and accurate disease diagnosis under field conditions are of great importance for the early intervention of diseases in humans, animals, and plants. However, in-field preparation of high-quality nucleic acids from various specimens for downstream analyses, such as amplification and sequencing, is challenging. Thus, developing and adapting sample lysis and nucleic acid extraction protocols suitable for portable formats have drawn significant attention. Similarly, various nucleic acid amplification techniques and detection methods have also been explored. Combining these functions in an integrated platform has resulted in emergent sample-to-answer sensing systems that allow effective disease detection and analyses outside a laboratory. Such devices have a vast potential to improve healthcare in resource-limited settings, low-cost and distributed surveillance of diseases in food and agriculture industries, environmental monitoring, and defense against biological warfare and terrorism. This paper reviews recent advances in portable sample preparation technologies and facile detection methods that have been / or could be adopted into novel sample-to-answer devices. In addition, recent developments and challenges of commercial kits and devices targeting on-site diagnosis of various plant diseases are discussed.

Evaluation of Xpert MTB/RIF Ultra for the Diagnosis of Extrapulmonary Tuberculosis: A Retrospective Analysis in Saudi Arabia

The incidence of extrapulmonary tuberculosis (EPTB) in low- and middle-income countries, as well as, high-income countries has increased over the last two decades. The acid-fast bacillus (AFB) smear test is easy to perform and cost-effective with a quick turnaround time but the test has low sensitivity. Culture remains the gold standard for detecting TB; however, it has low sensitivity and slow bacterial growth patterns, as it may take up to 6 to 8 weeks to grow. Therefore, a rapid detection tool is crucial for the early initiation of treatment and ensuring an improved therapeutic outcome. Here, the Xpert Ultra system was developed as a nucleic acid amplification technique to accelerate the detection of MTB in paucibacillary clinical samples and endorsed by the World Health Organization. From March 2020 to August 2021, Xpert Ultra was evaluated for its sensitivity and specificity against EPTB and compared with those of the routinely used Xpert, culture, and AFB tests in 845 clinical samples in Saudi Arabia. The results indicate the overall sensitivity and specificity of Xpert Ultra to be 91% and 95%, respectively, compared with the Xpert (82% and 99%, respectively) and AFB smear (18% and 100%, respectively) tests. The results also indicated that despite the low microbial loads that were categorized as trace, very low, or low on Xpert Ultra, yet, complete detection was achieved with some sample types (i.e., 100% detection). Consequently, Xpert Ultra has great potential to replace conventional diagnostic approaches as a standard detection method for EPTB.

Tuberculous meningitis in Bhubaneswar, Odisha, during 2016 to 2022

BackgroundThe burden of tuberculous meningitis varies substantially by location, and is influenced by the overall burden of tuberculosis in that geographical area, and the age structure of the population. Conventional methods for diagnosis of tuberculous meningitis include acid fast bacilli (AFB) smear and culture. AFB smear has low sensitivity and culture techniques are time consuming. During the last few years, rapid and sensitive molecular methods such as cartridge based nucleic acid amplification technique (CBNAAT) is being used for the diagnosis of tuberculous meningitis. In this study, we evaluated the burden of tuberculosis and rifampicin resistance in suspected cases of tuberculous meningitis in Bhubaneswar, Odisha, during February 2016 to December 2022 using CBNAAT. MethodsUnder the National Strategic Plan 2012–2017, CBNAAT machines were rolled out in 2016, of which 1 was installed in the National Reference Laboratory (NRL) for Tuberculosis in Bhubaneswar. For this study, retrospective data on CBNAAT testing of cerebrospinal fluid (CSF) samples at the NRL was collected and analyzed from February 2016 to December 2022. All the demographic and laboratory data were entered in Excel 2010 (Microsoft office, USA) for data analysis. The age and sex distribution of the presumptive TB patients, proportion of samples referred from public and private health care facilities, proportion of M. tuberculosis positive CSF samples along with sensitivity to rifampicin was evaluated. The study was approved by the Institutional Human Ethics Committee. ResultsDuring February 2016 to December 2022, a total of 1627 CSF samples from presumptive TB meningitis patients were received in the NRL for CBNAAT testing. 60.7% (988/1627) of the presumptive TB patients were males. Of the 1627 patients, 3.1% (50/1627) were positive for M. Tuberculosis by CBNAAT. 56% (28/50) of the patients positive for tuberculous meningitis were males. The positivity for M. tuberculosis varied from 2.1% in 2017 to 5.1% in 2021. The CSF positivity for M. tuberculosis ranged from 0.7% in patients aged >60 years to 6.1% in 15–30 years age group. Of the 50 M. tuberculosis positive samples, 6% (3/50) were resistant for rifampicin. ConclusionOur study which included more than 1600 samples over a period of approximately 7 years found a 3.1% positivity for M. tuberculosis in CSF samples using CBNAAT. 6% of the M. tuberculosis positive samples were resistant to rifampicin. Future studies involving data from other districts of Odisha will help provide a more accurate information on the prevalence of tuberculous meningitis in Odisha state.

Paradigm shift of respiratory viruses causing lower respiratory tract infection in children during COVID-19 pandemic in India.

Acute lower respiratory tract infections (ALRTIs) are the commonest cause of mortality in children mostly attributed to respiratory viruses. During the coronavirus disease 2019 (COVID-19) pandemic, the dynamics and transmission of infections changed worldwide due to widespread public health measures. This study aimed to understand the pattern of respiratory viruses associated with ALRTIs in children pre and during COVID-19 pandemic in India. Respiratory samples were collected from ALRTI patients during pre-pandemic period (October 2019 to February 2020; n = 166), Delta (July 2021 to December 2021; n = 78) and Omicron wave (January 2022 to July 2022; n = 111). Samples were screened for Influenza (Inf) A pdmH1N1, InfA H3N2, InfB, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human bocavirus (hBoV), human rhinovirus (hRV), and parainfluenza virus (PIV-2 and PIV-3) by nucleic acid amplification techniques (NAATs). Significantly higher proportion of children with ALRTIs had virus/es isolated during pre-pandemic period than during mid-pandemic period [78.9% (131/166) vs. 52.9% (100/189); p < 0.001). RSV positivity was significantly higher (51.2%) in pre-pandemic period than 10.3% and 0.9% during the Delta and Omicron waves respectively. No significant difference in positivity rate of Inf A pdmH1N1, Inf A H3N2 and Inf B was seen. The increase in positivity of hRV (39.2% vs 42.3% vs 56.8%) and hBOV (1.2% vs 5.1% vs 9%) was documented in pre-pandemic, delta wave and omicron wave respectively. The COVID-19 pandemic significantly impacted the frequency and pattern of respiratory viruses among hospitalized children with ALRTIs in India.

Performance evaluation of the Aptima CMV quant assay using plasma and non-plasma samples.

Cytomegalovirus (CMV) infection has a major negative impact on transplantation and is associated with increased morbidity and mortality in this patient population. Quantitation of CMV infections using a molecular test is the preferred method for monitoring patients post-transplant. For this analysis, we compared the Aptima CMV Quant Assay (Aptima CMV) on the Panther system to the ELITech MGB Alert® CMV 3.0 ASR (MGB CMV) run on the ELITe InGenius®. The analytical performance of the assay was assessed using commercially available CMV reference panels that meet the 1st WHO International Standard for Human Cytomegalovirus for nucleic acid amplification techniques. The clinical performance of the assay was determined using 249 plasma and non-plasma samples. The 95% LOD of the Aptima assay was determined to be 50 IU/mL and 200 IU/mL for the MGB CMV assay. A strong linear correlation with the reference panel (R2=0.9945), excellent reproducibility, and accuracy (R2=0.986) over the detection range of the assay was observed. Of the 249 clinical samples tested, only 17 (6.8%) yielded discordant results which were at or near the lower limit of quantification of the assays. Although the Aptima CMV assay demonstrated excellent concordance of qualitative results to the MGB CMV assay for all samples, the MGB CMV quantified CMV DNA at an average of 0.5LogIU/mL higher than Aptima CMV. The Aptima CMV assay is both sensitive and accurate in quantifying CMV in both plasma and non-plasma specimens on the fully automated Panther system.

Manejo desde atención primaria de la infección por la viruela del mono (MPOX) en humanos

Monkeypox (MPOX) is a viral zoonosis endemic in West or Central African countries that is sporadically exported to another area. In May 2022, a global outbreak of MPOX smallpox began to occur in several countries in Europe and North America. Most of the reported cases are identified at the outpatient level and mainly affect men who have sex with men (MSM). Transmission is by close contact with lesions, body fluids, respiratory secretions or contaminated material from an infected person or animal. The clinical picture is similar to human smallpox, with less severity. Mild, self-limiting skin involvement predominates after 2-4 weeks. In MSM, atypical skin lesions appear due to the mode of infection. Severe forms or complications may appear in certain risk groups. The case fatality rate is 3%-6% depending on the clade responsible. The diagnosis of suspicion is confirmed by detection of the virus from exudates of lesions or scabs, with nucleic acid amplification techniques by conventional or real-time PCR. Clinical management in most cases is performed in primary care (PC), by monitoring the main symptoms. Between 5-10% require hospital management and there are some specific antiviral treatment options. Human smallpox vaccines protect against MPOX and are used as pre- and post-exposure prophylaxis for persons at risk. Measures to reduce exposure to the virus are the main MPOX prevention strategy. In addition, the role of the family physician is key to controlling the spread of MPOX through active surveillance and early diagnosis of the disease.