Human microbiota, a complex consortium of microorganisms co-evolved with the host, profoundly influences tissue development, immune regulation, and disease progression. Growing evidence shows that microbial metabolites and signaling molecules modulate key stem cell pathways-such as Hedgehog, Wnt/β-catenin, and Notch-thereby reprogramming stem cell fate toward tumor-suppressive or tumor-promoting outcomes. Specific taxa within oral, intestinal, and urogenital niches have been linked to cancer initiation, therapy resistance, and recurrence. In parallel, clinical studies reveal that microbiota composition affects infection dynamics: bacterial isolates from symptomatic urinary tract infections inhibit commensal growth more strongly than the reverse, with Gram-positive and Gram-negative strains displaying distinct interaction profiles. Collectively, these findings highlight microbiota's dual role in regulating cellular plasticity and pathogenicity. Elucidating host-microbe and microbe-microbe mechanisms may guide microbiota-targeted interventions to improve cancer and infectious disease management.

INTRODUCTION:Sexually Transmitted Infections (STIs) have become a global health problem, causing severe diseases and, in some cases, death. This study aimed to highlight the prevalence and spread of STIs in the Apulian population in the urban area of Bari. This would provide a clearer picture of the onset, persistence, social status, and pathophysiology of STIs, and allow health authorities to identify high-risk groups and personalize prevention and treatment programs. METHODS:This is a retrospective analysis of a study conducted between May 21, 2018, and May 31, 2024, on 6,931 patients. Enrolled patients were of both sexes; swabs, fluid samples, and biopsies were sent to our clinical microbiology laboratory at the Aldo Moro University Hospital in Bari for diagnosis and tested by specific, targeted quantitative Polymerase Chain Reaction (qPCR). RESULTS:The most frequent infection is caused by Ureaplasma parvum, with over 1400 cases. Our data suggest a possible epidemiological correlation between the transmissibility of STIs, cultural and social status, sex, and age. Patients were predominantly women (about 74.9% versus 25.1%) and aged between 25 and 50 years. The prevalence of individuals affected by STI-causing microorganisms was significantly higher among younger adults aged 25-55 years, with Ureaplasma urealyticum, Ureaplasma parvum, and Mycoplasma hominis as the major co-infecting agents. In pregnant women, two types of moderate positive correlation were observed: Mycoplasma hominis with Ureaplasma parvum and Chlamydia trachomatis, followed by Ureaplasma parvum and Chlamydia trachomatis. DISCUSSION:The study highlights the need for comprehensive care, sensitive to cultural and developmental differences, to address sexuality-related issues among disadvantaged groups, particularly adolescents and young adults. These criteria should be applied nationwide and could serve as a guideline for officials and public health professionals to promote access to sexual health services among these population groups and provide useful information, strengthening preventive measures among younger age groups, and designing sexuality education programs. CONCLUSIONS:By analyzing data (age, gender, race, socioeconomic status, etc.) alongside STI diagnoses, we sought to identify patterns and trends in STI prevalence. These data will be crucial for the adoption of evidence-based STI control programs in the Puglia Region. They would eventually help in understanding the specific populations most vulnerable to STIs and the factors contributing to their increased risk.

Sjögren's syndrome (SS) is an autoimmune disease and its management is palliative. There is no specific dietary protocol for SS patients. A gluten-free diet has been tested in SS patients with celiac disease (CD) and indicated modest improvements. Whether gluten-free diets per sè could alleviate autoimmune inflammatory processes in the salivary glands of SS patients with associated CD or even in SS patients without CD is an interesting hypothesis and warrants clinical studies. Hypokalemia in SS patients is among the most frequent sequelae of renal tubular acidosis. Supplementation with potassium (K)-rich diets can reduce inflammation and oxidative stress. K level in CD patients is highly abnormal at diagnosis and gluten-free diets help to normalize its serum level in CD patients. Furthermore, treatment of severe cases of SS requires concomitant glucocorticoid therapy and K supplementation. Results of two separate clinical trials in (i) patients with rheumatoid arthritis (RA) –a disease with similar pathology to SS- indicated that the enhanced serum cortisol followed K supplementation, and in (ii) celiac patients, serum K levels were normalized after the administration of a gluten-free diet. We reviewed the literature extensively on this topic to propose a hypothesis to address this problem and suggest a novel potential for K-rich, gluten-free diets in SS patients. Based on causal associations, we propose that higher K absorption and cortisol secretion following gluten-free diets accompanied by K-rich diets in SS patients with low serum potassium levels, may confer a higher therapeutic potential. Clinical trials are needed to test this hypothesis.

Coronavirus disease-2019 (COVID-19) is a respiratory disease in which Spike protein from SARS-CoV-2 plays a key role in transferring virus genomic code into target cells. Spike protein, which is found on the surface of the SARS-CoV-2 virus, latches onto angiotensin-converting enzyme 2 receptors (ACE2r) on target cells. The RNA genome of coronaviruses, with an average length of 29 kb, is the longest among all RNA viruses and comprises six to ten open reading frames (ORFs) responsible for encoding replicase and structural proteins for the virus. Each component of the viral genome is inserted into a helical nucleocapsid surrounded by a lipid bilayer. The Spike protein is responsible for damage to several organs and tissues, even leading to severe impairments and long-term disabilities. Spike protein could also be the cause of the long-term post-infectious conditions known as Long COVID-19, characterized by a group of unresponsive idiopathic severe neuro- and cardiovascular disorders, including strokes, cardiopathies, neuralgias, fibromyalgia, and Guillaume- Barret's like-disease. In this paper, we suggest a pervasive mechanism whereby the Spike proteins either from SARS-CoV-2 mRNA or mRNA vaccines, tend to enter the mature cells, and progenitor, multipotent, and pluripotent stem cells (SCs), altering the genome integrity. This will eventually lead to the production of newly affected clones and mature cells. The hypothesis presented in this paper proposes that the mRNA integration into DNA occurs through several components of the evolutionarily genetic mechanism such as retrotransposons and retrotransposition, LINE-1 or L1 (long interspersed element-1), and ORF-1 and 2 responsible for the generation of retrogenes. Once the integration phase is concluded, somatic cells, progenitor cells, and SCs employ different silencing mechanisms. DNA methylation, followed by histone modification, begins to generate unlimited lines of affected cells and clones that form affected tissues characterized by abnormal patterns that become targets of systemic immune cells, generating uncontrolled inflammatory conditions, as observed in both Long COVID-19 syndrome and the mRNA vaccine.

The amniotic membrane has recently garnered attention as a potential scaffold for regenerating damaged tissues such as burns, corneal surfaces and orthopedic injuries. Our study utilized collagen membranes derived from amniotic membranes as a scaffold for mesenchymal stem cells obtained from umbilical cord blood to produce keratinocyte-like cell sheets. These cell sheets were employed to treat skin loss lesions in a mouse model. Analysis of the results indicated that the group treated with cell sheets exhibited the most rapid tissue healing, with wound sizes healed at 83.60%, while the untreated group showed the slowest tissue healing at 77.63%. Histological examination revealed that utilizing cell sheets for regenerating damaged skin tissue resulted in superior healing, with the structure of the regenerated tissue closely resembling that of mouse skin epithelium, surpassing the outcomes of the other two groups of mice. This research underscores the potential of keratinocyte like cell sheets as a viable graft for autologous keratinocyte sheets or autologous skin grafts in treating patients with skin loss lesions. Particularly beneficial for patients lacking a sufficient supply of autologous skin grafts or requiring immediate intervention, cell sheets can serve as a tissue graft product, offering a viable alternative to conventional grafts.

Sepsis remains a critical medical emergency caused by a dysregulated immune response to infection, with timely detection and intervention being essential for improving survival rates. Traditional methods often rely on clinician intuition and structured scoring systems, which may be time-intensive and prone to variability. To address these limitations, Machine Learning (ML) offers a powerful alternative, bringing precision and efficiency to sepsis detection. This study investigates both white-box and complex black-box ML models applied to patient data collected across the continuum of care, including monitoring at the urgent care, en route in ambulances, and diagnostics conducted within hospital emergency department settings themselves. White-box models, such as logistic regression and decision trees, are valued for their interpretability, allowing healthcare providers to understand and trust the reasoning behind predictions. Meanwhile, black-box models like deep neural networks and support vector machines deliver superior accuracy but pose challenges in clinical transparency. This trade-off between explainability and performance is explored in detail, supported by experimental results aimed at identifying the most effective computational strategies for early sepsis recognition across diverse healthcare environments.

Mesenchymal stem cells (MSCs), particularly their secreted exosomes, small microvesicles, represent a major focus in regenerative medicine due to their therapeutic potential. Exosomes exhibit growth factors and cytokines and are loaded with microRNAs (miRNA) and short interfering RNA (siRNA) that can be transferred to other cells, potentially affecting their function. Exosomes are crucial mediators of intercellular communication, are immunomodulatory, and are promoters of tissue regeneration. Despite their promise, the standardized methods for exosome isolation and characterization remain weak. This exploratory study addresses this gap by detailing an effective method for isolating exosomes from adipose tissue mesenchymal stem cells (AT-MSCs), emphasizing precipitation as a technique yielding a high efficiency and purity compared to other methods. Functionally, we aimed to confirm the AT-MSC exosomes’ ability to exert an effective protective activity on the skin and its main components, such as fibroblasts, collagen, and elastin. To achieve this goal, we had to demonstrate that AT-MSC exosomes are safe and free of toxic substances. They can express specific proteins such as CD9, CD63, and CD81, which are well-known exosome markers. These exosomes also contain key miRNAs, including miRNA-203 A, miRNA-203 B, and miRNA-3196, important for skin regeneration, as well as enhancers of cell integrity and proliferation. We eventually confirmed the ability of exosomes to exert protective and recovery effects on fibroblasts after H2O2-induced damage in vitro, as well as on mouse skin after UVB-induced damage in vivo. These effects were verified by measuring levels of reactive oxidative species (ROS), assessing SA-β-Galactosidase (SA-β-Gal) activity, analyzing the cell cycle, evaluating the telomere length of fibroblasts by RT-PCR, and conducting histological assessments of collagen and elastin structure in murine skin after UVB exposure. This exploratory work provides valuable insights into the isolation, characterization, and bioactive and reparative properties of exosomes from AT-MSCs, supporting their development for future studies and therapeutic applications.

INTRODUCTION:To evaluate the effectiveness of selective laser trabeculoplasty (SLT) in lowering intraocular pressure (IOP) in Vietnamese patients with pseudoexfoliation glaucoma (PXG). METHODS:This prospective, comparative study included a total of 64 eyes from PXG patients who had not previously received any antiglaucomatous medication. Patients were categorized into two groups: Group 1 consisted of 32 eyes treated with SLT as the initial therapy, while Group 2 comprised 32 eyes treated with Tafluprost 0.0015%. RESULTS:The baseline IOP before treatment ranged from 22 to 26 mmHg. There were no significant differences between the two groups in terms of mean cup/disc (C/D) ratio and retinal nerve fiber layer (RNFL) thickness. At the final visit, the mean percentage reduction of IOP was 26.73% in Group 1 and 27.15% in Group 2. DISCUSSION:SLT significantly reduced IOP in PXG patients, with outcomes comparable to Tafluprost. Treatment was more successful in eyes with higher trabecular pigmentation and early- stage glaucoma. Structural and functional parameters remained stable over 12 months. Side effects, including mild itchiness, IOP spikes, and anterior chamber reactions, were transient and manageable. No vision-threatening complications occurred, confirming SLT as a safe and effective first-line option for PXG in Vietnamese patients. CONCLUSION:SLT can be effectively used as a primary treatment for PXG. In cases of recurrent IOP elevation, a second SLT procedure can be administered alone or in combination with other medications.

BACKGROUND:Due to its outstanding biological properties, the Human Amniotic Membrane (HAM) has been a topic of medical research for many years to treat, repair, and regenerate damages related to the epithelial surface, corneal epithelial, orthopedic, and infected body parts. The HAM shows crucial anti-inflammatory, anti-bacterial, and anti-viral properties and immunomodulating qualities. Therefore, the HAM is one of the most used natural biomaterials in tissue engineering to generate bio-scaffolds. OBJECTIVE:This study used HAM as a raw material to produce a collagen membrane that fully meets the criteria for creating a bioscaffold with suitable mechanical strength and limited risk of graft rejection. This collagen membrane would be a bioscaffold to carry Human Umbilical Cord Blood Mesenchymal Stem Cells (hUCB-MSCs) to generate human epithelial-like cell sheets. Shortly, these cell sheets can repair and regenerate the epithelial layer in trauma cases, severe damage from accidents, burns, and other post-operational conditions. METHODS:Among the varieties of stem cells currently being researched, MSCs are the most studied and applied due to their outstanding bio-qualities and regenerative features. We used International Society for Cells and Gene Therapy (ISCT) standards to identify and characterize the hUCB-MSCs obtained from a donor's umbilical cord blood who provided consent. We used an inverted phase contrast microscope to evaluate morphological changes and epithelial stratification of cultured growing cells on the collagen scaffold. Further, staining techniques, such as H&E and Trichrome, were used to assess the morphology and structure of the new epithelial construct; histochemical staining with p63 and ck 1/10 markers was used to evaluate the cellular characteristics and specific traits. Transmission electron microscopy was used to validate cell junctions. RESULTS:This study successfully obtained human epithelial-like cell sheets with multiple cell layers, morphology, and characteristics comparable to human epithelial tissue. CONCLUSION:The results of this study will constitute a potential alternative to autologous skin grafting in heavy skin loss and deep lesions.

Medical decision-support and advising systems are critical for emergency physicians to quickly and accurately assess patients' conditions and make diagnosis. Artificial Intelligence (AI) has emerged as a transformative force in healthcare in recent years and Large Language Models (LLMs) have been employed in various fields of medical decision-support systems. We studied responses of a group of different LLMs to real cases in emergency medicine. The results of our study on five most renown LLMs showed significant differences in capabilities of Large Language Models for diagnostics acute diseases in medical emergencies with accuracy ranging between 58

The aim of this prospective comparative study was to determine whether alterations in ocular microbiota composition affect dry eye disease (DED) in newly married couples. Methods: This study comprised 392 spouses with confirmed dry eye disease (DED) who were either insomniac or hypersomniac, and 196 spouses as controls. We prospectively collected data on ocular surface parameters including tear osmolarity, Schirmer’s test and Ocular Surface Disease Index (OSDI), sleep parameters and ocular microbiota composition on day 1 and after six months follow up. Results: The frequency of moderate-to-severe DED in spouses who had married with an insomniac person was significantly higher than healthy controls and hypersmoniacs (P<0.001). The DED phenotype was positively significantly correlated with species differences in each of the three groups (P value <0.001). At baseline, frequency of both mild and moderate-to-severe DED phenotype was significantly highest in insomniacs. Interestingly, in support of our hypothesis, the frequency of both mild and moderate-to-severe DED phenotype in healthy spouses was significantly increased after six months of marriage with an insomniac person (but not with a hypersomniac person), suggesting that there is a possibility that ocular microbiota alteration might have played a role in occurrence of DED in this group. Conclusions: We for the first time contribute to the existing literature by discovering an association between ocular gut microbiota and DED, possibly through person-to-person contact. In the framework of family medicine and personalized medicine, the practical and theoretical implications of this study cover vast areas of sleep therapy and microbiota-related ocular diseases.

Background: The current manuscript’s aim was to determine the human papillomavirus (HPV) genotype-specific prevalence and distribution among individuals, males, and females, of different ages in the region of Apulia, Italy, highlighting the possible variables involved in the carcinogenicity mechanism. In addition, we proposed two hypothetical models of HPV’s molecular dynamics, intending to clarify the impact of prevention and therapeutic strategies, explicitly modeled by recent survey data. Methods: We presented clinical data from 9647 participants tested for either high-risk (HR) or low-risk (LR) HPV at the affiliated Bari Policlinic University Hospital of Bari from 2011 to 2022. HPV DNA detection was performed using nested-polymerase chain reaction (PCR) and multiplex real-time PCR assay. Statistical analysis showed significant associations for all genders and ages and both HR- and LR-HPV types. A major number of significant pairwise associations were detected for the higher-risk types and females and lower-risk types and males. Results: The overall prevalence of HPV was 50.5% (n-4.869) vs. 49.5% (n-4.778) of the study population, of which 74.4% (n-3621) were found to be HPV high-risk (HR-HPV) genotypes and 57.7% (n-2.807) low-risk HPV (LR-HPV) genotypes, of which males were 58% and females 49%; the three most prevalent HR-HPV genotypes were HPV 53 (n707-15%), 16 (n704-14%), and 31 (n589-12%), and for LR-HPV, they were 42 (19%), 6 (16%), and 54 (13%); 56% of patients screened for HPV were ≤ 30 years old, 53% were between 31 and 40 years old, 46% were 41–50 and 51–60 years old, and finally, 44% of subjects were >60 years old. Conclusions: Our study provided comprehensive epidemiological data on HPV prevalence and genotype distribution among 9647 participants, which could serve as a significant reference for clinical practice, and it implied the necessity for more effective screening methods for HPV carcinogenesis covering the use of more specific molecular investigations. Although this is a predominantly descriptive and epidemiological study, the data obtained offer not only a fairly unique trend compared to other studies of different realities and latitudes but also lead us to focus on the HPV infection within two groups of young people and adults and hypothesize the possible involvement of dysbiosis, stem cells, and the retrotransposition mechanism.

The emergence of cancer-causing bacteria and viruses that have become resistant to either antibiotics or viral treatment is often attributed to their overuse in people and livestock. Intriguingly, the class of antidepressant drugs, such as selective serotonin reuptake inhibitors (SSRIs), may also contribute to generating resistant microorganisms with a profound disturbing role in local cervix-vagina homeostasis. Therefore, we proposed the possible involvement of SSRIs in the onset and growth of cervical cancer (CC), which is based on some empirical considerations, (i) the number of women diagnosed with post-menopausal CC is almost equivalent to the number of women diagnosed with depressive problems, (ii) the proven affection of SSRIs on both vaginal and intestinal microbiota, (iii) the essential role of the microbiota in the immune response against oncogenic pathogens, including human papillomavirus and Chlamydia trachomatis (C. trachomatis), (IV) the role of SSRIs in favoring the rise of antibiotic-resistant microorganisms, and ( V) the inhibitory effect of SSRIs in the estradiol-serotonin-prolactin axis. These features are important in the overproduction of reactive oxygen species, responsible for different harmful activities such as higher cell membrane permeability and porosity, hyperstimulation of transcription, and translational activities, with overall stress on check-point genes involved in ATP synthesis, DNA breaks, and abnormal mutations. This paper's results suggested reconsidering the efficacy of SSRIs on long-term use since the overall increase in prescriptions is mainly due to long-term treatment rather than the diagnoses per se, secondly little is known about the consequences of long-term use. The evidence that supports the role of antidepressant drugs in increasing the risk of CC was searched and discussed.

The application of AI (Artificial Intelligence) in emergency medicine helps significantly improve the quality of diagnostics under limitations of resources and time constraints in emergency cases. We have designed a comprehensive AI-based diagnostic and treatment plan decision-support system for emergency medicine by integrating the available LLMs (Large Language Models), like ChatGPT, Gemini, Claude, and others, and tuning them up with additional training on actual emergency cases. There is a special focus on early detection of life-threatening and time-sensitive diseases like sepsis, stroke, and heart attack, which are the major causes of death in emergency medicine. Additional training was conducted on a total of 600 cases (300 sepsis; 300 non-sepsis). The collective capability of the integrated LLMs is much stronger than each individual engine. Emergency cases can be predicted based on information from multiple sensors and streaming sources combining traditional IT (Information Technology) infrastructure with Internet of Things (IoT) schemes. Medical personnel compare and validate the AI models used in this work.

Due to its outstanding biological properties human amniotic membrane (hAM) has been a topic of medical research for many years in trying to treat, repair and regenerate difficult damages related to the epithelial surface, corneal epithelial, orthopedic, and infected body parts. The hAM’s showed also to possess crucial anti-inflammatory, anti-bacterial, and anti-viral properties and immunomodulating qualities. As a consequence, the hAM is one of the most used natural bio-materials in tissue engineering for the construction of bio-scaffolds. We used human hAM (hAM) as a raw material source to home umbilical cord mesenchymal stem cells (hUCB-MSCs) to manufacture collagen membranes that could fully meet the standards for making a skin scaffold, with suitable mechanical strength whilst limiting the risk graft rejection. Among the varieties of stem cells currently being researched MSCs are the most studied and applied due to their outstanding bio-qualities and regenerative features. We used International Society for Cells and Gene Therapy (ISCT) standards to identify and characterize the hUCB-MSCs obtained from a consent donor’s umbilical cord blood. In this study, we used hAM as a scaffold to culture domestic hUBC-MSCs to create human-like epithelial tissues aimed at repairing and regenerating the epithelial layer following trauma, deep damage from accidents, burns, and/or conditions post-operative. We used an inverted phase contrast microscope to observe the cultured growing cells on the amniotic specimens to evaluate morphological changes and epithelial stratification. Staining techniques such as H&E and Trichrome were used to evaluate the morphology and structure of the new epithelial construct, histochemical staining with p63 and ck 1/10 markers was instead used to evaluate the cellular characteristics, the transmission electron microscopy (TEM) was used to validate cell junctions. Ultimately, the results successfully showed the possibility of obtaining human-like epithelial tissue with multiple cell layers with morphology and characteristics similar to human epithelial-like tissue. The results of this study will constitute a potential alternative to autologous skin grafting in cases of heavy skin loss and deep lesions.

AIMS:Medication-related osteonecrosis of the jaw (MRONJ) is a drug-related adverse reaction characterized by bone destruction and necrosis in the jaw. This case series aims to evaluate the treatment approaches and outcomes in MRONJ patients.MATERIALS AND METHODS:The retrospective study was conducted at the Dental Unit of the University of Bari, Italy. Patients with MRONJ were treated and followed up for 60 months. The treatment approach involved piezosurgery and concentrated growth factor (CGF). Six clinical cases from this group are described in detail.RESULTS:None of the patients showed recurrence of necrotic MRONJ lesions during the follow-up period. The surgical interventions, including bone resections and the application of CGF, resulted in successful mucosal healing and the prevention of disease progression.CONCLUSIONS:This study highlights the complexity of managing MRONJ and the importance of a multidisciplinary approach. Conservative treatment options and minimally invasive surgery have shown efficacy in controlling symptoms and improving patients' quality of life. However, the optimal treatment approach remains a challenge, and further studies are needed to evaluate alternative therapies and resective surgery. A comprehensive preoperative evaluation and collaboration among dental, endocrinology, and oncology specialists are crucial for personalized and multidisciplinary management. Ongoing research efforts are necessary to explore new therapeutic modalities and improve our understanding of MRONJ management, providing better support to patients dealing with this complex condition.

It is well established the importance of stem cells (SCs) in tissue growth, regeneration and repair, given their ability to self-renew and differentiate into mature cells. Stem cells are present in all individuals and are potentially active to the end of life. However, less is known about their unique function within the immune system as immune regulators and their important task in viral protection. Antiviral resistance is a common mechanism in all cells though stem cells utilize an antiviral RNA interference (RNAi) mechanism, while adult cells react by using the interferondependent repression pathway via interferon-associated protein-based response to induce an antiviral response. Therefore, the idea behind this review is to highlight the mechanisms of viral evasion of host defense, which would then allow us to highlight the rationale use of autologous stem cells and their biochemical and immunological ability to reset the subverted immune responses. Recently, scientists have highlighted their use in the field of immune-therapy, establishing the possibilities of using them outside the conventional protocol with the advancement in manipulating these cells in such a way that specific body activity can be restored. This paper describes the remarkable SCs profile and discusses some ideas regarding their promising use in vivo.

MRONJ is a serious drug-related side effect that is most common in people using antiresorptive and/or angiogenic medications. Therapy options for this condition include conservative treatments, surgical procedures with varied degrees of invasiveness, and adjuvant therapies. The aim of the present study is to identify the most successful and promising therapy alternatives available to clinicians. PubMed, Cochrane, Scopus, Web of Science, and Embase were searched for works on our topic published between 8 January 2006 and 8 January 2023. The search was restricted to randomized clinical trials, retrospective studies, clinical studies, and case series involving human subjects with at least five cases and no age restriction on participants. A total of 2657 was found. After the selection process, the review included 32 publications for qualitative analysis. Although conservative treatments (pharmacological, laser, and minimally invasive surgery) are effective in the early stages of MRONJs or as a supplement to traditional surgical resection therapy, most studies emphasize the importance of surgical treatment for the resolution or downstaging of advanced lesions. Fluorescence-guided surgery, PRP, PRF, CGF, piezosurgery, VEGF, hyaluronic acid, and ozone therapy all show significant potential for improving treatment outcomes.

Shortly after its emergence, Omicron and its sub-variants have quickly replaced the Delta variant during the current COVID-19 outbreaks in Vietnam and around the world. To enable the rapid and timely detection of existing and future variants for epidemiological surveillance and diagnostic applications, a robust, economical real-time PCR method that can specifically and sensitively detect and identify multiple different circulating variants is needed. The principle of target- failure (TF) real-time PCR is simple. If a target contains a deletion mutation, then there is a mismatch with the primer or probe, and the real-time PCR will fail to amplify the target. In this study, we designed and evaluated a novel multiplex RT real-time PCR (MPL RT-rPCR) based on the principle of target failure to detect and identify different variants of SARS-CoV-2 directly from the nasopharyngeal swabs collected from COVID-19 suspected cases. The primers and probes were designed based on the specific deletion mutations of current circulating variants. To evaluate the results from the MPL RT-rPCR, this study also designed nine pairs of primers for amplifying and sequencing of nine fragments from the S gene containing mutations of known variants. We demonstrated that (i) our MPL RT-rPCR was able to accurately detect multiple variants that existed in a single sample; (ii) the limit of detection of the MPL RT-rPCR in the detection of the variants ranged from 1 to 10 copies for Omicron BA.2 and BA.5, and from 10 to 100 copies for Delta, Omicron BA.1, recombination of BA.1 and BA.2, and BA.4; (iii) between January and September 2022, Omicron BA.1 emerged and co-existed with the Delta variant during the early period, both of which were rapidly replaced by Omicron BA.2, and this was followed by Omicron BA.5 as the dominant variant toward the later period. Our results showed that SARS-CoV-2 variants rapidly evolved within a short period of time, proving the importance of a robust, economical, and easy-to-access method not just for epidemiological surveillance but also for diagnoses around the world where SARS-CoV-2 variants remain the WHO's highest health concern. Our highly sensitive and specific MPL RT-rPCR is considered suitable for further implementation in many laboratories, especially in developing countries.