Multiplex Real-time PCR Assay Research Articles

Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus

Avian metapneumovirus (aMPV) represents a significant threat to the poultry industry, exhibiting a high degree of genetic diversity. Of these, the aMPV types A (aMPV-A), B (aMPV-B) and C (aMPV-C) are frequently detected in Chinese waterfowl and live poultry markets. Therefore, the rapid and accurate identification of these subtypes is of paramount importance in order to halt the spread of the disease. In this study, we have developed a multiplex real-time PCR assay endowed with the capacity to simultaneously discriminate aMPV-A, aMPV-B, and aMPV-C. This method demonstrates remarkable specificity, selectively amplifying aMPV-A, aMPV-B, and aMPV-C without cross-reactivity with other common avian pathogens. Furthermore, this method exhibits high sensitivity, with a detection threshold of 8.5 × 102 copies/μL for aMPV-A, aMPV-B, and aMPV-C. Moreover, the assay demonstrates reproducibility, as evidenced by intra- and inter-assay variability, with a coefficient of variation between 0.21% and 1.91%. Additionally, the receiver operating characteristic (ROC) curve analysis demonstrated that the multiplex real-time PCR assay exhibited high specificity and sensitivity (100.0% and 100.0% for aMPV-A, 90.9% and 100.0% for aMPV-B, 100% and 96.8% for aMPV-C) when compared with the classical aMPV real-time RT-PCR. Analyses of field samples (n=105) using the multiplex real-time PCR assay indicated that 35.2% (37/105) of samples were positive for aMPV, of which 29.7% (11/37) for aMPV-A, 32.4% (12/37) for aMPV-B and 37.8% (14/37) for aMPV-C. These data demonstrated that the multiplex real-time PCR assay can be used for epidemiological investigations of tree subtypes of aMPV and that aMPV had been observed to exhibit a proclivity for multiple types of co-infection in the Zhejiang province of China.

Accurate Multiplex qPCR Detection of Epstein–Barr Virus/Cytomegalovirus/BK Virus in Kidney Transplant Patients: Pilot Study

Chronic kidney disease is a really important heath issue, and transplantation is an intervention that can greatly increase patient quality of life and survival. The aim of this study was to perform a comprehensive evaluation of the BK virus, CMV, and EBV in kidney transplant recipients (KTRs); to assess the prevalence of infections; and to test if our detection method would be feasible for use in follow-ups with KTRs. A total of 157 KTRs registered at the Clinical Hospital “Dr. C. I. Parhon”, Iași, Romania, were selected using specific inclusion/exclusion criteria. We tested the blood samples from each patient for BK, EBV, and CMV using a multiplex real-time PCR (qPCR) assay and the TaqMan PCR principle. The highest prevalence was detected for BKV (11/157, 7%), followed by CMV (9/157, 5.7%) and EBV (5/157, 3.2%). By simultaneously detecting three possible nephropathic viruses and oncogenes in KTRs using multiplex real-time PCR, we aimed to optimize their monitoring and follow-up. The prevalence of the tested nephropathogenic viruses—BKV, CMV, and EBV—was comparable to that analyzed in other studies. We demonstrate that the use of qPCR for viral detection in KTRs is a robust, cost-effective method for case monitoring.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Impact of Wastewater Use for Irrigation and Contamination of Lettuce by Enteric Viruses: Case of Ouagadougou Market Gardening Sites, Burkina Faso.

Raw vegetables irrigated with polluted water that may contain enteric viruses can be associated with foodborne viral disease outbreaks. The objective of this study is to investigate the possible transmission of enteric viruses from irrigation water to lettuce. Therefore, we performed a commercial multiplex real-time PCR assay to monitor the occurrence of enteric viruses in irrigation water samples and in raw vegetables that were cultivated at market gardening sites in Ouagadougou, Burkina Faso. Samples were collected from six market gardening sites located in Ouagadougou. RT-PCR was performed to detect norovirus GI, norovirus GII, rotavirus, enteric adenoviruses F (Serotype 40/41), astrovirus and sapovirus (Genogroups G1, 2, 4, 5). From the 10 irrigation water samples and the 80 lettuce samples, three (30%) and twenty-two (27.5%) were positive for enteric viruses, respectively. Norovirus GII, astrovirus and enteric adenoviruses F (Serotype 40/41) were the most frequently detected viruses in lettuce and irrigation water samples. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.

Systemic Acanthamoeba T17 infection in a free-ranging two-toed sloth: case report and literature review of infections by free-living amebas in mammals.

A free-ranging, adult female two-toed sloth (Choloepus hoffmanni) was brought to a wildlife rescue center in Costa Rica with ocular and auricular myiasis and numerous skin lesions. After one month of unsuccessful systemic and topical antimicrobial treatment, the patient died. A postmortem examination was performed, and tissues were examined histologically, confirming disseminated amebic infection with intralesional trophozoites and cysts in the lungs, liver, eye, heart, spleen, and stomach. Immunohistochemistry identified the ameba as Acanthamoeba sp. A multiplex real-time PCR assay, 18S ribosomal DNA PCR, and sequencing performed on formalin-fixed, paraffin-embedded lung tissue confirmed the Acanthamoeba T17 genotype. The Acanthamoeba genus is in the group of free-living amebas that cause infection in humans and animals, and it is ubiquitous in the environment. Acanthamoeba T17 has been isolated from water and soil, but to our knowledge, this genotype has not been implicated in infections of animals previously and has not been reported from Costa Rica. Systemic Acanthamoeba infection has not been described in sloths previously. We provide a comprehensive literature review describing infections by free-living amebas of the genus Acanthamoeba spp., Balamuthia spp., and Naegleria spp. in domestic, zoo, and wild mammals.

Evaluation of a multiplex real-time PCR targeting the β-tubulin gene for the detection and differentiation of Sporothrix schenckii and Sporothrix brasiliensis.

Sporothrix sp. is a thermally dimorphic genus of fungi known to cause subacute or chronic subcutaneous lesions in humans and animals and is the cause of increasing public health concern due to spread of feline-associated cases. Here, we adapted and evaluated a recently described real-time PCR assay targeting the β-tubulin gene to rapidly detect and differentiate two related species, S. schenckii and S. brasiliensis. The assay was tested with 55 S. brasiliensis, 19 S. schenckii, and 85 isolates from other clinically relevant fungi, and showed 100% concordance with reference identification methods. The assay showed high analytical sensitivity with a limit of detection of 1 pg of DNA per microliter of sample for both targets. The assay was further evaluated with 11 fresh and 17 formalin-fixed, paraffin-embedded (FFPE) tissues. This multiplex real-time PCR assay successfully detected the DNA from both S. brasiliensis and S. schenckii isolates as well as S. schenckii from fresh and FFPE tissues. Our results demonstrate this assay performs well and could be a helpful molecular tool to support rapid species identification in cultures and primary specimens.IMPORTANCEHaving available molecular tools to identify and differentiate closely related species will allow clinical, veterinarians, and public health labs to provide diagnostic results with accuracy and short turnaround time for the routine and outbreak response activities.

Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA

Quantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probe-based multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143 bp; nDNA = 71 and 181 bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69 bp mtDNA target and the 71 bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71 bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice.

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.

Abnormal Promoter Methylation of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Gene in the Pathogenesis of Crohn’s Disease

Background: Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Epigenetic modifications, including DNA methylation, are considered an important mechanism in the suppression of gene activity. In this study, we investigated the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of Crohn’s disease (CD). Methods: Colonic mucosa samples were obtained from 15 Iranian patients with IBD and 15 age- and sexmatched healthy controls with no history of autoimmune disease. After the bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR (QM-MSP) assay. Results: Using this approach, we identified that IBD patients showed a decreased level of methylation of the NOD2 promoter in the colonic mucosa than did the healthy controls. (Unmethylated DNA in Crohn's disease vs. healthy controls; 0.128±0.093 vs. 0.025±0.016, P<0.000). Conclusion: According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD. Furthermore, aberrant DNA methylation levels are expected to serve as a clinically useful risk marker.

Terbinafine Resistance in Trichophyton Strains Isolated from Humans and Animals: A Retrospective Cohort Study in Italy, 2016 to May 2024.

Background: In recent decades, globalization and international migration have increased the spread of infectious agents, including dermatophytes. Although considered minor infections, dermatophytoses are highly contagious, and they significantly reduce the quality of life, inducing itching, burning, sleep disturbances, and even depressive states. Moreover, the increasing resistance to antifungals threats the public health and burdens the costs for the healthcare system. Methods: DermaGenius® Resistance Multiplex real-time PCR assay allowed to analyze the terbinafine susceptibility/resistance of 172 Trichophyton strains, which were isolated from human and animal samples collected from 2016 to May 2024 and previously identified by Sanger sequencing. Results: All the 11 animal strains belonged to the T. interdigitale/T. mentagrophytes complex and tested terbinafine sensitive. Out of 161 human strains, 9 (5.6%) showed terbinafine resistance and 7 (4.3%) were identified as T. indotineae. Conclusions: This study provides preliminary data about behavior toward antifungals in animals and finalizes the scientific information currently available about human strains, highlighting the importance of the One Health concept. Moreover, it supports the relevant role of T. indotineae as an emerging dermatophyte with high proportion of terbinafine resistance.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam

ObjectiveThis study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation.ResultsThe rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Development of a Multiplex Real-Time PCR to Disambiguate Culicoides sonorensis within Culicoides variipennis Complex, the Proven Vector of Bluetongue and Epizootic Hemorrhagic Disease Viruses in North America.

Species delimitation of Culicoides complex species can be challenging. Among species within the Culicoides variipennis complex, C. sonorensis is considered the primary vector of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in North America. Morphological identification of C. sonorensis within the C. variipennis complex is laborious, time-consuming, and requires entomology expertise. Therefore, in this study we developed and validated a multiplex real-time PCR for rapid detection and differentiation of C. sonorensis from the two other main cryptic species (C. variipennis and C. occidentalis) within the C. variipennis complex. The assay targets the EF1α gene and has a built-in internal control targeting 18 S. The specificity and the sensitivity of the multiplex real-time PCR were evaluated using morphologically identified reference and field-collected specimens. The multiplex PCR was 100% specific when nucleic acid extracted from C. variipennis, sonorensis, and occidentalis specimens was tested. When nucleic acid extracted from pools of midges was tested, the multiplex PCR was able to detect all three Culicoides species with comparable sensitivity. The multiplex assay, however, failed to detect eight morphologically identified C. sonorensis specimens collected from Alberta in 2014. The EF1α gene sequences of these specimens formed a distinct phylogenetic cluster, amongst those from C. variipennis, sonorensis, and occidentalis, suggesting that they belong to a different species. We hypothesize that those specimens might be C. albertensis, the only other species remaining in the C. variipennis complex with known geographical distribution in North America. We believe that this highly sensitive and specific multiplex real-time PCR assay could be an effective tool for rapid detection and differentiation of C. sonorensis, the known vector of BTV and EHDV, in trap collections in future vector surveillance programs.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose‑d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

Development of a TaqMan-based multiplex real-time PCR for simultaneous detection of porcine epidemic diarrhea virus, Brachyspira hyodysenteriae, and Lawsonia intracellularis.

PEDV, Brachyspira hyodysenteriae, and Lawsonia intracellularis, are highly contagious diarrheal pathogens that have caused significant harm to the global swine industry. Co-infections with multiple pathogens are common, making it challenging to identify the actual causative agents depending only on clinical information. It is crucial to develop a reliable method to simultaneously detect and differentiate these pathogens. Based on the conserved regions of the M gene of PEDV, NADH oxidase gene of B. hyodysenteriae, and the 16S rDNA gene of L. intracellularis, specific probes and primers for the multiplex real-time PCR assay were designed. The concentrations of primers and probes were optimized using a matrix method. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to diarrheal diseases. It showed high sensitivity with a detection limit of 10 copies/μL for B. hyodysenteriae and L. intracellularis, and 100 copies/μL for PEDV, respectively. It also demonstrated high reproducibility and stability with low coefficients of variation. Results from the multiplex real-time PCR method were in complete agreement with the commercial singleplex real-time PCR kit for detecting PEDV, B. hyodysenteriae and L. intracellularis. Clinical data revealed single infection rates of 31.46% for PEDV, 58.43% for B. hyodysenteriae, and 98.6% for L. intracellularis. The co-infection rates were 16.85% for PEDV + B. hyodysenteriae, 31.46% for PEDV + L. intracellularis, 57.86% for B. hyodysenteriae + L. intracellularis, and 16.85% for PEDV + B. hyodysenteriae + L. intracellularis, respectively. The new multiplex real-time PCR method can simultaneously differentiate PEDV, B. hyodysenteriae and L. intracellularis, making it a valuable diagnostic tool for preventing and controlling infectious diseases, as well as aiding in epidemiological investigations.

Longer ICU stay and invasive mechanical ventilation accelerate telomere shortening in COVID-19 patients 1 year after recovery

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes virus-induced-senescence. There is an association between shorter telomere length (TL) in coronavirus disease 2019 (COVID-19) patients and hospitalization, severity, or even death. However, it remains unknown whether virus-induced-senescence is reversible. We aim to evaluate the dynamics of TL in COVID-19 patients 1 year after recovery from intensive care units (ICU). Longitudinal study enrolling 49 patients admitted to ICU due to COVID-19 (August 2020 to April 2021). Relative telomere length (RTL) quantification was carried out in whole blood by monochromatic multiplex real-time quantitative PCR (MMqPCR) assay at hospitalization (baseline) and 1 year after discharge (1-year visit). The association between RTL and ICU length of stay (LOS), invasive mechanical ventilation (IMV), prone position, and pulmonary fibrosis development at 1-year visit was evaluated. The median age was 60 years, 71.4% were males, median ICU-LOS was 12 days, 73.5% required IMV, and 38.8% required a prone position. Patients with longer ICU-LOS or who required IMV showed greater RTL shortening during follow-up. Patients who required pronation had a greater RTL shortening during follow-up. IMV patients who developed pulmonary fibrosis showed greater RTL reduction and shorter RTL at the 1-year visit. Patients with longer ICU-LOS and those who required IMV had a shorter RTL in peripheral blood, as observed 1 year after hospital discharge. Additionally, patients who required IMV and developed pulmonary fibrosis had greater telomere shortening, showing shorter telomeres at the 1-year visit. These patients may be more prone to develop cellular senescence and lung-related complications; therefore, closer monitoring may be needed.

Utilization of multiplex polymerase chain reaction for simultaneous and rapid detection of viral infections from different ocular structures

The impact of viral keratitis (VK) on individuals and society is notable. Early diagnosis and treatment are crucial in managing viral keratitis effectively. Timely intervention with antiviral medications and supportive care can help mitigate the severity of the infection and improve visual outcomes. We examined the prevalence of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), adenovirus (AdV) and herpes simplex virus type 2 (HSV-2) in patients suspected for ocular infections. Patients included in the study exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions. Four different types of tear fluid, corneal samples epithelium, aqueous humor and vitreous humor were taken. After genome extraction, multiplex real-time PCR was used for diagnosis of viruses. 48 (29.6%) out of the total of 162 (100%) eye specimen were positive. The dominant prevalence was VZV (12.3%) and HSV-1 (11.7%) followed by AdV (4.9%) and HSV-2 (0.6%). There were 4 (8.3%) coinfections within the samples (HSV-1 and VZV). Aqueous humor samples demonstrated superior virus detection ability and our only HSV-2 positive sample was from aqueous humor. The utilization of multiplex real-time PCR assays in differential diagnosis of VK holds promise for expeditious diagnoses while also preventing unwarranted antibiotic prescriptions. Moreover, the aqueous humor appears to be a more sensitive site for detecting viral keratitis.

Combined Use of Phenotypic Screening and of a Novel Commercial Assay (REALQUALITY Carba-Screen) for the Rapid Molecular Detection of Carbapenemases: A Single-Center Experience.

Carbapenem resistance is a serious public health threat, causing numerous deaths annually primarily due to healthcare-associated infections. To face this menace, surveillance programs in high-risk patients are becoming a widespread practice. Here we report the performance of the combined use of a recently approved commercial multiplex real-time PCR assay (REALQUALITY Carba-Screen kit) with conventional phenotypic screening. In this three-month study, 479 rectal swabs from 309 patients across high-risk units were evaluated by combining the two approaches. Although the molecular assay showed a higher positivity rate than phenotypic screening (7.1% vs. 5%), it should be noted that the molecular method alone would have missed eight carbapenem-resistant isolates, while using only phenotypic screening would not have detected sixteen isolates. This demonstrates the complementary strengths of each method. Our study confirms the need for a combined approach to maximize the possible clinical impact of this kind of screening, ensuring a more comprehensive detection of resistant strains.

High Prevalence of Colistin-Resistant Encoding Genes Carriage among Patients and Healthy Residents in Vietnam.

Background and Objectives: We aimed to investigate the carriage of colistin-resistant genes among both patients with a history of antibiotic exposure and apparently healthy adults with no recent healthcare contact. Materials and Methods: Stool swabs were collected from healthy people, and specimens were collected at the infection foci from the patients. Eleven primer/probe sets were used to perform the Multiplex Real-Time PCR assay with the QuantiNova Multiplex Probe PCR kit for screening the carriage of colistin-resistant genes (mcr-1 to mcr-10) and 16S rRNA gene as internal control. Results: In total, 86 patients and 96 healthy residents were included. Twenty two patients (25.9%) were positive with at least one colistin-resistance encoding gene. The mcr-1 gene was the most frequent (16.5%), followed by mcr-9, mcr-6, and mcr-4 genes, where the prevalence was 11.8%, 10.6%, and 9.4%, respectively. No patient was positive with mcr-3, mcr-7, and mcr-8 genes. Eight patients (9.4%) were positive with multiple colistin-encoding genes. Twenty-three healthy people (24.0%) were positive with at least one colistin-resistance encoding gene, and the mcr-10 gene was the most frequent (27.0%), followed by the mcr-1, mcr-8, and mcr-9 genes, where the prevalence was 24.3%, 21.6%, and 13.5%, respectively. No person was positive with the mcr-2 and mcr-5 genes. Conclusions: Our findings underscore the urgent need for enhanced surveillance, infection control measures, and stewardship interventions to mitigate the spread of colistin resistance in Vietnam.

Epidemiological characteristics and molecular identification of Plasmodium species among cases of imported malaria in Kuwait during the COVID-19 pandemic.

Cases of imported malaria are reported each year in several malaria non-endemic countries, including Kuwait. PCR testing is the ideal method for identification of the infecting Plasmodium spp. The present study documented the epidemiologic characteristics of molecularly confirmed cases of imported malaria in Kuwait during the first year of COVID-19 pandemic. During the period from February 2020 to February 2021, 100 travelers with suspected malaria who had come from malaria-endemic countries of Africa (n = 60) and Asia (n = 40) were examined. Malaria diagnosis was made by microscopy of blood-stained smears and confirmed by a multiplex real-time PCR assay. Samples with discordant species identification results were sequenced. A total of 27 cases (27%) [P. falciparum, 14; P. vivax, 11; P. ovale, 1; mixed P. falciparum and P. malariae, 1] were detected, of whom 12 came to Kuwait for the first time and 15 were returning after visiting their home countries. Most of the returning travelers (12 out of 15 cases, 80%) had not received malaria chemoprophylaxis. Most cases of falciparum malaria (13/15) were Africans while most of the vivax cases (9/11) were Asians. Malaria was more common among subjects entering Kuwait for the first time (OR = 4.025, CI 1.07,15.1) and illiterates (OR = 13.8, CI 1.8,101.4). In conclusion, imported malaria caused mainly by P. falciparum and P. vivax was an ongoing problem during the COVID-19 pandemic. Travel history and education level were significant predictors of malaria among suspected cases.

Evaluating the efficiency of ELISA, monoplex and multiplex probe-based real-time reverse-transcription PCR assays in the detection of SARS-CoV-2 (COVID-19) and influenza A and B viruses: A cross-sectional study.

The current study aimed to evaluate the efficiency of Enzyme-linked immunosorbent assay (ELISA) assay and monoplex and multiplex real-time reverse-transcription PCR (rRT-PCR) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A and B viruses (Flu A and Flu B). The SARS-CoV-2 -specific IgG and IgM antibodies, as well as, Flu A (H1N1 and H3N2 serotypes) and Flu B virus antibodies were determined by ELISA assay. The one-step qRT-PCR method was used to detect the SARS-CoV-2 in nasopharyngeal swab samples. Furthermore, the presence of Flu A and B viruses was evaluated using probe-based RT-PCR. Simultaneous detection of SARS-CoV-2, Flu A and B viruses was performed by multiplex rRT-PCR assay. SARS CoV-2 IgM and IgG antibodies were detected in 33.3% and 58.3% of patients, respectively. In contrast, the SARS CoV-2 genome was detected in 50% of patients using the one-step monoplex RT-PCR assay. Flu A serotypes H1N1 and H3N2 were found in 16.7% and 8.3% of patients. Probe-based RT-PCR revealed that 39.3% of patients were positive for the Flu A virus. Multiplex rRT-PCR detect the SARS-CoV-2, Flu A, and Flu B in 50%, 39.3%, and 19% of samples, respectively. The sensitivity and specificity of multiplex rRT-PCR assay in comparison to monoplex RT-PCR were 100% and 55%, respectively. Coinfection with SARS-CoV-2, Flu A, and Flu B viruses was found in 9.5% of patients. Multiplex rRT-PCR can be used as a repaid, cost-effective and suitable tool for molecular surveillance of SARS-CoV-2 and Flu A/B viruses.