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Background: Fungi are common opportunistic pathogens that pose a significant threat to immunocompromised patients, particularly when late detection occurs. Methods: In this study a multiplex real-time PCR has been developed for simultaneous detection of common fungal pathogens associated with invasive mycoses in a diagnostic setting. Results: The specificity of the assay was rigorously tested on 40 types of organisms (n = 65), demonstrating 100% specificity. The limit of detection was determined to be 100 pg/μl (106 copies/μl), achievable within a rapid 3-h timeframe. The PCR assay efficiency exhibited a range between 89.77% and 104.30% for each target organism, with linearity falling between 0.9780 and 0.9983. Conclusion: This multiplex real-time PCR assay holds promise for enhancing the timely and accurate diagnosis of invasive mycoses, particularly in immunocompromised patient populations. [ABSTRACT FROM AUTHOR]

Introduction: 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. Methods: 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. Results: The assay’s detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no crossreactivity 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%). Discussion: 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. [ABSTRACT FROM AUTHOR]

Infectious diseases caused by fungal sources are of great interest owing to their increasing prevalence. Invasive fungal infections, including invasive pulmonary aspergillosis caused by Aspergillus fumigatus, and Pneumocystis pneumonia caused by Pneumocystis jirovecii, are significant causes of morbidity and mortality among immunocompromised patients. The accurate and timely detection of these pathogens in this high-risk population is crucial for effective patient management. We developed a multiplex real-time polymerase chain reaction (PCR) assay, RF2 mRT-PCR, specifically designed to detect two respiratory fungi, P. jirovecii and A. fumigatus, and evaluated its performance in specimens of patients with lower respiratory tract infection. The performance was evaluated using 731 clinical samples, 55 reference species, and one synthetic DNA. The reproducibility test yielded a probit curve with a lower limit of detection of 19.82 copies/reaction for P. jirovecii and 64.20 copies/reaction for A. fumigatus. The RF2 mRT-PCR assay did not cross-react with non-A. fumigatus Aspergillus species or other common bacterial and viral species, and showed 100% in vitro sensitivity and specificity with reference assays. Additionally, it simultaneously detected A. fumigatus and P. jirovecii in co-infected samples. Therefore, the RF2 mRT-PCR assay is an efficient and reliable tool for in vitro diagnosis of A. fumigatus and P. jirovecii pulmonary infections. [ABSTRACT FROM AUTHOR]

Cervical cancer related to high-risk human papillomavirus (HR-HPV) is the second female cancer in Mauritania (Northwest Sahelian Africa). We assessed the distribution of HPV genotypes in Mauritanian women with high-grade cervical intraepithelial neoplasia (CIN2/3) or invasive cervical cancer (ICC). A prospective study was conducted in the Centre Hospitalier National, Nouakchott, Mauritania, to collect cervical biopsies among women suspected of CIN2/3 or cancer. HPV DNA detection and genotyping were carried out from formalin-fixed, paraffin-embedded biopsies using multiplex PCR (Human Papillomavirus Genotyping Real-Time PCR Kit, Bioperfectus Technologies Co., Taizhou, China). Fifty biopsies were included from women (mean age: 56.7 years) suffering from CIN2/3 (28.0%) and ICC (72.0%) which corresponded to 32 (64.0%) squamous cell carcinomas (SCC) and 4 (8.0%) adenocarcinomas (ADC). HPV DNA detection was successful in 47 (94.0%) samples. The most prevalent HR-HPV genotypes were HPV-45 (40.4%), HPV-16 (38.3%), HPV-39 and HPV-52 (23.4%), HPV-33 (17.0%), HPV-18 (14.9%), HPV-35 (4.2%), and HPV-56 (2.1%). The majority (93.6%) of HPV-positive biopsies contained at least one HPV type covered by the 9-valent Gardasil-9® vaccine, and 40.9% were infected by multiple vaccine HPV genotypes. To eradicate cervical cancer in Mauritania, prophylactic HPV vaccination must be combined with primary molecular screening of cervical HR-HPV infection. [ABSTRACT FROM AUTHOR]

Introduction: Although diarrheal diseases are highly preventable, they remain one of the leading causes of mortality and morbidity in the pediatric population in developing countries. Knowledge of etiological agents is essential to implement public health programs. Viral infections are the most common cause of acute infectious diarrhea among children. There is a paucity of information regarding viral enteropathogens responsible for diarrhea in India, as many hospitals do not routinely carry out diagnostic tests for viral detection. Aim and Objectives: To estimate the prevalence of viral enteropathogens in children under five years of age presenting with acute diarrhea using real-time multiplex polymerase chain reaction (PCR) and its correlation with clinico-demographic features. Methodology: A cross-sectional study was carried out in a tertiary care center in Maharashtra from January 2016 to June 2019. Our study included 300 children who were under five years old with acute diarrhea. Standard protocols were advocated for the collection and transportation of stool samples. All samples were subjected to real-time multiplex PCR (Light cycler 480 Roche) using Fast Track Diagnostic kits for viral gastroenteritis. Data analysis was done using descriptive and analytical methods. Results: In our study, 57% of samples were positive for viral enteropathogens. Rotavirus (32%) was the most common isolate, followed by Norovirus Genogroup II (GII) (17%). The majority of pathogens were isolated from children less than 2 years of age. Conclusion: There is a need to test stool specimens of clinically confirmed diarrheal patients for enteric virus. This will avoid blind antibiotic treatment.

ObjectiveTo study the prevalence and antimicrobial resistance characteristics of Salmonella enterica serovar typhimurium（S. typhimurium）and monophasic S. typhimurium from clinical patients and food in Henan Province from 2015 to 2022.MethodsThree hundred and ninety-nine strains of Salmonella from food and clinical patients in Henan Province were serotyped according to the Kauffmann White scheme by slide agglutination and multiplex real-time PCR method. Microbroth dilution method was used to test antimicrobial resistance of S. typhimurium. Statistical analysis was carried out using Chi‑square test.ResultsThree hundred and ninety-nine strain were composed of two hundred twenty-five monophasic S. typhimurium （56.39%） and one hundred seventy-four S. typhimurium. The imipenem and trimethoprim/ sulfamethoxazole resistant isolates were found since 2017. The antimicrobial resistance rate of two Salmonella strains to ampicillin， chloramphenicol and tetracycline was higher， reaching more than 50%. The antimicrobial resistance rate of monophasic S. typhimurium to ampicillin and gentamicin was significantly higher than that of S. typhimurium （P

Introduction: 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. Methods: 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. Results: 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 realtime 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. Discussion: 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. [ABSTRACT FROM AUTHOR]

Introduction: Although diarrheal diseases are highly preventable, they remain one of the leading causes of mortality and morbidity in the pediatric population in developing countries. Knowledge of etiological agents is essential to implement public health programs. Viral infections are the most common cause of acute infectious diarrhea among children. There is a paucity of information regarding viral enteropathogens responsible for diarrhea in India, as many hospitals do not routinely carry out diagnostic tests for viral detection. Aim and Objectives: To estimate the prevalence of viral enteropathogens in children under five years of age presenting with acute diarrhea using real-time multiplex polymerase chain reaction (PCR) and its correlation with clinico-demographic features. Methodology: A cross-sectional study was carried out in a tertiary care center in Maharashtra from January 2016 to June 2019. Our study included 300 children who were under five years old with acute diarrhea. Standard protocols were advocated for the collection and transportation of stool samples. All samples were subjected to real-time multiplex PCR (Light cycler 480 Roche) using Fast Track Diagnostic kits for viral gastroenteritis. Data analysis was done using descriptive and analytical methods. Results: In our study, 57% of samples were positive for viral enteropathogens. Rotavirus (32%) was the most common isolate, followed by Norovirus Genogroup II (GII) (17%). The majority of pathogens were isolated from children less than 2 years of age. Conclusion: There is a need to test stool specimens of clinically confirmed diarrheal patients for enteric virus. This will avoid blind antibiotic treatment. [ABSTRACT FROM AUTHOR]

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Background Fungi are common opportunistic pathogens that pose a significant threat to immunocompromised patients, particularly when late detection occurs. Methods In this study a multiplex real-time PCR has been developed for simultaneous detection of common fungal pathogens associated with invasive mycoses in a diagnostic setting. Results The specificity of the assay was rigorously tested on 40 types of organisms (n = 65), demonstrating 100% specificity. The limit of detection was determined to be 100 pg/μl (106 copies/μl), achievable within a rapid 3-h timeframe. The PCR assay efficiency exhibited a range between 89.77% and 104.30% for each target organism, with linearity falling between 0.9780 and 0.9983. Conclusion This multiplex real-time PCR assay holds promise for enhancing the timely and accurate diagnosis of invasive mycoses, particularly in immunocompromised patient populations.

Viruses infecting plants of the genus Rubus are among the pathogens that can cause significant economic losses to growers. As there is no effective treatment, it is necessary to plant healthy propagation material to prevent their spreading. To test plant material of the genus Rubus, real-time PCR-based multiplex detection systems have been developed for the diagnostics of known viruses: Black raspberry necrosis virus (BRNV), Raspberry ringspot virus (RpRSV), Raspberry bushy dwarf virus (RBDV), Strawberry latent ringspot virus (SLRSV), Rubus yellow net virus (RYNV); and the newly discovered Raspberry rubodvirus 1 (RaRV1) and Raspberry enamovirus 1 (RaEV1). The aim of the work was to verify the functionality of these systems on samples from living plants and to evaluate the presence of each virus in five categories corresponding to different origins of samples obtained in the Czech Republic. The results of the testing show that out of a total of 297 samples tested, 72 % of the plants were found to be infected with either a single virus or co-infected with multiple viruses. The most frequently occurring virus was RYNV, which was found in 51 % of the plants tested.

Background: Ureaplasma spp. is an endemic microorganism that causes placental chorioamnionitis or preterm delivery in pregnant women, and the occurrence of bronchopulmonary dysplasia or intraventricular hemorrhaging in preterm infants after birth, although the pathogenicity of Ureaplasma remains controversial. The association between Ureaplasma exposure and the symptoms or outcomes of infected mothers or their infants born at term remains poorly understood. We investigated the clinical characteristics of preterm and term infants with or without Ureaplasma in their gastric fluid. Methods: Gastric fluid samples were collected from 47 newborns in the neonatal intensive-care unit immediately after birth and tested using multiplex polymerase chain reaction (PCR) assays targeting Ureaplasma spp., Ureaplasma parvum, and Ureaplasma urealyticum. The clinical findings and outcomes of the neonates and their mothers were retrospectively evaluated. Results: Ureaplasma spp. were detected in 9/47 samples (19%) by multiplex PCR assays. In all cases, the subspecies was U. parvum. The Ureaplasma-positive group had a significantly higher incidence of chorioamnionitis in utero than the Ureaplasma-negative group. Regarding preterm infants, the IgM levels in the Ureaplasma-positive group were significantly higher than in the Ureaplasma-negative group. In contrast, in term infants, the rates of a non-reassuring fetal status, a maternal fever, and maternal leukocyte counts and maternal C-reactive protein levels within five days before delivery in the Ureaplasma-positive group were significantly higher than those in the Ureaplasma-negative group. All three extremely-low-birth-weight infants with Ureaplasma developed bronchopulmonary dysplasia. The length of hospitalization in the Ureaplasma-positive group was almost same as that in the Ureaplasma-negative group for term infants. Conclusion: Mothers or their fetuses with exposure to Ureaplasma expressed characteristic clinical features during pregnancy and after birth.

The Salmonella enterica serovars Typhimurium (S. Typhimurium), Heidelberg (S. Heidelberg), and their monophasic variants (S. 1,4,[5],12:i:-, S. 1,4,[5],12:r:- and S. 1,4,[5],12:-:1,2) are highly disseminated in poultry farming and can contaminate chicken meat, eggs, and other foods of avian origin. A time-consuming bacteriological and serological analysis is usually required to identify serovars by traditional methods. Incomplete and inconclusive serological results are frequent in routine analysis, mainly due to the occurrence of bacterial isolates presenting similar antigenic profiles. Molecular biology assays have been developed to improve the detection of specific Salmonella serovars and strains. This study aimed to develop a multiplex real-time PCR (SHTAmp) for the rapid DNA detection of S. Typhimurium, S. Heidelberg, and their monophasic variants from poultry samples. The methodology was used in the analysis of 147 field isolates from Brazilian poultry flocks previously evaluated with serological analysis. The results demonstrated that it was able to specifically and rapidly detect 21 S. Typhimurium and 57 S. Heidelberg isolates with complete antigenic formulae. Furthermore, SHTAmp was able to differentiate nine S. Typhimurium and 44 S. Heidelberg isolates with incomplete serological formulae (monophasic and aphasic variants). The complete methodology was also successfully used to detect these bacteria directly from 34 poultry samples after pre-enrichment in buffered peptone water (BPW). In conclusion, SHTAmp is a fast and accurate method to detect the two frequent and concerning serovars S. Typhimurium and S. Heidelberg directly from poultry samples.

Introduction: Porcine viral diarrhea is a common clinical disease, which results in highmortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. Methods: A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. Results: This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/µL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. Discussion: The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations. [ABSTRACT FROM AUTHOR]

In Vietnam, due to the lack of facilities to detect respiratory viruses from patients' specimens, there are only a few studies on the detection of viral pathogens causing pneumonia in children, especially respiratory syncytial virus (RSV) and adenovirus (Adv). Here, we performed a cross-sectional descriptive prospective study on 138 children patients from 2 to 24 months old diagnosed with severe pneumonia hospitalized at the Respiratory Department of Children's Hospital 1 from November 2021 to August 2022. The number of patients selected in this study was based on the formula n = ([Z(1 − α/2)]2 × P [1 − P])/d2, with α = 0.05, p = 0.5, and d = 9%, and the sampling technique was convenient sampling until the sample size was met. A rapid test was used to detect RSV and Adv from the nasopharyngeal swabs and was conducted immediately after the patient's hospitalization. Laboratory tests were performed, medical history interviews were conducted, and nasotracheal aspirates were collected for multiplex real-time PCR (MPL-rPCR) to detect viral and bacterial pathogens. The results of the rapid test and the MPL-rPCR in the detection of both pathogens were the same at 31.9% (44/138) for RSV and 8.7% (7/138) for Adv, respectively. Using MPL-rPCR, the detection rate was 21% (29/138) for bacterial pathogens, 68.8% (95/138) for bacterial–viral co-infections, and 6.5% (9/138) for viral pathogens. The results showed few distinctive traits between RSV-associated and Adv-associated groups, and the Adv group children were more prone to bacterial infection than those in the RSV group. In addition, the Adv group experienced a longer duration of treatment and a higher frequency of re-hospitalizations compared to the RSV group. A total of 100% of Adv infections were co-infected with bacteria, while 81.82% of RSV co-infected with bacterial pathogens (p = 0.000009). This study might be one of the few conducted in Vietnam aimed at identifying viral pathogens causing severe pneumonia in children. [ABSTRACT FROM AUTHOR]

Severe acute respiratory tract infections (SARIs) has been well described in South Africa with seasonal patterns described for influenza and respiratory syncytial virus (RSV), while others occur year-round (rhinovirus and adenovirus). This prospective syndromic hospital-based surveillance study describes the prevalence and impact of public interventions on the seasonality of other respiratory pathogens during the coronavirus disease-19 (COVID-19) pandemic. This occurred from August 2018 to April 2022, with 2595 patients who met the SARS case definition and 442 controls, from three sentinel urban and rural hospital sites in South Africa. Naso/oro-pharyngeal (NP/OP) swabs were tested using the FastTrack Diagnostics® Respiratory pathogens 33 (RUO) kit. Descriptive statistics, odds ratios, and univariate/multivariate analyses were used. Rhinovirus (14.80%, 228/1540) and Streptococcus pneumoniae (28.50%, 439/1540) were most frequently detected in NP/OP swabs and in children <1 years old (35%, 648/1876). Among others, pathogens associated with SARI cases causing disease were influenza A&B, HRV, RSV, hCoV 229e, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pre-COVID-19, seasonal trends of these pathogens correlated with previous years, with RSV and influenza A seasons only resuming after the national lockdown (2021). It is evident that stringent lockdown conditions have severe impacts on the prevalence of respiratory tract infections. [ABSTRACT FROM AUTHOR]

Respiratory infections are one of the world's most common infectious diseases. Following the species, numbers, and seasonal distribution of acute respiratory agents is important for the protection of public health. Our study aimed to determine the effect of the COVID-19 pandemic on the circulation and seasonal distribution of non-SARS-CoV-2 respiratory tract agents and research on non-SARS-CoV-2 agents. The results of the Multiplex PCR respiratory panel of 3702 nasopharyngeal swab samples sent between January 2018 and December 2021 were evaluated retrospectively. Scientific articles on acute respiratory infections between 2010 and 2021 from Turkey were analyzed in Scopus for bibliometric analysis. 1.382 pathogens were detected. During the pandemic, the number of non-SARS-CoV-2 pathogens was found to be statistically significantly lower than before the pandemic. It was determined that while the most frequent agent before the pandemic was the Adenovirus, the most frequent agent was the RSV-A during the pandemic. Our network analysis of keywords indicated that academic interest in 2020-21 was directed toward COVID-19, which coincides with the pandemic period. Our study determined the fact that the incidence, species, and seasonal distribution of non-SARS-COV-2 respiratory agents changed after the onset of the pandemic. Increasing the identification and following-up of these pathogens in health organizations and also presenting these data to literature and sharing with academics is important. We are of the opinion that the results of our study shall shed light on the epidemiology of changing respiratory infections and the prevention and following-up of future health problems. [ABSTRACT FROM AUTHOR]

The Salmonella enterica serovars Typhimurium (S. Typhimurium), Heidelberg (S. Heidelberg), and their monophasic variants (S. 1,4,[5],12:i:-, S. 1,4,[5],12:r:- and S. 1,4,[5],12:-:1,2) are highly disseminated in poultry farming and can contaminate chicken meat, eggs, and other foods of avian origin. A time-consuming bacteriological and serological analysis is usually required to identify serovars by traditional methods. Incomplete and inconclusive serological results are frequent in routine analysis, mainly due to the occurrence of bacterial isolates presenting similar antigenic profiles. Molecular biology assays have been developed to improve the detection of specific Salmonella serovars and strains. This study aimed to develop a multiplex real-time PCR (SHTAmp) for the rapid DNA detection of S. Typhimurium, S. Heidelberg, and their monophasic variants from poultry samples. The methodology was used in the analysis of 147 field isolates from Brazilian poultry flocks previously evaluated with serological analysis. The results demonstrated that it was able to specifically and rapidly detect 21 S. Typhimurium and 57 S. Heidelberg isolates with complete antigenic formulae. Furthermore, SHTAmp was able to differentiate nine S. Typhimurium and 44 S. Heidelberg isolates with incomplete serological formulae (monophasic and aphasic variants). The complete methodology was also successfully used to detect these bacteria directly from 34 poultry samples after pre-enrichment in buffered peptone water (BPW). In conclusion, SHTAmp is a fast and accurate method to detect the two frequent and concerning serovars S. Typhimurium and S. Heidelberg directly from poultry samples. [ABSTRACT FROM AUTHOR]

Ureaplasma spp. is an endemic microorganism that causes placental chorioamnionitis or preterm delivery in pregnant women, and the occurrence of bronchopulmonary dysplasia or intraventricular hemorrhaging in preterm infants after birth, although the pathogenicity of Ureaplasma remains controversial. The association between Ureaplasma exposure and the symptoms or outcomes of infected mothers or their infants born at term remains poorly understood. We investigated the clinical characteristics of preterm and term infants with or without Ureaplasma in their gastric fluid. Gastric fluid samples were collected from 47 newborns in the neonatal intensive-care unit immediately after birth and tested using multiplex polymerase chain reaction (PCR) assays targeting Ureaplasma spp. , Ureaplasma parvum, and Ureaplasma urealyticum. The clinical findings and outcomes of the neonates and their mothers were retrospectively evaluated. Ureaplasma spp. were detected in 9/47 samples (19%) by multiplex PCR assays. In all cases, the subspecies was U. parvum. The Ureaplasma -positive group had a significantly higher incidence of chorioamnionitis in utero than the Ureaplasma -negative group. Regarding preterm infants, the IgM levels in the Ureaplasma -positive group were significantly higher than in the Ureaplasma -negative group. In contrast, in term infants, the rates of a non-reassuring fetal status, a maternal fever, and maternal leukocyte counts and maternal C-reactive protein levels within five days before delivery in the Ureaplasma -positive group were significantly higher than those in the Ureaplasma -negative group. All three extremely-low-birth-weight infants with Ureaplasma developed bronchopulmonary dysplasia. The length of hospitalization in the Ureaplasma -positive group was almost same as that in the Ureaplasma -negative group for term infants. Mothers or their fetuses with exposure to Ureaplasma expressed characteristic clinical features during pregnancy and after birth. [ABSTRACT FROM AUTHOR]

Background and Aims: Second to COVID‐19 pandemic, other viral respiratory infections are still important causes of human diseases or co‐infections. Hence, the present study was carried out to investigate the common respiratory viruses in patients with respiratory illness diagnosed negative for severe acute respiratory syndrome coronavirus‐2 in primary screening. Methods: In a cross‐sectional study, a real‐time PCR was carried out using HiTeq. 17 Viro Respiratory pathogen One Step RT‐PCR Kit (Genova, Bonda Faravar, Bioluence, Tehran, Iran). Results: A total of 311 individuals (mean age ± SD: 48.2 ± 21.7 years, range: 1–97 years) underwent second PCR. Among these, 161 (51.7%) were female. In total, 55 (17.6%) cases (mean age ± SD: 45.7 ± 18.1 years) were found positive for respiratory viruses panel in the second PCR. The HCoV‐OC43/HKU1 was in 5.4% (17/311), Flu A in 4.5% (14/311), HCoV‐229E/NL63 in 2.8% (9/311), HMPV in 1.9% (6/311), HPiV 1, 2, 3 in 1.2% (4/311), HRSV in 0.9% (3/311), and HAdV in 0.6% (2/311) of the cases studies. Also, co‐infection was detected in 4 samples (1.2%). In addition, sore throat (0.028), headache (p = 0.016), and body pain (p = 0.0001) were statistically the most significant symptoms in studied cases. Conclusion: According to the findings of our study, respiratory virus infections and co‐infections were 17.6% and 1.2% frequent, respectively. Interestingly, nearly half of our positive cases (47.2%) were identified by coronaviruses (ОС43, Е229, NL63, and HKUI), followed by influenza A virus (25.4%). However, for more comprehensive results, we recommend using greater sample size. [ABSTRACT FROM AUTHOR]

Cervical cancer related to high-risk human papillomavirus (HR-HPV) is the second female cancer in Mauritania (Northwest Sahelian Africa). We assessed the distribution of HPV genotypes in Mauritanian women with high-grade cervical intraepithelial neoplasia (CIN2/3) or invasive cervical cancer (ICC). A prospective study was conducted in the Centre Hospitalier National, Nouakchott, Mauritania, to collect cervical biopsies among women suspected of CIN2/3 or cancer. HPV DNA detection and genotyping were carried out from formalin-fixed, paraffin-embedded biopsies using multiplex PCR (Human Papillomavirus Genotyping Real-Time PCR Kit, Bioperfectus Technologies Co., Taizhou, China). Fifty biopsies were included from women (mean age: 56.7 years) suffering from CIN2/3 (28.0%) and ICC (72.0%) which corresponded to 32 (64.0%) squamous cell carcinomas (SCC) and 4 (8.0%) adenocarcinomas (ADC). HPV DNA detection was successful in 47 (94.0%) samples. The most prevalent HR-HPV genotypes were HPV-45 (40.4%), HPV-16 (38.3%), HPV-39 and HPV-52 (23.4%), HPV-33 (17.0%), HPV-18 (14.9%), HPV-35 (4.2%), and HPV-56 (2.1%). The majority (93.6%) of HPV-positive biopsies contained at least one HPV type covered by the 9-valent Gardasil-9® vaccine, and 40.9% were infected by multiple vaccine HPV genotypes. To eradicate cervical cancer in Mauritania, prophylactic HPV vaccination must be combined with primary molecular screening of cervical HR-HPV infection.

Purpose: We aimed to evaluate the performance of the FilmArray (FA) meningitis/encephalitis (ME) panel. Secondarily, we analyzed the false positive (FP) and false negative (FN) results, as well as the predictive values of the technique, regarding the cerebrospinal fluid (CSF) characteristics. Methods: FA is a multiplex real-time PCR detecting 14 of the most common ME pathogens in CSF. All FA performed at our hospital (2018–2022) were retrospectively reviewed. FA was compared to conventional techniques and its performance was assessed based on the final diagnosis of the episode. Results: FA was performed in 313 patients with suspicion of ME. Most patients had altered mental status (65.2%) and fever (61%). Regarding CSF characteristics, 49.8% and 53.7% presented high CSF proteins and pleocytosis, respectively. There were 84 (26.8%) positive FA results, mainly for HSV-1 (10.9%), VZV (5.1%), Enterovirus (2.6%), and S. pneumoniae (1.9%). In the 136 cases where both FA and routine methods were performed, there was a 25.7% lack of agreement. We identified 6.6% FN results, but 28.6% FP, mainly due to HSV-1. This resulted in a high negative predictive value (NPV) of 93.4%, but a positive predictive value (PPV) of 73%. Remarkably, PPV as low as 36.9%, and 70.2%, were found in cases without pleocytosis, or lack of high CSF protein levels, respectively. Conclusion: FA was associated with high NPV, but frequent FP results and low PPV, particularly for HSV-1, and especially in patients without high CSF protein levels or pleocytosis. [ABSTRACT FROM AUTHOR]

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7–100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9–99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

BackgroundIt has been reported that differential diagnosis of bacterial or viral pneumonia and tuberculosis (TB) in infants and young children is complex. This could be due to the difficulty in microbiological confirmation in this age group. In this study, we aimed to assess the utility of a real-time multiplex PCR for diagnosis of respiratory pathogens in children with pulmonary TB.MethodsA total of 185 respiratory samples [bronchoalveolar lavage (15), gastric aspirates (98), induced sputum (21), and sputum (51)] from children aged 3–12 years, attending tertiary care hospitals, Chennai, India, were included in the study. The samples were processed by N acetyl L cysteine (NALC) NAOH treatment and subjected to microbiological investigations for Mycobacterium tuberculosis (MTB) diagnosis that involved smear microscopy, Xpert® MTB/RIF testing, and liquid culture. In addition, DNA extraction from the processed sputum was carried out and was subjected to a multiplex real-time PCR comprising a panel of bacterial and fungal pathogens.ResultsOut of the 185 samples tested, a total of 20 samples were positive for MTB by either one or more identification methods (smear, culture, and GeneXpert). Out of these 20 MTB-positive samples, 15 were positive for one or more bacterial or fungal pathogens, with different cycle threshold values. Among patients with negative MTB test results (n = 165), 145 (87%) tested positive for one or more than one bacterial or fungal pathogens.ConclusionThe results suggest that tuberculosis could coexist with other respiratory pathogens causing pneumonia. However, a large-scale prospective study from different geographical settings that uses such simultaneous detection methods for diagnosis of childhood tuberculosis and pneumonia will help in assessing the utility of these tests in rapid diagnosis of respiratory infections.

Abstract Background and Aims Second to COVID‐19 pandemic, other viral respiratory infections are still important causes of human diseases or co‐infections. Hence, the present study was carried out to investigate the common respiratory viruses in patients with respiratory illness diagnosed negative for severe acute respiratory syndrome coronavirus‐2 in primary screening. Methods In a cross‐sectional study, a real‐time PCR was carried out using HiTeq. 17 Viro Respiratory pathogen One Step RT‐PCR Kit (Genova, Bonda Faravar, Bioluence, Tehran, Iran). Results A total of 311 individuals (mean age ± SD: 48.2 ± 21.7 years, range: 1–97 years) underwent second PCR. Among these, 161 (51.7%) were female. In total, 55 (17.6%) cases (mean age ± SD: 45.7 ± 18.1 years) were found positive for respiratory viruses panel in the second PCR. The HCoV‐OC43/HKU1 was in 5.4% (17/311), Flu A in 4.5% (14/311), HCoV‐229E/NL63 in 2.8% (9/311), HMPV in 1.9% (6/311), HPiV 1, 2, 3 in 1.2% (4/311), HRSV in 0.9% (3/311), and HAdV in 0.6% (2/311) of the cases studies. Also, co‐infection was detected in 4 samples (1.2%). In addition, sore throat (0.028), headache (p = 0.016), and body pain (p = 0.0001) were statistically the most significant symptoms in studied cases. Conclusion According to the findings of our study, respiratory virus infections and co‐infections were 17.6% and 1.2% frequent, respectively. Interestingly, nearly half of our positive cases (47.2%) were identified by coronaviruses (ОС43, Е229, NL63, and HKUI), followed by influenza A virus (25.4%). However, for more comprehensive results, we recommend using greater sample size.

Infectious diseases caused by fungal sources are of great interest owing to their increasing prevalence. Invasive fungal infections, including invasive pulmonary aspergillosis caused by Aspergillus fumigatus, and Pneumocystis pneumonia caused by Pneumocystis jirovecii, are significant causes of morbidity and mortality among immunocompromised patients. The accurate and timely detection of these pathogens in this high-risk population is crucial for effective patient management. We developed a multiplex real-time polymerase chain reaction (PCR) assay, RF2 mRT-PCR, specifically designed to detect two respiratory fungi, P. jirovecii and A. fumigatus, and evaluated its performance in specimens of patients with lower respiratory tract infection. The performance was evaluated using 731 clinical samples, 55 reference species, and one synthetic DNA. The reproducibility test yielded a probit curve with a lower limit of detection of 19.82 copies/reaction for P. jirovecii and 64.20 copies/reaction for A. fumigatus. The RF2 mRT-PCR assay did not cross-react with non-A. fumigatus Aspergillus species or other common bacterial and viral species, and showed 100% in vitro sensitivity and specificity with reference assays. Additionally, it simultaneously detected A. fumigatus and P. jirovecii in co-infected samples. Therefore, the RF2 mRT-PCR assay is an efficient and reliable tool for in vitro diagnosis of A. fumigatus and P. jirovecii pulmonary infections.

Abstract Grapevine downy mildew (GDM) caused by Plasmopara viticola is a recurrent disease of wine grapes in the Ningxia Hui Autonomous Region (Ningxia) of northwestern China. However, the primary infectious pathogen in this region has not been thoroughly investigated. In this study, a multiplex real-time PCR assay was utilized to quantify P. viticola in soil, leaf residues, and asymptomatic leaf samples from ten commercial vineyards in two consecutive years to better understand the epidemiological significance of overwintering primary inoculum and its inoculum potential before the appearance of the first visual GDM symptoms. The DNA primers and multiplex real-time PCR assays that had been established exhibited specificity towards P. viticola within the test samples. The majority of the asymptomatic leaves (60%), leaf residues (80%), and soil samples (100%) tested positive for P. viticola. In addition, the amount of primary inoculum of P. viticola was found to be lower in soil than in leaf residues. The area under the disease progress curve in terms of the molecular disease index (AUDPC-MDI) was used to evaluate the overall latent P. viticola infection in asymptomatic leaves. Asymptomatic leaves were found to have different levels of P. viticola infection, and high AUDPC-MDIs correlated with a high AUDPC in terms of disease index (AUDPC-DI), with a significant correlation relationship between them (P

Introduction: Increased carbapenem resistance in Klebsiella spp. strains causes high morbidity and mortality. The genes encoded for carbapenemaseare transferrable between different bacterial species. In the present study, we aimed to investigate carbapenem resistance genes in Klebsiella spp. strains. Methodology: Fifty Klebsiella spp. strains were isolated from rectal swabs of patients hospitalized in the neonatal intensive care unit (NICU). All strains were identified with API20E. The minimum inhibitory concentrations (MICs) of carbapenems were determined by the broth dilution method. The major five carbapenem genes (OXA-48, NDM, VIM, KPC, and IMP) were detected by the multiplex real-time PCR method. Results: It was found that 49 (98%) of the strains were resistant to ertapenem (MIC = 2µg/mL) and imipenem(MIC = 4 µg/mL), and 47 (94%) of the strains were resistant to doripenem (MIC = 4 µg/mL)and meropenem(MIC = 4 µg/mL). NDM was detected in 42%, OXA-48 in 16%, and VIM in one (2%) isolate, and NDM + OXA-48 co-existed in 36% of the isolates. The KPC and IMP genes were not detected. Conclusions: NDM and NDM co-existing with OXA-48 were prevalent in the NICU of Istanbul Medical Faculty Hospital. Paying attention to the hand hygiene of healthcare workers, screening of rectal swabs of hospitalized patients for the presence of carbapenem resistance strains, and isolation of infected patients can effectively control the spread of carbapenem-resistant strains. [ABSTRACT FROM AUTHOR]

Access to clean and safe drinking water still remains a major challenge in the developing world, causing public health risks in terms of waterborne infections, especially in rural areas of sub-Saharan Africa. This study aimed to track and detect enteric pathogens (Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Shigella flexneri, and Campylobacter jejuni subsp. jejuni) in rural water sources. It also sought to establish a correlation between these pathogens and the sources of faecal pollution. Multiplex qPCR and specific primers and probes were used for detection and tracking. The study successfully correlated the occurrence of target pathogens with sources of human and animal faecal contamination using host-specific genetic markers (BacHum and HF183 for humans, BacCow for cows, Pig-2-Bac for pigs, Cytb for chickens, and BacCan for dogs). The study revealed that enteric pathogens were found in 47.69% and 32.80% of samples during the wet and dry seasons, respectively. These pathogens were associated with human or animal faecal contamination. Correlations between pathogens and contamination sources were significant (p ≤ 0.05), with varying strengths during the wet and dry seasons. The findings emphasize the importance of identifying faecal contamination sources to protect rural communities from waterborne infections. [ABSTRACT FROM AUTHOR]

In Vietnam, due to the lack of facilities to detect respiratory viruses from patients’ specimens, there are only a few studies on the detection of viral pathogens causing pneumonia in children, especially respiratory syncytial virus (RSV) and adenovirus (Adv). Here, we performed a cross-sectional descriptive prospective study on 138 children patients from 2 to 24 months old diagnosed with severe pneumonia hospitalized at the Respiratory Department of Children’s Hospital 1 from November 2021 to August 2022. The number of patients selected in this study was based on the formula n = ([Z(1 − α/2)]2 × P [1 − P])/d2, with α = 0.05, p = 0.5, and d = 9%, and the sampling technique was convenient sampling until the sample size was met. A rapid test was used to detect RSV and Adv from the nasopharyngeal swabs and was conducted immediately after the patient’s hospitalization. Laboratory tests were performed, medical history interviews were conducted, and nasotracheal aspirates were collected for multiplex real-time PCR (MPL-rPCR) to detect viral and bacterial pathogens. The results of the rapid test and the MPL-rPCR in the detection of both pathogens were the same at 31.9% (44/138) for RSV and 8.7% (7/138) for Adv, respectively. Using MPL-rPCR, the detection rate was 21% (29/138) for bacterial pathogens, 68.8% (95/138) for bacterial–viral co-infections, and 6.5% (9/138) for viral pathogens. The results showed few distinctive traits between RSV-associated and Adv-associated groups, and the Adv group children were more prone to bacterial infection than those in the RSV group. In addition, the Adv group experienced a longer duration of treatment and a higher frequency of re-hospitalizations compared to the RSV group. A total of 100% of Adv infections were co-infected with bacteria, while 81.82% of RSV co-infected with bacterial pathogens (p = 0.000009). This study might be one of the few conducted in Vietnam aimed at identifying viral pathogens causing severe pneumonia in children.

Severe acute respiratory tract infections (SARIs) has been well described in South Africa with seasonal patterns described for influenza and respiratory syncytial virus (RSV), while others occur year-round (rhinovirus and adenovirus). This prospective syndromic hospital-based surveillance study describes the prevalence and impact of public interventions on the seasonality of other respiratory pathogens during the coronavirus disease-19 (COVID-19) pandemic. This occurred from August 2018 to April 2022, with 2595 patients who met the SARS case definition and 442 controls, from three sentinel urban and rural hospital sites in South Africa. Naso/oro-pharyngeal (NP/OP) swabs were tested using the FastTrack Diagnostics® Respiratory pathogens 33 (RUO) kit. Descriptive statistics, odds ratios, and univariate/multivariate analyses were used. Rhinovirus (14.80%, 228/1540) and Streptococcus pneumoniae (28.50%, 439/1540) were most frequently detected in NP/OP swabs and in children Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pre-COVID-19, seasonal trends of these pathogens correlated with previous years, with RSV and influenza A seasons only resuming after the national lockdown (2021). It is evident that stringent lockdown conditions have severe impacts on the prevalence of respiratory tract infections.

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2–10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study. [ABSTRACT FROM AUTHOR]

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Grapevine downy mildew (GDM) caused by Plasmopara viticola is a recurrent disease of wine grapes in the Ningxia Hui Autonomous Region (Ningxia) of northwestern China. However, the primary infectious pathogen in this region has not been thoroughly investigated. In this study, a multiplex real-time PCR assay was utilized to quantify P. viticola in soil, leaf residues, and asymptomatic leaf samples from ten commercial vineyards in two consecutive years to better understand the epidemiological significance of overwintering primary inoculum and its inoculum potential before the appearance of the first visual GDM symptoms. The DNA primers and multiplex real-time PCR assays that had been established exhibited specificity towards P. viticola within the test samples. The majority of the asymptomatic leaves (60%), leaf residues (80%), and soil samples (100%) tested positive for P. viticola. In addition, the amount of primary inoculum of P. viticola was found to be lower in soil than in leaf residues. The area under the disease progress curve in terms of the molecular disease index (AUDPC-MDI) was used to evaluate the overall latent P. viticola infection in asymptomatic leaves. Asymptomatic leaves were found to have different levels of P. viticola infection, and high AUDPC-MDIs correlated with a high AUDPC in terms of disease index (AUDPC-DI), with a significant correlation relationship between them (P < 0.01). Additionally, a well-correlated relationship was observed between the disease progress in the previous year and the MDIs of leaf residues and asymptomatic leaves in the following year, as well as the AUDPC-DI (Spearman's correlation coefficient ρ = 0.643, 0.498, and 0.595, respectively) (P < 0.01). These findings provide valuable information for quantifying the primary infection of P. viticola in commercial vineyards. [ABSTRACT FROM AUTHOR]

Wenjing Liu,1 Min Li,2 Yingchun Xu,1 Fengchao Wang,3 Jing Wang,4 Huizhu Wang,2 Xinmin Xu,2 Yajie Wang,2 Hongli Sun1 1Department of Laboratory Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijing, 100730, People’s Republic of China; 2Department of Clinical Laboratory, Beijing Ditan Hospital, Capital Medical University, Beijing, 100015, People’s Republic of China; 3The First Affiliated Hospital of Bengbu Medical College, Anhui, 233004, People’s Republic of China; 4Chongqing Public Health Medical Center, Chongqing, 400036, People’s Republic of ChinaCorrespondence: Hongli Sun, Peking Union Medical College Hospital (Dongdan Campus), No. 1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, 100730, People’s Republic of China, Tel +86-1069159788, Fax +86-1069159766, Email sunhl2010@sina.com Yajie Wang, Department of Clinical Laboratory, Beijing Ditan Hospital, Capital Medical University, Beijing, 100015, People’s Republic of China, Tel +86-13611269270, Email wangyajie@ccmu.edu.cnPurpose: To evaluate the performance of a multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii from sputum.Patients and Methods: Sputum samples (n=537) from patients with suspected invasive fungal infection (IFI) were collected from four centers; they were tested by both multiplex real-time PCR assay and DNA sequencing. DNA sequencing was considered as the reference method, and the performance of the multiplex real-time assay was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The interference experiment, repeatability, reproducibility, and stability of the multiplex real-time PCR assay were also evaluated.Results: The detection performance of the multiplex real-time assay, compared with that of DNA sequencing, for the three pathogens was as follows: sensitivity, specificity, PPV, and NPV for Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were 99.40%, 98.64%, 97.09%, and 99.73%; 100%, 99.59%, 96.36%, and 100.00%; and 99.28%, 98.50%, 95.80%, and 99.75%, respectively. The consistency of the two methods was almost perfect: the kappa value was between 0.97 and 0.98. The minimum detection limit of the multiplex real-time assay for each of the three pathogens was 1250 copies/mL. Interference experiment showed that blood and normally used antifungal drugs had no effect on the results. No cross-reactivity was detected for any bacteria or fungi. In 40 patients, mixed infections by Aspergillus and/or Cryptococcus neoformans and/or Pneumocystis jirovecii were detected by the multiplex real-time assay. Among these patients, those with acquired immune deficiency syndrome (AIDS) ranked first, with Aspergillus and Pneumocystis mixed infection accounting for 75%.Conclusion: The multiplex real-time PCR assay is fast, sensitive, and specific and has good clinical application prospects.Keywords: Aspergillus, Cryptococcus neoformans, Pneumocystis jirovecii, multiplex real-time PCR, sequencing, invasive fungal infection

Background and purpose: Pneumonia is the most common infectious cause of death. This study aimed at investigating the causative agent of pneumonia in nasopharyngeal secretions by PCR method. Materials and methods: This case-control study was carried out in patients (older than 19 years of age) suspected of pneumonia admitted to Qaemshahr Razi Hospital and Sari Imam Khomeini Hospital, Iran 2019. The control group included inpatient and outpatient cases with non-respiratory diseases. The case group included patients who met the clinical and radiological criteria. FTD respiratory pathogens 21 plus kit (Multiplex RT-PCR) was used which covers a significant number of microorganisms. Data were analyzed in SPSS V18. Results: The study included 60 patients with the mean age of 52.13±16.84 years (19-87 years old) and the mean age of the control group was 47.8±16.63 years (21-83 years old). Etiological agents were significantly different between the two groups (P

Background: Acute gastroenteritis is one of the major causes of morbidity and mortality worldwide, especially in children and the elderly. The identification of various diarrhea-causing bacteria using multiplex polymerase chain reaction (PCR) and rapid antigen testing has enabled a more detailed analysis of diarrhea-causing pathogens. Previous reports have a limitation in that they do not include data on multiple infections in which two or more infectious agents are simultaneously detected, and there are no data on clinical information. We investigated various diarrhea-causing bacteria and viruses detected by multiplex real-time PCR for one year at a single institution. Methods: This study included 766 subjects who underwent multiplex real-time PCR testing of direct stool specimens for the purpose of diagnosis from April 2019 to February 2020. The multiplex PCR test used in our study can simultaneously detect 16 types of bacteria and five types of viruses. When two or more pathogens were detected by multiplex real-time PCR, they were confirmed using single conventional PCR or real-time PCR. Demographic, clinical, and laboratory data were collected from electronic medical records (EMR). The detected bacteria and viruses were analyzed according to age and season. Results: Out of a total of 352 stool samples with pathogen detection, 265 (75.3%) were detected as single and 87 (24.7%) showed co-detection. The highest rates of single and co-detection were for Clostridium perfringens, and the highest combination of co-infections was for C. perfringens and Staphylococcus aureus. Conclusions: We demonstrated that different age groups showed varying pathogen distributions. While no special seasonality was found in the monthly distribution, it should be noted that the total number of cases peaked in September. The data presented in our study serves as epidemiologically important basic data. [ABSTRACT FROM AUTHOR]

Simple Summary: At present, mixed infection with multiple pathogens is an important reason for the complexity of swine diseases in China. Among them, African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are extremely important DNA viruses that cause reproductive abnormalities in sows. Therefore, there is an urgent need for rapid and sensitive detection methods that can diagnose three kinds of DNA viruses in the clinic. Herein, a multiplex real-time PCR (qPCR) assay based on TaqMan probes was developed for the simultaneous determination of ASFV, PCV2, and PRV. To ascertain the use of the multiplex real-time qPCR, 383 field specimens from the pig farms of four provinces in East China were collected. The survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. The assay established in this study could be used as a differential diagnostic tool for the monitoring and control of ASFV, PCV2, and PRV in the field. African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 101 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases. [ABSTRACT FROM AUTHOR]

This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China. [ABSTRACT FROM AUTHOR]

Background: The etiological diagnosis of community-acquired pneumonia (CAP) is still a challenge. We compared the conventional culture method and real-time polymerase chain reaction (RT-PCR) for the identification of Streptococcus pneumoniae in severe pediatric CAP.Methods: A retrospective hospital-based study was conducted. From 2012 to 2018, we have selected patients who had peripheral blood and/or pleural fluid collected for etiological investigation by RT-PCR.Results: We included 113 children (median age: 3 years; interquartile range 1-6 years). RT-PCR increased the detection rate of S. pneumoniae by 6.5 times using blood samples and eight times using pleural fluid samples. Patients subjected to RT-PCR showed more prolonged hospitalization (p = 0.006), fewer comorbidities (p = 0.03), presence of pleural effusion (p = 0.001), presence of young forms of leukocytes (p = 0.001) and radiograph with characteristics of pneumonia (p = 0.002). The presence of pleural effusion [odds ratio (OR) = 14.7, 95% confidence interval (CI) 1.6-133.9; p = 0.01] and young forms of leukocytes (OR = 8.9, 95% CI 0.9-84.4; p = 0.05) were risk factors for positive RT-PCR pneumococcal when multivariate analysis was performed.Conclusions: RT-PCR is a reliable method for diagnosing severe CAP using sterile materials and a potentially applicable method in patients with clinical, radiological and non-specific laboratory characteristics of lower respiratory tract infection, especially in complicated cases with pleural effusion. [ABSTRACT FROM AUTHOR]

To determine the distribution of two important virulence factors [lipooligosaccharide (LOS) and capsular polysaccharide (CPS)] in Campylobacter jejuni (C. jejuni) isolated from different sources in China and to develop a rapid screening method for Guillain–Barré syndrome (GBS)-associated strains. Whole-genome sequencing was carried out for 494 C. jejuni strains. The OrthoMCL software was used to define the LOS/CPS gene clusters. CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software. Real-time Polymerase chain reaction (PCR) was developed with the unique sequences of specific CPS types. Nine novel and 29 previously confirmed LOS classes were identified. LOS classes A, B, and C were the most common (48.2%, 238/494) among the 494 strains. Twenty-six capsular types were identified in 448 strains. HS2, HS4c, HS5/31, HS19, and HS8/17 were the most frequent CPS genotypes (58.7%, 263/448). Strains of 17 CPS genotypes (strain number > 5) had one or two prevalent LOS classes (P < 0.05). Multiplex real-time PCR for rapid identification of HS2, HS19, and HS41 was developed and validated with strains of known serotypes. Our results describe the genetic characteristics of the important virulence factors in C. jejuni strains in China. The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China. [ABSTRACT FROM AUTHOR]

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R2 values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.

ABSTRACT The incidence of tickborne infections in the United States has risen significantly. Automation is needed for the increasing demand for testing. The Panther Fusion (Fusion) has an Open Access functionality to perform lab developed tests (LDTs) on a fully automated system. Our laboratory adapted two LDTs on Fusion; a multiplex real-time PCR for Anaplasma phagocytophilum and Ehrlichia chaffeensis (AP/EC) and a Babesia microti (BM) PCR. Limits of detection (LODs) were performed with target region plasmid panels spiked into whole blood. The LODs for AP, BM, and EC on the Fusion were 11, 17, and 10 copies/reaction, respectively. The performance of AP/EC was evaluated with 80 whole blood specimens, including 50 specimens previously positive for AP by our test of record (TOR) and 30 specimens (including 20 AP positive) spiked with EC plasmid. AP was detected in 49 out of 50 positive specimens and EC was detected in all 30 spiked specimens. BM PCR on Fusion was evaluated with 75 whole blood samples, including 16 specimens previously shown to be positive for BM and 59 negative specimens, of which 29 were spiked with BM plasmid DNA. BM was detected in 45 samples as expected. AP/EC and BM PCRs were successfully developed and optimized on the Panther Fusion with performance characteristics comparable to our TOR. These assays complement each other and allow for a modular testing approach for tickborne diseases which have differing clinical presentation. Furthermore, automation of these assays will help the lab meet the increasing demand for testing. IMPORTANCE Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. Here, we describe the development of PCR tests on the highly automated Panther Fusion for three tickborne diseases. The Panther Fusion assays were evaluated using 155 archived whole blood (WB) specimens previously tested for Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Babesia microti, while WB spiked with DNA from plasmid clones of the target regions were used for analytical sensitivity. We demonstrated that the Panther Fusion assays performed similar to the manual PCR tests used clinically in our laboratory and that automation of these tests had no adverse effect on the performance.

[Objectives]Based on self-screening specific target gene ASL18_04625(sa19)and two important toxin genes(type A and type U enterotoxins, sea344 and seu222), a multiplex real-time PCR method was developed for the simultaneous detection of Staphylococcus aureus and its enterotoxins. [Methods]The concentrations of primers and other factors were optimized for the best multiplex real-time PCR reaction system, the kit was assembled in terms of the established detection system, and artificial contaminated samples and actual samples were detected. [Results]The 1.5×Syto9 Green was used as fluorescent dye. The optimal reaction conditions were as follows:Taq enzyme concentration 0.015 U·μL-1; dNTP concentration 0.10 mmol·L-1; the final concentrations of three pairs of primers were 0.05, 0.10 and 0.20 μmol·L-1, respectively. The genomic sensitivity of sa19 was 40.7 pg·μL-1, and that of sea344 and seu222 was 4.07 pg·μL-1. The colony concentration sensitivity of sa19 was 1.19×103 CFU·mL-1, and that of sea344 and seu222 was 1.19×104 CFU·mL-1. The intra-group and inter-group repetition rates of the kit were 100%. Moreover, the results of the kit test showed that there was no significant change after 60 cycles of freezing and thawing, 72 hours of storage in foam box, 90 days of storage at -20 ℃ and 45 days of storage at 4 ℃. Meanwhile, it had good specificity and anti-interference ability. [Conclusions]In this study, the multiplex real-time PCR method for the detection of the foodborne pathogen S.aureus was established with the advantages of rapidity, good specificity and high sensitivity, which was assembled into a commercial kit suitable for practical detection. It had a broad application prospect in the field of food safety determination. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp., Entamoeba histolytica (E. histolytica), and Giardia intestinalis (G. intestinalis) are prevalent in developed and developing countries, including Egypt. These enteric protozoa usually provoke indistinguishable manifestation including diarrhea. There is scarce studies of their true prevalence in Egypt. We aimed to identify the accurate detection of these enteric pathogenic protozoa from human diarrheic samples using a multiplex-PCR (MT-PCR) assay and to evaluate the diagnostic performance of coproscopy and monoplex nested PCR (nPCR). Fecal specimens were collected from 100 diarrheic patients and were examined coproscopically and molecularly using MT-PCR and nPCR assays. Diagnostic yield using MT-PCR, nPCR, and microscopy revealed 25, 24, and 15 cases, respectively, for G. intestinalis and 12, 9, and 2 cases, respectively, for Cryptosporidium spp. Coproscopy showed six positive samples for E. histolytica complex, while E. histolytica copro-DNA was not recovered in any sample. Coproscopy showed lower sensitivity than copro-MT-PCR in identifying Cryptosporidium spp. (17%) and G. intestinalis (60%) with 100% specificity. Coproscopy has limited specificity for detection of E. histolytica complex. Compared to MT-PCR, nPCR showed a sensitivity of (75%) for Cryptosporidium spp. and (96%) for G. intestinalis, with 100% specificity for both parasites. G. intestinalis and Cryptosporidium spp. were the prevailing diarrhea-causing protozoa, while E. histolytica was absent among examined Egyptians. Copro-PCR assays could distinguish between pathogenic E. histolytica and non-pathogenic Entamoeba dispar avoiding misdiagnosis and overmedication. Subsequently, MT-PCR allows simultaneous detection of the three protozoa in a single, affordable, and less time-consuming reaction with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

In this article, we present a multiplex real-time PCR method for a simultaneous, sensitive and specific detection and semi-quantitative estimation of the allergenic species peanut, hazelnut, walnut and cashew in food. Due to the use of multicopy target sequences, a very sensitive detection of the allergenic ingredients was possible. The method was validated in-house as well as by a collaborative trial with 12 laboratories. Within the ring trial, 0.64 mg/kg (i.e. approx. 0.1–0.2 mg of peanut and tree nut-derived protein/kg) could still be detected in a processed cookie matrix, confirmed by results of incurred, processed samples spiked at very low levels between 0.9 and 50 mg/kg of the corresponding allergenic ingredient (peanut, tree nut). In addition, the method revealed good precision data. With regard to quantitative analysis though, insufficient recovery data (bias) were determined in some cases, resulting in measurement uncertainties of more than 50%. [ABSTRACT FROM AUTHOR]

Access to clean and safe drinking water still remains a major challenge in the developing world, causing public health risks in terms of waterborne infections, especially in rural areas of sub-Saharan Africa. This study aimed to track and detect enteric pathogens (Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Shigella flexneri, and Campylobacter jejuni subsp. jejuni) in rural water sources. It also sought to establish a correlation between these pathogens and the sources of faecal pollution. Multiplex qPCR and specific primers and probes were used for detection and tracking. The study successfully correlated the occurrence of target pathogens with sources of human and animal faecal contamination using host-specific genetic markers (BacHum and HF183 for humans, BacCow for cows, Pig-2-Bac for pigs, Cytb for chickens, and BacCan for dogs). The study revealed that enteric pathogens were found in 47.69% and 32.80% of samples during the wet and dry seasons, respectively. These pathogens were associated with human or animal faecal contamination. Correlations between pathogens and contamination sources were significant (p ≤ 0.05), with varying strengths during the wet and dry seasons. The findings emphasize the importance of identifying faecal contamination sources to protect rural communities from waterborne infections.

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2–10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study.

Persistent infection and prolonged shedding of human bocavirus 1 (HBoV1) in children have been reported, and the role of HBoV1 as a sole causative pathogen in acute respiratory infection (ARI) is yet to be established. While the reported prevalence of HBoV infection varies due to different detection methods and sampling criteria, determining the viral and bacterial etiology of HBoV infection using multiplex real-time PCR is yet to be reported. Herein, we aimed to further explore the pathogenicity of HBoV in patients with ARI by screening the viral and bacterial infections in children with ARI in Qingdao and comparing the epidemiological, clinical characteristics, and etiological results. Human bocavirus was identified in 28.1% of the samples, and further sequencing analysis of the detected HBoV confirmed 96.4% as HBoV1. The rate of HBoV as a single viral infection was 75%, and the rate of coinfection with bacteria was 66.1%, suggesting the need for continued monitoring of HBoV in children with ARIs. Clinical characterization suggested that HBoV infection may affect the function of organs, such as the liver, kidney, and heart, and the blood acid–base balance. Additionally, it is essential to promote awareness about the importance of disinfection and sterilization of the hospital environment and standardizing operations. The interactions between HBoV and other pathogens remain to be investigated in further detail in the future. [ABSTRACT FROM AUTHOR]

Background and purpose: Pneumonia is the most common infectious cause of death. This study aimed at investigating the causative agent of pneumonia in nasopharyngeal secretions by PCR method. Materials and methods: This case-control study was carried out in patients (older than 19 years of age) suspected of pneumonia admitted to Qaemshahr Razi Hospital and Sari Imam Khomeini Hospital, Iran 2019. The control group included inpatient and outpatient cases with non-respiratory diseases. The case group included patients who met the clinical and radiological criteria. FTD respiratory pathogens 21 plus kit (Multiplex RT-PCR) was used which covers a significant number of microorganisms. Data were analyzed in SPSS V18. Results: The study included 60 patients with the mean age of 52.13±16.84 years (19-87 years old) and the mean age of the control group was 47.8±16.63 years (21-83 years old). Etiological agents were significantly different between the two groups (P<0.05). According to PCR results, the prevalence of viral and bacterial etiology was 56.7% and 26.7%, respectively, and in 16.7% the PCR was negative. Conclusion: Multiplex RT-PCR is associated with great specificity and specificity and is easily performed. The assay is not low cost but is of great benefit in detecting causative agents and avoiding inappropriate treatments and can reduce further bacterial resistance. [ABSTRACT FROM AUTHOR]