Multiplex PCR reveals unique trends in pathogen and parasitoid infestations of alfalfa leafcutting brood cells.

The alfalfa leafcutting bee Megachile rotundata Fabricius (HYMENOPTERA: Megachilidae) is an important pollinator for multiple agricultural seed commodities in the United States. Megachile rotundata is a solitary bee that forms brood cocoons where its larvae can develop. During the developmental stages of growth, broods can be preyed upon by multiple different fungal and bacterial pathogens and insect predators and parasitoids, resulting in the loss of the developing larvae. Larval loss is a major concern for alfalfa (Medicago sativa L.) seed producers because they rely on pollinator services provided by Megachile rotundata and reduced pollination rates result in lower yields and increased production costs. In the present study, we examined the taxonomic composition of organisms found within M. rotundata brood cells using a multiplex PCR assay which was developed for the detection of the most common bacterial, fungal, and invertebrate pests and pathogens of M. rotundata larvae. Known pests of M. rotundata were detected, including members of the fungal genus Ascosphaera, the causative agent of chalkbrood. Co-infection of single brood cells by multiple Ascosphaera species was confirmed, with potential implications for chalkbrood disease management. The multiplex assay also identified DNA from more than 2,400 total species including multiple new predators and pathogenetic species not previously documented in associated with M. rotundata brood cells.

Systematic Evaluation of a Novel 6-dye Direct and Multiplex PCR-CE-Based InDel Typing System for Forensic Purposes

Insertion/deletion (InDel) polymorphisms, combined desirable characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are considerable potential in the fields of forensic practices and population genetics. However, most commercial InDel kits designed based on non-Asians limited extensive forensic applications in East Asian (EAS) populations. Recently, a novel 6-dye direct and multiplex PCR-CE-based typing system was designed on the basis of genome-wide EAS population data, which could amplify 60 molecular genetic markers, consisting of 57 autosomal InDels (A-InDels), 2 Y-chromosomal InDels (Y-InDels), and Amelogenin in a single PCR reaction and detect by capillary electrophoresis, simultaneously. In the present study, the DNA profiles of 279 unrelated individuals from the Hainan Li group were generated by the novel typing system. In addition, we collected two A-InDel sets to evaluate the forensic performances of the novel system in the 1,000 Genomes Project (1KG) populations and Hainan Li group. For the Universal A-InDel set (UAIS, containing 44 A-InDels) the cumulative power of discrimination (CPD) ranged from 1–1.03 × 10–14 to 1–1.27 × 10–18, and the cumulative power of exclusion (CPE) varied from 0.993634 to 0.999908 in the 1KG populations. For the East Asia-based A-InDel set (EAIS, containing 57 A-InDels) the CPD spanned from 1–1.32 × 10–23 to 1–9.42 × 10–24, and the CPE ranged from 0.999965 to 0.999997. In the Hainan Li group, the average heterozygote (He) was 0.4666 (0.2366–0.5448), and the polymorphism information content (PIC) spanned from 0.2116 to 0.3750 (mean PIC: 0.3563 ± 0.0291). In total, the CPD and CPE of 57 A-InDels were 1–1.32 × 10–23 and 0.999965, respectively. Consequently, the novel 6-dye direct and multiplex PCR-CE-based typing system could be considered as the reliable and robust tool for human identification and intercontinental population differentiation, and supplied additional information for kinship analysis in the 1KG populations and Hainan Li group.

Optimal encephalitis/meningitis roadmap via precise diagnosis and treatment (IMPROVE): a study protocol for a randomized controlled trial

Background Encephalitis/meningitis brings a heavy disease burden, and the origin of disease remains unknown in 30–40% of patients. It is greatly significant that combinations of nucleic acid amplification and autoimmune antibody testing improves the diagnosis and treatment of encephalitis/meningitis. Moreover, though several diagnostic methods are in clinical use, a recognized and unified diagnosis and treatment process for encephalitis management remains unclear. Methods IMPROVE is a multicenter, open label, randomized controlled clinical trial that aims to evaluate the diagnostic performance, applications, and impact on patient outcomes of a new diagnostic algorithm that combines metagenomic next-generation sequencing (mNGS), multiplex polymerase chain reaction (PCR) and autoimmune antibody testing. The enrolled patients will be grouped into two parallel groups, multiplex PCR test plus autoimmune antibody group (Group I) or the mNGS plus autoimmune antibody group (Group II) with a patient ratio of 1:1. Both groups will be followed up for 12 months. The primary outcomes include the initial time of targeted treatment and the modified Rankin scale score on the 30th day of the trial. The secondary outcomes are the cerebrospinal fluid index remission rate on the 14th day, mortality rate on the 30th day, and an evaluation of diagnostic efficacy. The two groups are predicted to comprise of 484 people in total. Discussion To optimize the roadmap of encephalitis/meningitis, precise diagnosis, and treatment are of great significance. The effect of rapid diagnosis undoubtedly depends on the progression of new diagnostic tests, such as the new multiplex PCR, mNGS, and examination of broad-spectrum autoimmune encephalitis antibodies. This randomized-controlled study could allow us to obtain an accurate atlas of the precise diagnostic ability of these tests and their effect on the treatment and prognosis of patients. Trial registration ClinicalTrial.gov, NCT04946682. Registered 29 June 2021, ‘Retrospectively registered’, https://clinicaltrials.gov/ct2/show/NCT04946682?term=NCT04946682&draw=2&rank=1

Investigation of Children with Acute Gastroenteritis by Multiplex PCR Method in Central Part of Turkey

Objective Gastroenteritis is a disease that affects all age groups, especially children, and causes high mortality and morbidity in all countries. The most common agents of acute gastroenteritis are viral agents. As a result, millions of diarrhea attacks and hospital admissions occur worldwide every year due to viral gastroenteritis. This study uses the multiplex polymerase chain reaction (PCR) method to investigate the viruses that are the causative agents of viral gastroenteritis in the pediatric patient group in Konya, Turkey. Methods Stool samples of 94 patients aged 0 to 18 years sent from Emergency clinics and Pediatric outpatient clinics, Meram Medical Faculty Hospital Pediatric clinics, Konya Necmettin Erbakan University to Medical Microbiology Laboratory with a diagnosis of gastroenteritis between February and December 2018 were included in the study. Stool samples were stored at –80°C until the time of the analysis. Deoxyribonucleic acid/ribonucleic acid isolation from stool samples was performed with EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany) using an automatic extraction system (BioRobot EZ1 system, Qiagen). The presence of astrovirus, rotavirus, adenovirus, norovirus (GI, GII), and sapovirus agents was investigated by the multiplex PCR method (Fast Track Diagnostics, Luxembourg) viral gastroenteritis kit. Results Viral gastroenteritis agents were detected in 56.3% of the patients. One viral agent was detected in 47 (50%) of these patients and at least two viral agents in 6 (6.3%) of them. Norovirus GII was detected in 20 (21.2%) of the children included in the study, adenovirus in 13 (13.8%), rotavirus in 11 (12.8%), astrovirus in 11 (11.7%), sapovirus in 4 (4.2%), and norovirus GI in 1 (1.06%). When the distribution of viral agents was examined by months, the most number of agents were observed (21; 35%) in May, followed by April and June (12; 20%). Considering the distribution of the prevalence of the agents by age, it was seen to be mainly between 0 and 12 months (42%). Conclusion Considering that the most common viral agent in our region is norovirus GII, it will be useful to investigate the norovirus that is not routinely examined in children who are admitted to clinics with the complaint of gastroenteritis. It will be appropriate to examine routinely adenovirus, rotavirus, and norovirus in the laboratory, especially in children with diarrhea and vomiting in the winter and spring months.

Genotyping and Molecular Diagnosis of Hepatitis A Virus in Human Clinical Samples Using Multiplex PCR-Based Next-Generation Sequencing

Hepatitis A virus (HAV) is a serious threat to public health worldwide. We used multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) to derive information on viral genetic diversity and conduct precise phylogenetic analysis. Four HAV genome sequences were obtained using multiplex PCR-based NGS. HAV whole-genome sequence of one sample was obtained by conventional Sanger sequencing. The HAV strains demonstrated a geographic cluster with sub-genotype IA strains in the Republic of Korea. The phylogenetic pattern of HAV viral protein (VP) 3 region showed no phylogenetic conflict between the whole-genome and partial-genome sequences. The VP3 region in serum and stool samples showed sensitive detection of HAV with differences of quantification that did not exceed <10 copies/μL than the consensus VP4 region using quantitative PCR (qPCR). In conclusion, multiplex PCR-based NGS was implemented to define HAV genotypes using nearly whole-genome sequences obtained directly from hepatitis A patients. The VP3 region might be a potential candidate for tracking the genotypic origin of emerging HAV outbreaks. VP3-specific qPCR was developed for the molecular diagnosis of HAV infection. This study may be useful to predict for the disease management and subsequent development of hepatitis A infection at high risk of severe illness.

Mannheimia haemolytica serovars associated with respiratory disease in cattle in Great Britain

Background Mannheimia haemolytica is commonly associated with respiratory disease in cattle worldwide as a cause of fibrinous pneumonia, bronchopneumonia and pleuritis. M. haemolytica is further subdivided into 12 serovars, however not all are considered to be pathogenic in cattle. The study aim was to determine the most common serovars of M. haemolytica associated with respiratory disease in cattle in Great Britain, which is currently unknown and could be useful information for clinicians when considering preventative strategies. Results One hundred four M. haemolytica isolates isolated from bovine clinical pathology and post-mortem samples from pneumonia cases between 2016 and 2018 were tested using a multiplex PCR assay to identify M. haemolytica serovars A1, A2 and A6. 46 isolates (44.2%) typed as M. haemolytica serovar A1, 31 (29.8%) as M. haemolytica serovar A2 and 18 isolates (17.3%) as M. haemolytica serovar A6. Nine isolates (8.7%) were not A1, A2 or A6 so were considered to belong to other serovars or were not typable. Conclusion This study highlights the importance of M. haemolytica serovars other than A1 which may be responsible for respiratory disease in cattle and could help guide the veterinarian when making choices on preventative vaccination programmes.

Epidemiology of Respiratory Pathogens in Children with Severe Acute Respiratory Infection and Impact of the Multiplex PCR Film Array Respiratory Panel: A 2-Year Study

Sever acute respiratory infections (SARIs) are a public health issue that are common in children and are associated with an important morbidity and mortality rate worldwide. Although SARI are mainly caused by viruses, they are still a cause of antibiotic overuse. The use of molecular methods especially real-time multiplex PCR allowed to detect a wide range of respiratory viruses and their subtype as well as some atypical bacteria. The aim of this study was to investigate the epidemiology of respiratory pathogens detected in children admitted with SARI and to highlight the role of real-time multiplex PCR in the rapid diagnosis of viral and bacterial SARI. This work is a descriptive observational study from January 2018 to December 2019 including nasopharyngeal secretions collected from 534 children hospitalised in paediatric department. The detection of respiratory viruses and bacteria was performed by the FilmArray® Respiratory Panel. A total of 387 (72.5%) children were tested positive for at least one respiratory pathogen, and 23.3% of them were coinfected with more than one pathogen. Viral aetiology was found in 91.2% (n = 340). The most common viruses detected were HRV (n = 201) and RSV (n = 124), followed by PIV (n = 35) influenza A (n = 29) and human metapneumovirus (n = 27). Bacteria was found in 8.8% (n = 47), and Bordetella pertussis was the most detected. Respiratory syncytial virus and Bordetella pertussis were significantly higher in infants less than 6 months old. The detection of RSV and influenza A presented a pic in winter, and HMPV was statistically significant in spring ( p < 0.01 ). This study described the epidemiology of respiratory pathogens involved in severe respiratory infections in children that were affected by several factors such as season and age group. It also highlighted the importance of multiplex PCR in confirming viral origin, thus avoiding irrational prescription of antibiotics in paediatric settings.