Molecular Survey of Tick-Borne Haemoparasites of Dogs by Multiplex Polymerase Chain Reaction from Punjab, India.

PURPOSE: Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection. METHODS: Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied. RESULTS: Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection. CONCLUSION: The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs.

Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Background: Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. Aim: It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. Methods: A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. Results: The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. Conclusion: This method provides a sensitive and specific approach to detect issues related to well-being and safety.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

Upper and/or Lower Respiratory Tract Infection Caused by Human Metapneumovirus After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Human metapneumovirus (hMPV) epidemiology, clinical characteristics and risk factors for poor outcome after allogeneic stem cell transplantation (allo-HCT) remain a poorly investigated area. METHODS: This retrospective multicenter cohort study examined the epidemiology, clinical characteristics, and risk factors for poor outcomes associated with human metapneumovirus (hMPV) infections in recipients of allo-HCT. RESULTS: We included 428 allo-HCT recipients who developed 438 hMPV infection episodes between January 2012 and January 2019. Most recipients were adults (93%). hMPV infections were diagnosed at a median of 373 days after allo-HCT. The infections were categorized as upper respiratory tract disease (URTD) or lower respiratory tract disease (LRTD), with 60% and 40% of cases, respectively. Patients with hMPV LRTD experienced the infection earlier in the transplant course and had higher rates of lymphopenia, neutropenia, corticosteroid use, and ribavirin therapy. Multivariate analysis identified lymphopenia and corticosteroid use (>30 mg/d) as independent risk factors for LRTD occurrence. The overall mortality at day 30 after hMPV detection was 2% for URTD, 12% for possible LRTD, and 21% for proven LRTD. Lymphopenia was the only independent risk factor associated with day 30 mortality in LRTD cases. CONCLUSIONS: These findings highlight the significance of lymphopenia and corticosteroid use in the development and severity of hMPV infections after allo-HCT, with lymphopenia being a predictor of higher mortality in LRTD cases.

Multicenter evaluation of the Acaruiter Respiratory Panel for diagnosis of respiratory tract infections in Chinese children.

IMPORTANCE: Compared to multiplex PCR assays that are available in the Chinese market, the Acaruiter Respiratory Panel (fluorescent PCR) covers a wider range of pathogens including eight viruses, five bacteria, Mycoplasma pneumoniae and Chlamydia pneumoniae, and has high accuracy and effectiveness in the detection of pathogens. This is the first study to evaluate the performance of the Acaruiter Respiratory Panel. This regent may be a promising assay for comprehensive testing for respiratory pathogens from nasopharyngeal swab specimens in Chinese children.

Development and intralaboratory validation of three Arcidae species using a multiplex polymerase chain reaction assay combined with capillary electrophoresis.

Recently, various commercial ark shell products were being sold, and in the case of processed foods, the loss of morphological traits makes species identification visually challenging, which can lead to seafood fraud. Therefore, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously identify three ark shells. The specific PCR amplicon sizes of the generated species-specific primer pairs were observed to be 99 bp for Anadara kagoshimensis, 148 bp for Anadara broughtonii, and 207 bp for Tegillarca granosa. Specificity was confirmed for 17 fish and shellfish, and only the target was amplified without cross-reactivity. The detection limit for the multiplex PCR assay was 1 pg. Furthermore, 31 commercial products were evaluated to assess the developed assay's applicability. Therefore, the analytical approach used in this study can rapidly and accurately identify ark shells in commercial food, and may be used as an authentication tool in the seafood industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01269-2.

Simultaneous and rapid detection of avian respiratory diseases of small poultry using multiplex reverse transcription-Polymerase Chain Reaction assay.

Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput.

Real-life impact of respiratory panel PCR assay on antibiotic prescription in geriatric acute care in the pre-COVID-19 era.

OBJECTIVES: In this era of bacterial resistance, avoiding inappropriate use of antibiotic treatments is of major importance. Respiratory tract infections are frequent among older patients, and differentiating viral from bacterial infections is a challenge. The aim of our study was to evaluate the impact of recently available respiratory PCR testing on antimicrobial prescription in geriatric acute care. METHODS: We performed a retrospective study, including all hospitalized geriatric patients who had had multiplex respiratory PCR testing prescribed from 1st October 2018 to 30th September 2019. The PCR test comprised a respiratory viral panel (RVP) and a respiratory bacterial panel (RBP). PCR testing could be prescribed at any time during hospitalization by geriatricians. Our primary endpoint was antibiotic prescription after viral multiplex PCR testing results. RESULTS: All in all, 193 patients were included, 88 (45.6%) of whom had positive RVP, while none had positive RBP. Patients with positive RVP had significantly fewer antibiotic prescriptions following test results than patients with negative RVP (odds ratio (OR) 0.41, 95% confidence interval (CI) 0.22-0.77; p = 0.004). Among positive-RVP patients, factors associated with antibiotic continuation were presence of radiological infiltrate (OR 12.02, 95%CI 3.07-30.29), and detected Respiratory Syncytial Virus (OR 7.54, 95%CI 1.74-32.65). That said, discontinuation of antibiotic treatment seems safe. CONCLUSION: In this population, the impact of viral detection by respiratory multiplex PCR on antibiotic therapy was low. It could be optimized by means of clearly formulated local guidelines, qualified staff and specific training by infectious disease specialists. Cost-effectiveness studies are necessary.

A specific SNP-based multiplex PCR assay for the simultaneous identification of two biological ingredients for the Chinese patent medicine, Danggui Buxue pill.

Background: An increasing number of Chinese patent medicines (CPM) have been widely used in East Asian and North American countries, and the safety and efficacy of CPM have highly attracted public attention. However, it is difficult to supervise the authenticity of multiple biological ingredients within CPM based on microscopic inspection and physical and chemical detection. The raw materials may have similar characteristics of tissue structures and ergastic substances or similar chemical composition and contents when substitutes and/or adulterants are added. DNA molecular markers have been used to distinguish the biological ingredients within CPM based on conventional PCR assay. However, it was proved to be time- and labor-consuming and reagent-wasting, as multiple PCR amplification strategies were required for identifying the complex species composition within CPM. Here, we took the CPM (Danggui Buxue pill) as an example and aimed to establish a specific SNP-based multiplex PCR assay and simultaneously determine the authenticity of the two biological ingredients (Angelicae Sinensis Radix and Astragali Radix) within this CPM. Methods: We, respectively, designed the species-specific primers based on highly variable nrITS for discriminating Angelicae Sinensis Radix and Astragali Radix from their common substitutes and adulterants. The specificity of the primers was checked through conventional PCR assay and multiplex PCR assay. Furthermore, we used a handcrafted Danggui Buxue pill sample (DGBXP) to optimize annealing temperatures for the primers with multiplex PCR, and the sensitivity was also assessed. Finally, fourteen batches of commercial Danggui Buxue pills were used to verify the stability and practicability of the established multiplex PCR assay. Results: Two pairs of highly species-specific primers for amplifying Angelicae Sinensis Radix and Astragali Radix were screened, and our established multiplex PCR assay showed high specificity and sensitivity (lowest detection concentration: 4.0 × 10-3 ng/µL) at an optimal annealing temperature of 65°C. The method could simultaneously identify both biological ingredients within the Danggui Buxue pill. Conclusion: The specific SNP-based multiplex PCR provided a simple, time-, and labor-saving method for the simultaneous identification of the two biological ingredients within Danggui Buxue pills. This study was expected to provide a novel qualitative quality control strategy for CPM.

Characterisation of genes encoding for extended spectrum ß-lactamase in Gram-negative bacteria causing healthcare-associated infections in Mwanza, Tanzania.

Healthcare-associated infections (HCAIs) caused by extended spectrum ß-lactamase-producing Gram-negative bacteria (ESBL-GNB) increase morbidity and mortality. This cross-sectional study characterised ESBL genes (bla CTX-M, bla TEM and bla SHV) among 30 ceftriaxone-resistant GNB causing HCAIs between January 2022 and July 2022 by multiplex polymerase chain reaction assay at the zonal referral hospital in Mwanza, Tanzania. Twenty-five (83.3%) had at least one ESBL gene, of which 23/25 (92.0%) carried the bla CTX-M gene. Seventy-two percent (18/25) of the GNB-ESBL isolates carried more than one ESBL gene, of which the majority (88.8%; n = 16/25) carried the bla CTX-M and bla TEM genes. Extended spectrum ß-lactamase genes, particularly bla CTX-M, are common among ceftriaxone-resistant GNB causing HCAIs. What this study adds: This study revealed the distribution of genes (bla CTX-M, bla TEM and bla SHV) coding for ESBL production among ceftriaxone resistant GNB causing HCAIs However, all ESBL producing GNB were susceptible towards ceftriaxone-sulbactam indicating that ceftriaxone-sulbactam may be empirically prescribed for treating patients with HCAIs.

Utility of a Viral Vesicular Panel Multiplex Polymerase Chain Reaction Assay for the Diagnosis of Monkeypox, Herpes Simplex, and Varicella Zoster Viruses.

Mpox (monkeypox) represents a diagnostic challenge due to varied clinical presentations and multiple mimics. A commercially available multiplex polymerase chain reaction panel accurately detects mpox virus as well as common mimics (herpes simplex virus, varicella zoster virus) in clinical specimens and could be used in routine clinical, surveillance, and outbreak settings.

Developmental validation of a complementary Y-STR system for the amplification of forensic samples.

In this study, a new complementary Y-STR system that includes 31 loci was developed (DYS522, DYS388, DYF387S1a/b, DYS510, DYS587, DYS645, DYS531, DYS593, DYS617, GATA_A10, DYS622, DYS552, DYS508, DYS447, DYS527a/b, DYS446, DYS459a/b, DYS444, DYS557, DYS443, DYS626, DYS630, DYS526a, DYF404S1a/b, DYS520, DYS518, and DYS526b). This 31-plex Y-STR system, SureID® Y-comp, is designed for biological samples from forensic casework and reference samples from forensic DNA database. To validate the suitability of this novel kit, many developmental works including size precision testing, sensitivity, male specificity testing, species specificity, PCR inhibitors, stutter precision, reproducibility, suitability for use on DNA mixture and parallel testing of different capillary electrophoresis devices were performed. Mutation rates were investigated using 295 DNA-confirmed father-son pairs. The results demonstrate that the SureID® Y-comp Kit is time-efficient, accurate, and reliable for various case-type samples. It possessed a higher discrimination power and can be a stand-alone kit for male identification. Moreover, the simply acquired additional Y-STR loci will be conductive to construct a robust database. Even if various commercial Y-STR kits are used in distinct forensic laboratories, a wider trans-database retrieval will become feasible with the effort of the SureID® Y-comp Kit.

Multiplex PCR Detection of Respiratory Tract Infections in SARS-CoV-2-Negative Patients Admitted to the Emergency Department: an International Multicenter Study during the COVID-19 Pandemic.

Respiratory tract infection (RTI) is a common cause of visits to the hospital emergency department. During the ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), nonpharmaceutical intervention has influenced the rates of circulating respiratory viruses. In this study, we sought to detect RTI etiological agents other than SARS-CoV-2 in emergency department patients from 13 countries in Europe, the Middle East, and Africa from December 2020 to March 2021. We sought to measure the impact of patient characteristics and national-level behavioral restrictions on the positivity rate for RTI agents. Using the BioFire Respiratory Panel 2.0 Plus, 1,334 nasopharyngeal swabs from patients with RTI symptoms who were negative for SARS-CoV-2 were tested. The rate of positivity for viral or bacterial targets was 36.3%. Regarding viral targets, human rhinovirus or enterovirus was the most prevalent (56.5%), followed by human coronaviruses (11.0%) and adenoviruses (9.9%). Interestingly, age stratification showed that the positivity rate was significantly higher in the children's group than in the adults' group (68.8% versus 28.2%). In particular, human rhinovirus or enterovirus, the respiratory syncytial virus, and other viruses, such as the human metapneumovirus, were more frequently detected in children than in adults. A logistic regression model was also used to determine an association between the rate of positivity for viral agents with each country's behavioral restrictions or with patients' age and sex. Despite the impact of behavioral restrictions, various RTI pathogens were actively circulating, particularly in children, across the 13 countries. IMPORTANCE As SARS-CoV-2 has dominated the diagnostic strategies for RTIs during the current COVID-19 pandemic situation, our data provide evidence that a variety of RTI pathogens may be circulating in each of the 13 countries included in the study. It is now plausible that the COVID-19 pandemic will one day move forward to endemicity. Our study illustrates the potential utility of detecting respiratory pathogens other than SARS-CoV-2 in patients who are admitted to the emergency department for RTI symptoms. Knowing if a symptomatic patient is solely infected by an RTI pathogen or coinfected with SARS-CoV-2 may drive timely and appropriate clinical decision-making, especially in the emergency department setting.

[First report of invasive Pomacea snails in Shandong Province].

OBJECTIVE: To characterize the species of invasive Pomacea snails that were discovered for the first time in Shandong Province. METHODS: Pomacea snails samples were collected in the field of Jining City, Shandong Province on October 2021 for morphological identification. Pomacea snails were randomly sampled and genomic DNA was extracted from foot muscle tissues of Pomacea snails for multiplex PCR amplification. The PCR amplification product was sequenced. Then, the sequence was aligned and a phylogenetic tree was created using the software MegAlign 7.1.0. In addition, Angiostongylus cantonensis infection was detected in Pomacea snails with the lung microscopy. RESULTS: A total of 104 living Pomacea snails were collected, and all were characterized as Pomacea spp. based on morphological features. Of 12 randomly selected adult Pomacea snails, multiplex PCR assay and sequencing identified eleven snails as P. canaliculata and one as P. maculata. No A. cantonensis infection was detected in 104 Pomacea snails. CONCLUSIONS: This is the first report of invasive Pomacea snails in Shandong Province, where P. canaliculata and P. maculata are found.

Multiplex PCR Assay for the Identification of Four Species of the Anopheles Leucosphyrus Sub-Group in Malaysia.

The Leucosphyrus Group of mosquitoes are the major simian malaria vectors in Malaysia. Accurate species identification is required to help in curbing the spread of simian malaria. The aim of the study is to provide an accurate molecular method for identifying the four important Anopheles vector species found in Malaysia. Mosquito specimens were collected from various localities in Malaysia, where simian malaria cases were reported. DNA from 122 mosquito specimens was tested to develop a multiplex polymerase chain reaction (PCR) assay. The specificity of this assay was tested against other mosquito species. Molecular identification of the species was further confirmed by analysing the internal transcribed spacer 2 (ITS2) DNA region of the specimens. Anopheles balabacensis and An. latens showed two distinct clades in the phylogenetic tree. The multiplex PCR assay was developed based on the ITS2 region for the identification of Anopheles introlatus (298-299 bp), Anopheles latens (197-198 bp), Anopheles cracens (421-426 bp), and Anopheles balabacensis (224-228 bp). This method will be useful to accurately identify the major Anopheles Leucosphyrus Group species in Malaysia, which are difficult to identify morphologically, to determine the correct vector as well as its geographical distribution.

Multiplex detection of meningitis and encephalitis pathogens: A study from laboratory to clinic.

Introduction: Infectious meningitis and encephalitis (ME) are life-threatening conditions are caused by various pathogens. Conventional laboratory tests with low sensitivity and specificity cannot help with early diagnosis. Methods: A prospective study using the novel multiplex PCR detection for 18 pathogens of ME (MME-18) was conducted to investigate the clinical utilization and the epidemiology characteristics of ME in southwestern China. Patients with suspected intracranial infection were recruited between May and October 2019 at West China Hospital of Sichuan University. The MME-18 was used to detect cerebrospinal fluid, and conventional experiments including cryptococcal capsular antigen detection, GeneXpert, real-time PCR, and clinical feedback were used to verify the result of MME-18. Results: Among 581 tested patients, 139 eligible individuals were enrolled in the study. Among them, Mycobacterium tuberculosis was the most common pathogen in mono-infection. Viruses and Cryptococcus neoformans were also frequently detected. Of 139 infected patients, 12 cases were diagnosed by MME-18 only, 57 patients by conventional testing only, and 70 cases by both comparator tests and MME-18. There were 96.3% (79/82) diagnoses made by MME-18 had a favorable outcome, and two of twelve diagnoses, made solely by MME-18, had a likely unclear clinical significance. Discussion: The MME-18 showed satisfactory consistency with expert clinical consensus for patients presenting with ME. Combined with conventional testing and clinical suspicion, MME-18 may help clinicians with the early identification of pathogens.

First report of invasive Pomacea snails in Shandong Province / 中国血吸虫病防治杂志

Objective To characterize the species of invasive Pomacea snails that were discovered for the first time in Shandong Province. Methods Pomacea snails samples were collected in the field of Jining City, Shandong Province on October 2021 for morphological identification. Pomacea snails were randomly sampled and genomic DNA was extracted from foot muscle tissues of Pomacea snails for multiplex PCR amplification. The PCR amplification product was sequenced. Then, the sequence was aligned and a phylogenetic tree was created using the software MegAlign 7.1.0. In addition, Angiostongylus cantonensis infection was detected in Pomacea snails with the lung microscopy. Results A total of 104 living Pomacea snails were collected, and all were characterized as Pomacea spp. based on morphological features. Of 12 randomly selected adult Pomacea snails, multiplex PCR assay and sequencing identified eleven snails as P. canaliculata and one as P. maculata. No A. cantonensis infection was detected in 104 Pomacea snails. Conclusion This is the first report of invasive Pomacea snails in Shandong Province, where P. canaliculata and P. maculata are found.

Developmental validation study of a 32-plex STR direct amplification system for forensic reference samples.

The STRtyper-32G PCR Amplification Kit is a 6-dye multiplex system that combines the 30 autosomal STR loci with an Indel site (YIndel) and the sex-determinant locus Amelogenin. In addition to more loci, Master Mix has been optimized to amplify DNA on different substrates. The autosomal STR loci contained in this novel system meet the compatibility of requirements for databasing. In this study, the developmental validation study of the STRtyper-32G Kit followed the guidelines of SWGDAM (Scientific Working Group on DNA Analysis Methods), including PCR-based studies, species specificity, inhibitors, sensitivity, precision, repeatability, stutter, DNA mixtures, concordance studies, and population genetics studies. The validation results indicate that the new multiplex system is a robust tool for forensic database applications.

A Multicenter Study of Viral Aetiology of Community-Acquired Pneumonia in Hospitalized Children in Chinese Mainland.

Community-acquired pneumonia (CAP) is one of the leading causes of morbidity and mortality in children worldwide. In this study, we aimed to describe the aetiology of viral infection of pediatric CAP in Chinese mainland. During November 2014 to June 2016, the prospective study was conducted in 13 hospitals. The hospitalized children under 18 years old who met the criteria for CAP were enrolled. The throat swabs or nasopharyngeal aspirates (NPAs) were collected which were then screened 18 respiratory viruses using multiplex PCR assay. Viral pathogens were present in 56.6% (1539/2721) of the enrolled cases, with the detection rate of single virus in 39.8% of the cases and multiple viruses in 16.8% of the cases. The most frequently detected virus was respiratory syncytial virus (RSV) (15.2%, 414/2721). The highest detection rate of virus was in < 6-month-age group (70.7%, 292/413). RSV, human metapneumovirus (HMPV), human parainfluenza viruses (HPIVs) and influenza B virus (Flu B) showed the similar prevalence patterns both in north and south China, but HPIVs, Flu A, human bocavirus (HBoV), human adenovirus (HAdV) and human coronaviruses (HCoVs) showed the distinct circulating patterns in north and south China. Human enterovirus/human rhinovirus (HEV/HRV) (27.6%, 27/98), HBoV (18.4%, 18/98), RSV (16.3%, 16/98) and HMPV (14.3%, 14/98) were the most commonly detected viruses in severe pneumonia cases with single virus infection. In conclusion, viral pathogens are frequently detected in pediatric CAP cases and may therefore play a vital role in the aetiology of CAP. RSV was the most important virus in hospitalized children with CAP in Chinese mainland.

A multiplex PCR assay for six Aedini species, including Aedes albopictus.

BACKGROUND: Mosquitoes, as vectors of various human pathogens, are significant drivers of serious human illness. In particular, those species in the Aedini tribe, which typically transmit dengue virus, Chikungunya fever virus, and Zika virus, are increasing their range because of climate change and international commerce. In order to evaluate the risk of disease transmission, accurate mosquito species identification and monitoring are needed. The goal of this work was to develop a rapid and simple molecular diagnostic method for six morphologically similar Aedini species (Aedes flavopictus, Aedes albopictus, Ochlerotatus koreicus, Ochlerotatus japonicus, Ochlerotatus togoi and Ochlerotatus hatorii) in Korea. METHODS: A total of 109 samples were assayed in this study. The internal transcribed spacer 2 (ITS2) regions from all six species were amplified, sequenced and analyzed using Mega 6. Following the identification of regions that were consistently different in terms of sequence between all six species, multiplex primers were designed to amplify these regions to generate species-specific fragments distinguishable by their size. RESULTS: Uniquely sized fragments were generated in Ae. flavopictus (495 bp), Ae. albopictus (438 bp), Oc. koreicus (361 bp), Oc. togoi (283 bp), Oc. hatorii (220 bp) and Oc. japonicus (160 bp). Pairwise distance analysis showed that the difference was 35.0 ± 1.5% between Aedes spp. and Ochlerotatus spp., 17.4 ± 0.2% between Ae. albopictus and Ae. flavopictus and 11.1 ± 0.3% between Oc. koreicus and Oc. japonicus. CONCLUSIONS: In this study, a multiplex PCR assay for six species of the Aedini tribe was developed. This assay is more accurate than morphological identification and will be useful for monitoring and controlling these vector mosquitoes.