Multiplex Real-time Polymerase Chain Reaction Research Articles

Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction.

Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

Viral Aetiology of Severe Acute Respiratory Infections in Hospitalised Adult Patients in Abidjan, Côte d’Ivoire

Aims: Pneumonia is a leading cause of morbidity and a significant cause of mortality worldwide. Although information is available on pneumonia in children, the incidence in adults in many parts of Africa including Côte d'Ivoire is unknown. Knowledge of local etiologic agents of pneumonia is critical for making reasonable decisions about treatment as differences in etiology may result in poor response to therapy chosen to cover common pathogenic microbes in studies done in high countries of income.
 The objective of this study was to identify the viral etiology of pneumonia in adult patients with pneumonia in Abidjan, Côte d 'Ivoire.
 Study Design: This is a prospective experimental study conducted on the basis of the successive recruitment of patients admitted to hospital for severe pulmonary interstitial pneumonitis confirmed by radio or CT scan of the thorax.
 Place and Duration of Study: Pneumophtisiology department (PPH) of the University Hospital Center of Cocody (Côte d'Ivoire) and laboratory of Bacteriology- Virology of Pasteur Institut of Côte d’Ivoire, between February 2016 and October 2017.
 Methodology: Among all admitted patients in the unit of pneumophtisiology (PPH) of the hospital University, 90 patients aged at least 18 years were pre-included. A total of 33 bronchoalveolar lavage fluid (BAL) samples from adults suspected of pneumonia were analyzed. The viruses were identified by the real-time multiplex reverse polymerase chain reaction (RT-PCR).
 Results: Of the 33 BAL samples tested, 18.2% (6/33) viral agents were detected. Parainfluenza-3 PV-3 was the most prevalent virus (57.1%, 4/7), followed by coronavirus OC43 (14.3%, 1/7), coronavirus HKUI (14.3%, 1/7). ) and human rhinovirus (14.3%, 1/7).
 A virus and virus association was detected, which was PV-3 associated with coronavirus HKUI (14.3%, 1/7).
 Conclusion: The viral etiology of pneumonia is not very frequent in Côte d’Ivoire.

Prevalence of respiratory pathogens during two consecutive respiratory syncytial virus seasons at a tertiary medical care center.

To determine the etiological profiles of lower respiratory tract infection (LRI) in neonates during respiratory syncytial virus(RSV) season, and to define the clinical features of RSV-related infection and others. The retrospective study included newborn infants who were hospitalized for LRI during the two consecutive RSV seasons, and then tested for possible etiological agent by multiplex real-time polymerase chain reaction. All relevant data were reviewed, and the clinical characteristics of RSV-related infection were compared to those of others. Of 224 patients, 160 (71 %) were positive for at least one potentially causative agent. Of them, 65 % had RSV, and 15 % had more than on ecausative agent (co-infection). The RSV group had more the findings of respiratory distress (p< 0.01), abnormal chest radiography (p< 0.01), need for intensive care (p< 0.01), and duration of oxygen requirement (p< 0.01) but less fever on admission and duration of antibiotic use (for both, p< 0.01), and no longer hospital stay. Need of intensive care nursery was more common in patients with co-infection than others (25 % vs. 6.5 %, p< 0.01). This study highlighted that RSV was the most frequent agent in neonates hospitalized for LRI during the season, with a more severe clinical course than other detected pathogens. The disease severity of RSV infection may have seemed to be increased by the presence of coinfection and abnormal chest radiography.

A fast multiplex real-time PCR assay for simultaneous detection of pork, chicken, and beef in commercial processed meat products

As rapid and efficient methods for species identification were required, we developed a fast multiplex real-time polymerase chain reaction (PCR) assay based on TaqMan® probes to simultaneously detect pork, chicken, and beef in processed meat samples within 40 min. For multiplex PCR reactions, the 5’ ends of the pork-, chicken-, and beef-specific probes were labeled with FAM, VIC, and NED, respectively. The specificity of this assay was evaluated using 20 animal species and no cross-contamination was observed. The absolute detection limit was 0.1 pg of DNA extracted for all three target species, and the limit of detection (LOD) of reference meat mixtures was determined to be 0.5%. The optimized method was applied to 66 commercial processed products and specific targets were successfully amplified in under 40 min. Therefore, the fast multiple real-time PCR method can be utilized for easily and rapidly monitoring the presence of three meat species in various processed meat products.

Enterovirus D68 Causing Acute Respiratory Infection: Clinical Characteristics and Differences With Acute Respiratory Infections Associated With Enterovirus Non-D68.

Enterovirus (EV) D68 is mainly associated with acute respiratory infection (ARI). Since 2014, when outbreaks in different countries were observed, this emerging virus was considered a potential threat to public health. During 2015-2017, the presence of enterovirus RNA was investigated in all respiratory samples of children younger than 15 years of age with ARI, obtained for virologic studies in the Pediatric Emergency Care Units and wards of 2 hospitals in Gipuzkoa (Spain), using a commercial multiplex real-time polymerase chain reaction. When enterovirus was detected, a polymerase chain reaction to amplify a specific viral polyprotein (VP1) gene region of EV-D68 was performed. In 2016, EV-D68 circulation was associated to ARI, with the highest incidence in the spring months. EV-D68 was detected in 44 children, mean age 30.1 ± 31.7 months old, 23 (52.3%) of them females and 17 (38.6%) with underlying respiratory medical conditions. Thirty-two patients (72%) required hospital admission, receiving the discharge diagnosis of recurrent wheezing (37.5%), asthmatic crisis (37.5%) or bronchiolitis (12.5%). Seven children (15.9%) needed the support of the pediatric intensive care unit. When coinfections were excluded, children with EV-D68 infection presented with increased work of breathing, recurrent wheezing or asthmatic crisis, more frequently than those with ARI associated with EV non-D68. Moreover, clinical outcomes (hospitalization, respiratory support) were more severe. All 44 EV-D68 strains detected belonged to lineage B3. EV-D68 circulated widely in Gipuzkoa during 2016 and was associated with severe ARI. In children with severe ARI of unknown etiology, the presence of EV-D68 should be considered.

RSVpredict: An Online Tool to Calculate the Likelihood of Respiratory Syncytial Virus Infection in Children Hospitalized With Acute Respiratory Disease.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in young children. Early detection of RSV infection can avoid unnecessary diagnostic and therapeutic intervention and is required to prevent the nosocomial spread of RSV infection in pediatric hospitals. We developed a web tool to calculate the probability of RSV infection in children hospitalized with acute respiratory tract infection (ARTI) (RSVpredict). During winter seasons 2014/2015 to 2017/2018, 1545 children hospitalized with clinical symptoms of ARTI at the University Hospital Heidelberg/Germany were prospectively included. Medical information was reported on a standardized data sheet, and nasopharyngeal swabs were obtained for multiplex real-time polymerase chain reaction analyses. We applied logistic regression to develop a prediction model and developed a web-based application to predict the individual probability of RSV infection. Duration of clinical symptoms ≥2 days on admission, calendar month of admission, admission for lower respiratory tract infection, the presence of cough and rale and younger age were associated with RSV infection (P < 0.05). Those data were included in the prediction model (RSVpredict, https://web.imbi.uni-heidelberg.de/rsv/). RSVpredict is a web-based application to calculate the risk of RSV infection in children hospitalized with ARTI. The prediction model is based on easily accessible clinical symptoms and predicts the individual probability of RSV infection risk immediately. Pediatricians might use the RSVpredict to take informed decisions on further diagnostic and therapeutic intervention, including targeted RSV testing in children with relevant RSV infection risk.

Development of a rapid and reliable single-tube multiplex real-time PCR method for HLA-A*24:02 genotyping.

Aim:HLA-A*24:02 is significantly associated with cutaneous adverse drug reactions caused by aromatic antiepileptic drugs. Here, we aimed to establish a fast and reliable detection method for HLA-A*24:02 genotyping. Methods: A single-tube multiplex quantitative real-time polymerase chain reaction(qPCR) assay for HLA-A*24:02 genotyping was established by combining allele-specific primers with TaqMan probes. Results: A 100% concordance was observed between qPCR and SBT result in 106 Han subjects. The detection limit of the new method was 0.05ng DNA. The positive rate of HLA-A*24:02 in Tibetans (55.6%, n=81) was significantly higher than those in Han (34%, n=106), Uighur (27.5%, n=102), Bouyei (25.9%, n=116) and Miao populations (26.5%, n=113). Conclusion: The newly established qPCR assay was reliable for HLA-A*24:02 screening in clinical applications.

High Prevalence of Anal and Oral High-Risk Human Papillomavirus in Human Immunodeficiency Virus-Uninfected French Men Who Have Sex With Men and Use Preexposure Prophylaxis.

BackgroundWe assessed the prevalence and risk factors of anal and oral high-risk (HR) human papillomavirus (HPV) infection in human immunodeficiency virus–uninfected men who have sex with men (MSM) and take preexposure prophylaxis (PrEP) in France.MethodsAnal and oral samples were screened by multiplex real-time polymerase chain reaction (Anyplex II HPV 28; Seegene) for HPV DNA.ResultsA total of 61 unvaccinated MSM (mean age, 36.1 years) were enrolled. Anal HPV and HR-HPV prevalences were 93.4% and 81.9%, respectively, and oral HPV and HR-HPV prevalences, 33.9% and 19.6%, respectively. HR-HPV type 33 was the most detected genotype, in both anal and oral samples. Among MSM, 68.8% carried ≥1 anal HPV type targeted by the 9-valent Gardasil-9 vaccine; all oral HPV-positive samples carried ≥1 strain included in the vaccine. Condomless receptive anal intercourse and history of anal gonorrhea were the main factors associated with increased risk for anal HPV infection (adjusted odds ratio, 10.4) and anal infection with multiple HR-HPV genotypes (5.77), respectively. Conversely, having had <10 partners in the last 12 months was associated with decreased risk for anal carriage of both multiple HPV (adjusted odds ratio, 0.19) and HR-HPV (0.17) types.ConclusionFrench MSM using PrEP are at high risk for both anal and oral carriage of HR-HPV that could lead to HPV-related cancers.

The detection of DNA methylation of tumour suppressor genes in cervical high-grade squamous intraepithelial lesion: A prospective cytological-histological correlation study of 70 cases.

DNA methylation has been suggested as one of the epigenetic changes promoting carcinogenesis. The aim of this study was to prospectively evaluate the methylation status of CADM 1, MAL and hsa-miR-124 genes in high-grade squamous intraepithelial lesion (HSIL) liquid-based cytology (LBC) samples with a histological correlation. Seventy histologically confirmed cases of HSIL paired with prior screening LBC diagnosis of HSIL within a 3-month interval were selected. Histologically, the lesions were reviewed and assessed including: (a) number of blocks harbouring dysplastic squamous epithelium; (b) number of blocks containing glandular extension of dysplastic epithelium; and (c) the depth of glandular extension (which was assessed semi-quantitatively as graded 1-3). Human papillomavirus (HPV) subtyping was performed from residual LBC materials using the LINEAR ARRAY HPV Genotyping Test and in-house polymerase chain reaction targeting the HPV E1 gene. The detection of methylation silencing of tumour suppressor genes CADM1, MAL and hsa-miR-124 was performed by multiplex methylation-specific real-time polymerase chain reaction. A positive methylation status was detected in 41 cases (58.6%). The number of blocks with HSIL varied from one to 13. Glandular extension was seen in 44 cases with the number of blocks involved ranging from one to 10. The depth of HSIL glandular extension varied. The DNA methylation test allows HSIL lesions to be divided into two distinct groups of methylated HSIL in significantly older patients and unmethylated HSIL in younger patients. This study was not able to prove that methylation status in cervical HSIL correlates with the size of the lesion (measured by the number of blocks involved) or with HSIL propensity for endocervical glandular extension, nor with HPV type or multi-infection.

Down-regulatory effects of green coffee extract on las I and las R virulence-associated genes in Pseudomonas aeruginosa

Antibiotic resistant strains of Pseudomonas aeruginosa are the cause of Gram negative nosocomial infections especially among the immunosuppressed patients. The bacteria contains las I and las R genes that play very important roles in the pathogenesis and mechanisms of aggression. These genes can be influenced by the quorum sensing (QS) system and such mechanism is becoming clinically important worldwide. This study aimed to investigate the preventive effects of green coffee extract (GCE) on the expression of pathogenesis-related genes, las I and las R in P. aeruginosa. A total of fifty four P. aeruginosa strains were isolated out of 100 clinical samples collected from the infectious wards in different hospitals (Tehran province) using conventional microscopic and biochemical methods. Susceptibility of the isolates to different antibiotics, GCE and chlorogenic acid were elucidated. Multiplex polymerase chain reaction (PCR) and real-time PCR were performed to detect and quantify the expression levels of las I and las R genes. The presence of chlorogenic acid in GCE was confirmed by HPLC. Antibiotic susceptibility tests revealed multidrug resistance among the clinical isolates of those 40 strains were resistant to ciprofloxacin (74.07%), 43 to ceftazidime (79.26%), 29 to amikacin (53.7%), 42 to ampicillin (77.77%), 17 to colistin (31.48%), 40 to gentamicin (74.77%), and 50 to piperacillin (92.59%). PCR outcomes exhibited that the frequency of las I and las R genes were 100% in resistant and sensitive strains isolated from clinical and standard strains of P. aeruginosa (ATCC 15449). Real-time PCR analyses revealed that GCE significantly prevented the expression of las I and las R genes in P. aeruginosa. GCE at concentration level as low as 2.5mg/mL could prevent the expression of lasI and lasR genes in P. aeruginosa clinical isolates. The presence and expression levels of las I and las R genes in P. aeruginosa isolates were investigated when the bacteria was exposed to GCE. Our results tend to suggest that genes involved in pathogenesis of:Pseudomonas aeruginosa are down regulated by quorum sensing effect of chlorogenic acid and therefore GCE could be useful as an adjuvant in combating multidrug resistance strains of Pseudomonas aeruginosa.

Real-Time Multiplex PCR Analysis in Infectious Uveitis

ABSTRACTIntroduction: Infectious uveitis is a serious inflammatory condition that often causes grave ocular morbidity including permanent vision loss and damage to the structures of the eye. The most common causes of infectious uveitis include herpesviruses and Toxoplasma gondii. Traditionally, these infections have been identified and differentiated based on characteristic clinical examination findings; however, there is often overlap between these presentations and the unique cause of a given patient’s infection is not always clear. Therefore, a reliable and fast method for definitively diagnosing infectious uveitis would be helpful and potentially sight-saving. Several groups have recently found experimental success with real-time multiplex polymerase chain reaction (PCR) techniques.Methods: A comprehensive review of the literature was undertaken to further understand the current state of real-time multiplex PCR and its clinical use. Search terms including “real time multiplex PCR”, “infectious uveitis”, and “uveitis diagnosis” were used. Appropriate English-language articles were included in this review.Results: Publications from four main groups (two from the United States, one from Japan, and one from India) citing success with real-time multiplex PCR were compared and contrasted. All four groups used the same technique to develop a highly sensitive and specific multiplex PCR analysis and found that their tests maintained high sensitivity and specificity during validation testing. These tests confirmed clinical suspicions in the majority of cases of infectious uveitis, but there were also cases of clinical misdiagnosis that were corrected based on molecular pathogen detection. These patients were then initiated on appropriate antimicrobial therapy with subsequent clinical improvement.Discussion: Real-time multiplex PCR is a highly sensitive and specific laboratory assay that allows for rapid and reliable molecular diagnosis of causative agents in infectious uveitis. This in turn facilitates swift initiation of effective therapy and prevents long-term ocular damage and vision loss.

A 3-Year Retrospective Study of the Epidemiology of Acute Respiratory Viral Infections in Pediatric Patients With Cancer Undergoing Chemotherapy.

Acute viral respiratory infections are common causes of febrile episodes in children. There are still limited data about distribution of acute viral respiratory infections in children with cancer. The first aim of this study was to evaluate the viral etiology and seasonality of acute viral respiratory infection in pediatric patients with cancer in a 3-year study. Our second aim was to evaluate the impact of viral infections on delaying the patients' chemotherapy or radiotherapy. This cross-sectional study was conducted from January 2014 to July 2017. Nasopharyngeal aspirates were analyzed in patients younger than 21 years with acute respiratory infections. Patients were treated in the Pediatric Hematology and Oncology Department of Dr. Behçet Uz Children's Hospital with real-time multiplex polymerase chain reaction. Data were analyzed to determine the frequency and seasonality of infections. The χ or the Fisher exact tests were used. A total of 219 samples of nasopharyngeal aspirates and blood were analyzed. The mean patient age was 76.8±59.3 months, with 46.3% female and 53.7% male children in a total of 108 patients. Of this total, 55% (60/108 cases) had multiple acute respiratory infections. Acute lymphoblastic leukemia (48.1%) was the most prevalent disease. The 3 most prevalent viruses were human rhinovirus (HRV) (33.1%), parainfluenza (PI) (18.7%), and coronavirus (CoV) (14.8%). In terms of the seasonal distribution of viruses, PI was most common in winter 2014, HRV in spring 2014, HRV in fall 2014, PI in winter 2015 and summer 2015, CoV in spring 2015, HRV in fall 2015, both influenza and HRV in winter 2016, both human metapneumovirus and bocavirus in spring 2016, HRV in summer 2016, both HRV and PI in fall 2016, both respiratory syncytial virus and influenza in winter 2017, HRV in spring 2017, and both HRV and adenovirus in summer 2017. The mean duration of neutropenia for patients with viral respiratory infection was 17.1±13.8 (range: 2 to 90) days. The mean duration of symptoms of viral respiratory infection was 6.8±4.2 (range: 2 to 31) days. A delay in chemotherapy treatment owing to viral respiratory infection was detected in 73 (33.3%) patients. The mean duration of delay in chemotherapy treatment was 9.6±5.4 (range: 3 to 31) days. In conclusion, we report our 3-year experience about the frequency and seasonality of respiratory viruses in children with cancer.

Etiology of Pulmonary Infections in Human Immunodeficiency Virus-infected Inpatients Using Sputum Multiplex Real-time Polymerase Chain Reaction.

BackgroundThere are limited data on the etiology of respiratory infections in human immunodeficiency virus (HIV)–infected patients in resource-limited settings.MethodsWe performed quantitative multiplex real-time polymerase chain reaction (PCR) for Pneumocystis jirovecii and common bacterial and viral respiratory pathogens on sputum samples (spontaneous or induced) from a prospective cohort study of HIV-infected inpatients with World Health Organization danger signs and cough. Mycobacterial culture was done on 2 sputum samples, blood cultures, and relevant extrapulmonary samples.ResultsWe enrolled 284 participants from 2 secondary-level hospitals in Cape Town, South Africa: median CD4 count was 97 cells/μL, 64% were women, and 38% were on antiretroviral therapy. One hundred forty-eight had culture-positive tuberculosis, 100 had community-acquired pneumonia (CAP), 26 had P. jirovecii pneumonia (PJP), and 64 had other diagnoses. Probable bacterial infection (>105 copies/mL) was detected in 133 participants; the prevalence was highest in those with CAP (52%). Haemophilus influenzae and Streptococcus pneumoniae were the commonest bacterial pathogens detected; atypical bacteria were uncommon. Viruses were detected in 203 participants; the prevalence was highest in those with PJP (85%). Human metapneumovirus was the commonest virus detected. Multiple coinfections were commonly detected.ConclusionsSputum multiplex PCR could become a useful diagnostic tool for bacterial respiratory infections in HIV-infected inpatients, but its value is limited as quantitative cutoffs have only been established for a few bacterial pathogens and validation has not been done in this patient population. We found a high prevalence of respiratory viruses, but it is unclear whether these viruses were causing infection as there are no accepted quantitative PCR cutoffs for diagnosing respiratory viral infections.

Multiplex polymerase chain reaction to diagnose bloodstream infections in patients after cardiothoracic surgery

BackgroundSepsis and other infectious complications are major causes of mortality and morbidity in patients after cardiac surgery. Whereas conventional blood culture (BC) suffers from low sensitivity as well as a reporting delay of approximately 48–72 h, real-time multiplex polymerase chain reaction (PCR) based technologies like “SeptiFast” (SF) might offer a fast and reliable alternative for detection of bloodstream infections (BSI). The aim of this study was to compare the performance of SF with BC testing in patients suspected of having BSI after cardiac surgery.MethodsTwo hundred seventy-nine blood samples from 169 individuals with suspected BSI were analyzed by SF and BC. After excluding results attributable to contaminants, a comparison between the two groups were carried out. Receiver operating characteristic (ROC) curves were generated to determine the accuracy of clinical and laboratory values for the prediction of positive SF results.Results14.7% (n = 41) of blood samples were positive using SF and 17.2% (n = 49) using BC (n.s. [p > 0.05]). In six samples SF detected more than one pathogen. Among the 47 microorganisms identified by SF, only 11 (23.4%) could be confirmed by BC. SF identified a higher number of Gram-negative bacteria than BC did (28 vs. 12, χ2 = 7.97, p = 0.005). The combination of BC and SF increased the number of detected microorganisms, including fungi, compared to BC alone (86 vs. 49, χ2 = 13.51, p < 0.001). C-reactive protein (CRP) (21.7 ± 11.41 vs. 16.0 ± 16.9 mg/dl, p = 0.009), procalcitonin (28.7 ± 70.9 vs. 11.5 ± 30.4 ng/dl, p = 0.015), and interleukin 6 (IL 6) (932.3 ± 1306.7 vs. 313.3 ± 686.6 pg/ml, p = 0.010) plasma concentrations were higher in patients with a positive SF result. Using ROC analysis, IL-6 (AUC 0.836) and CRP (AUC 0.804) showed the best predictive values for positive SF results.ConclusionThe SF test represent a valuable method for rapid etiologic diagnosis of BSI in patients after cardiothoracic surgery. In particular this method applies for individuals with suspected Gram-negative blood stream. Due to the low performance in detecting Gram-positive pathogens and the inability to determine antibiotic susceptibility, it should be used in addition to BC only (Pilarczyk K, et al., Intensive Care Med Exp ,3(Suppl. 1):A884, 2015).

Investigation of Viral Respiratory Tract Infection Agents by Multiplex PCR Method in Autopsy Cases: A Five-Year Study

Viral respiratory infections are one of the leading causes of morbidity and mortality, especially in children, elderly and immunocompromised patients. The inclusion of post-mortem studies to diagnose the infection causing mortality could be beneficial in specifying new pathogens and determining strategies for treatment and prevention.The aim of this study was to research viral etiology by applying multiplex real-time polymerase chain reaction (Rt-PCR) method in autopsy cases who have been considered to have a respiratory infection and to assess whether the viruses detected are the primary cause of the infection and whether they have any contributory effect on the mortality together with histopathological evidence. In this study, we included a total of 834 cases consisting of sudden death cases from infantile-pediatric age group and autopsy cases from > 18 year age group with suspected respiratory tract infection in our laboratory between January 2013 and May 2017. Of 834 cases, 468 (56.1%) were male and 366 (43.9%) were female, there were 191 (22.9%) cases between 0-1 months, 593 (71.1%) cases between 1 month-18 years, and 50 (6%) cases in the > 18 years age group. In 728 of 834 (87.3%) cases nasopharyngeal/tracheal swab samples and in 106 (12.7%) of them paraffin-embedded lung tissue samples were studied by the use of "FTD Respiratory 21 (Fast-Tract Diagnostics Luxemburg)" kit, with multiplex Rt-PCR method. The post-mortem samples were evaluated for human rhinovirus (HRV), parainfluenza viruses (PIV) (1, 2, 3, 4), influenza virus type A and B (INF-A, INF-B), enterovirus (EV), human bocavirus (HBoV), adenovirus (AdV), human coronavirus (HCoV 229,63,HKU,43), human metapneumovirus A ve B (HMPV-A/B), parechovirus, respiratory syncytial virus (RSV A/B) and Mycoplasma pneumoniae. In our study, at least one respiratory virus was detected by Rt-PCR in 379 (45.4%) of total 834 cases, whereas no viral agent was identified in 455 (54.6%) of the cases. One viral agent was detected in 278 (33.3%), two viral agents were detected in 83 (9.94%) and three viral agents were detected in 18 (2.16%) cases. Overall, the most common viral agent was HRV 110 (13.2%) followed by AdV 39 (4.7%) and RSV A/B 33 (4%). In pediatric cases the rate of positive results for respiratory viruses was 31.8% and in adult group it was 20% (p= 0.032). The most common virus detected among children was HRV and INF-A in adult group. In 101 (12.1%) cases infections caused by two or three agents were diagnosed. Infections with two causative agents were detected as 2.6% (5/191) in 0-1 month age group, 13% (77/593) in 1 month-18 year age group and 2% (1/50) in > 18 age group. The most frequently observed co-infections with double causative agents were HRV and INF-B, HRV and PIV, HRV and HBoV, HRV and AdV combinations. Infections with three causative agents were detected completely among 1 month-18 year age [3% (18/593)] group. In our study, 318 (38.1%) cases had no signs of infection in the postmortem histopathological examination of the lung tissues, while the most common finding was lobular pneumonia/purulent bronchitis in 233 (28%) cases and the second was interstitial pneumonia in 168 (20.1%) cases. When all cases were evaluated in terms of infection, positive results were detected in 469 (56.2%) cases. As a result; postmortem microbiological diagnosis with autopsy and histopathological detection of the patients who are thought to have respiratory tract infection will also determine the infectious agents causing death.

Respiratory syncytial virus, human metapneumovirus, and influenza virus infection in Bangkok, 2016-2017.

Children and adults residing in densely populated urban centers around the world are at risk of seasonal influenza-like illness caused by respiratory viruses such as influenza virus, human metapneumovirus (hMPV), and respiratory syncytial virus (RSV). In a large metropolitan of Thailand’s capital city Bangkok, most respiratory infections are rarely confirmed by molecular diagnostics. We therefore examined the frequency of RSV, hMPV, and influenza virus in 8,842 patients who presented influenza-like illness and sought medical care at a large hospital in Bangkok between 2016 and 2017. Using a multiplex real-time reverse-transcription polymerase chain reaction (RT-PCR), 30.5% (2,699/8,842) of nasopharyngeal (NP) swab samples tested positive for one or more of these viruses. Influenza virus comprised 17.3% (1,528/8,842), of which the majority were influenza A/H3N2. Such infection was most prevalent among adults and the elderly. RSV was identified in 11.4% (1,011/8,842) and were mostly ON1 and BA9 genotypes. Of the hMPV-positive samples (3.6%, 318/8,842), genotypes A2, B1, and B2 were detected. A small number of individuals experienced co-infections (1.8%, 155/8,842), most commonly between RSV and influenza A/H3N2. RSV and hMPV co-infections were also found, but mainly in young children. Viral respiratory tract infection peaked locally in the rainy season (June to September). These findings support the utility of rapid nucleic acid testing of RSV, hMPV, and influenza virus in patients with ILI.

Presence of Human Bocavirus 1 and other Respiratory Viruses in Children with Lower Respiratory Tract Infection in Latvia

Abstract Lower respiratory tract infection (LRTI) is the major cause of morbidity and mortality of children in the world. In addition to respiratory syncytial virus, influenza virus types A and B, parainfluenza types 1, 2 and 3, and adenoviruses, several new respiratory viruses associated with LRTI were discovered in the 21st century. These are metapneumovirus, coronaviruses NL63 and HKU1, parainfluenza virus type four and human bocavirus one (HBoV1). HBoV1 was discovered in 2005 and is considered as the fourth most prevalent respiratory virus worldwide. However, the high frequency of co-infections detected together with HBoV1 raises doubt about whether HBoV1 is a true pathogen or just a bystander. This is the first study aimed to determine the presence of HBoV1 and 18 other respiratory viruses in nasopharyngeal aspirates (NPA) of children with LRTI in Latvia. Using multiplex real-time polymerase chain reaction method, the HBoV1 genomic sequence was detected in 60.0% of NPA samples, showing that HBoV1 prevalence is high among children with LRTI in Latvia. HBoV1 mono-infection was revealed in 6.67%. The most common co-infections associated with HBoV1 were rhinovirus, adenovirus, respiratory syncytial virus A and B, metapneumovirus, and enterovirus.

MULTIPLEX RT-PCR BASED DETECTION OF HUMAN BOCAVIRUS AND OTHER RESPIRATORY VIRUSES IN INFANTS AND YOUNG CHILDREN WITH LOWER RESPIRATORY TRACT INFECTION

Background: Human Bocavirus (hBoV) has recently been identified as a causative agent of acute respiratory infection (ARI) in infant and young children. However, there is little information about its frequency and importance as a cause of lower respiratory tract infection (LRTI) in Iraq. Objective:To assess the frequency of hBoV and co-infection rate in infants and young children with LRTI. Methods: Nasopharyngeal/throat swabs were collected during the peak winter months from 100 hospitalized infants and young children less than 5 years of age with LRTI in Baghdad, Iraq. Five viruses were investigated by Multiplex Real-Time Polymerase Chain Reaction (RT-PCR). Results: It was found that 71 (71%) had a viral infection either single or mixed. Six out of 100 samples (6%) were hBoV positive, 10 (10%) were parainfluenza virus positive, 12 (12%) tested positive for adenovirus, 58 (58%) were rhinovirus positive and none of the samples gave positive results for coronavirus. Sixteen samples showed mixed infection with rhinovirus, eight of these (8%) with parainfluenza virus, 6 (6%) with adenovirus, and 2 (2%) with hBoV. There were no significant differences between the viral infected and non-infected patients with respect to age, gender, crowding status, gestational age, diagnosis at presenting and hospital stay (the average stay of viral-infected patients in the hospital was three days). In contrast, exposure to smoking was significantly more associated with viral-infected children than virus-free children (80% vs. 44.83%, P&lt;0.001). In addition, viral-infected children showed significantly less proportion for need for intensive care unit than virus-free children (15.5% vs. 79.31%, P&lt;0.001). Breastfeeding was found to have significant adverse association with viral infection (P&lt;0.001). About one-third of viral-infected children had a medical history involving a disease other than respiratory infection (P&lt;0.001). Finally, viral-infected children were reported to have longer period of illness before hospital admission (6.32±5.49 days) than virus-free children (6.32±5.49 days vs. 3.38±2.37 days, P&lt;0.01). Conclusion: More than two third of children with respiratory tract infection have been viral infection either single or mixed. Multiplex RT-PCR has the potential for clinical use in the rapid and differential detection of viral infection. Keywords: Viral respiratory tract infection, Children, Multiplex Real Time PCR Citation: Rasheed ZS, Al-Shuwaikh AMA, Issa KR. Multiplex RT-PCR based detection of Human Bocavirus and other respiratory viruses in infants and young children with lower respiratory tract infection. Iraqi JMS. 2019; 17(1): 74-82. doi: 10.22578/IJMS.17.1.11

SNP Typing Using Multiplex Real-Time PCR Assay for Species Identification of Forensically Important Blowflies and Fleshflies Collected in South Korea (Diptera: Calliphoridae and Sarcophagidae).

Medicolegal entomology—a subfield of forensic entomology—is mainly used in medicolegal investigations to estimate the postmortem interval (PMI). The minimum PMI of a corpse invaded by necrophagous immature insects can be estimated because the PMI is near to or earlier than the oviposition time of the larvae that hatched and fed on the corpse. As the growth speeds of larvae differ depending on temperature and species, species-specific growth data are used to estimate the minimum PMI. While morphological identification of adult necrophagous flies can be done by a well-trained entomologist, identification of larvae is relatively difficult. Larvae can only be identified up to the family level and developmental stage by observing the posterior spiracles. For these reasons, the molecular biology method of DNA barcoding has been developed. DNA barcoding that targets the mitochondrial cytochrome c oxidase subunit I (COI) gene is commonly used. COI sequences are currently acquired using polymerase chain reaction (PCR) and Sanger sequencing, which are too time-consuming and complex for practical use in medicolegal investigations. To compensate for these limitations and facilitate the use of entomology for medicolegal investigation, we designed a multiplex real-time PCR system to identify nineteen forensically important species of Calliphoridae and Sarcophagidae flies collected in South Korea. In contrast to the Sanger nucleotide sequencing process, this technology only requires a one-step real-time PCR with melt curve analysis of amplicons generated by primers targeting species-specific single nucleotide polymorphisms (SNPs). Multiplex real-time PCR was performed for twelve species of Calliphoridae (four reactions) and for seven species of Sarcophagidae (three reactions). This assay is expected to make it easier and faster for investigating authorities to identify major species of necrophagous flies at beginning of investigation and to increase the utilization of entomological evidence in forensic investigations.

English

The incidence of meningoencephalitis in developing countries is high and its diagnosis being inadequate as it is mainly based on conventional methods with a limited diagnostic capacity for bacterial, fungal and viral detection. This results in prescription of a cocktail of antibiotics, antifungal, or anti-viral drugs, since the causative agent is often unknown. There is therefore need for timely and appropriate diagnosis to guide treatment. This study aims at evaluating the utility of molecular diagnostic over conventional methods in detection of etiologic agents of central nervous system infections in Rwanda. Using a cross sectional design, 845 hospitalized patients suspected to have meningoencephalitis were enrolled from 8 study sites across the country. Four sterile tubes of Cerebral Spinal Fluid (CSF) specimens were collected from each patient. Two tubes were analyzed on site for bacteriology, fungal, biochemistry and cytology while the other two were transported to the National Reference Laboratory (NRL) for culture and Real-Time Multiplex Polymerase Chain Reaction (PCR) assays. Data entry and analysis was done using Epi Info7, Excel; SPSS and STATA16. In the study, no viruses were detected using the conventional methods while a range of viruses, 152/845 (18%) were detected using real time multiplex PCR. In addition, 185/845 (22%) samples were detected as positive for different types of bacteria using the PCR compared with the 59/845(7%) that were positive using conventional methods. There was however no difference in the detection capacity of fungal agents between the two methods with a detection level of 6% (49). This study has shown increased capacity for detection of bacterial and viral causative agents of Central Nervous System (CNS) infections using RT multiplex PCR compared with conventional methods. This result facilitated the development of a novel algorithm for both conventional and molecular diagnostic methods for CNS infections. Key words: Meningoencephalitis, RT- Multiplex PCR, conventional methods, causative agents, diagnostic capacity, cerebral spinal fluid.