Multiplex Real-time Polymerase Chain Reaction Research Articles

Multiplex Real-Time Polymerase Chain Reaction on Sputum for the Diagnosis of Pneumocystis jirovecii Pneumonia in Children: A Retrospective Study.

BackgroundPneumocystis jirovecii pneumonia (PCP) is a serious opportunistic infection in immunocompromised children. Real-time polymerase chain reaction (PCR) is widely used for the diagnosis of PCP due to its good accuracy. However, the diagnostic performance of multiplex real-time PCR on sputum in children with PCP has never been explored.MethodsMedical records of 63 consecutive pediatric patients were analyzed retrospectively, including 13 cases with PCP and 50 with non-PCP pneumonia. Pneumocystis jirovecii (P. jirovecii) and other co-pathogens detected by multiplex real-time PCR in sputum samples were summarized. Using clinical composite diagnosis as the reference standard, we further compared the diagnostic performance of multiplex real-time PCR to combined serological markers (1,3)-β-D-glucan plus lactate dehydrogenase. Additionally, modifications of antimicrobial treatment for pediatric PCP patients after the report of multiplex real-time PCR results were reviewed.ResultsIn children with PCP, nonproductive cough and shortness of breath were more common, lymphocyte count in peripheral blood was markedly lower, and serum levels of (1,3)-β-D-glucan and lactate dehydrogenase were much higher than non-PCP group. Multiplex real-time PCR reached a sensitivity of 100% in diagnosing PCP, which was better than serum (1,3)-β-D-glucan plus lactate dehydrogenase (76.9%). Its specificity (98.0%) significantly surpassed serum (1,3)-β-D-glucan plus lactate dehydrogenase (84.4%). Furthermore, multiplex real-time PCR showed a good performance in identifying co-pathogens in sputum of pediatric PCP patients. Cytomegalovirus, Epstein–Barr virus and Streptococcus pneumoniae were the most common co-pathogens in these patients. Initial antimicrobial treatment was modified in 76.9% of children with PCP after the report of PCR results.ConclusionMultiplex real-time PCR on sputum is a diagnostic tool with good performance for the identification of P. jirovecii as well as co-pathogens in children with PCP. Sputum may be an alternative to bronchoalveolar lavage fluid for PCR assay in children when bronchoscopic examination is not feasible.

Human Papillomavirus Prevalence and Genotype Distribution in Cervical Swab Samples in Istanbul, Turkey.

Human papillomavirus (HPV) is the most common sexually transmitted infection agent worldwide and, with high-risk (HR) HPV genotypes, is the main factor for development of cervical cancer. This study aimed to assess the prevalence of HPV and distribution of HR-HPV genotypes in cervical swab samples and compare them with demographic and clinical data. Cervical swab samples of 2,285 women between the age of 17 and 76 were assessed between January 2018 and October 2020 in order to obtain the data of Turkey. Fifteen different HR-HPV genotypes were determined using multiplex real-time polymerase chain reaction test. HPV was positive in 36.3% (829/2,285) of DNA samples. Prevalence of multiple HR-HPV infection was 40.7%. Of the women, 30.9% (256/829) were infected with HPV16, 14.6% (121/829) with HPV39, and 14.2% (118/829) with HPV51. The most frequently detected genotypes with HPV16 were HPV31, HPV39 and HPV52, respectively. In women with cervical dysplasia, HPV16, 31, and 39 were the most common, and in women with genital warts, HPV16, 59 and 66 were most common, respectively. The highest HR-HPV prevalence was detected in the 17-34 age group (44.1%) (p < 0.001). The prevalence of HR-HPV was 36.3% in this study. High prevalence (44.1%) especially in young women was consistent with findings in literature. The most common HR-HPV genotypes were HPV16, 39 and 51, respectively. Determining the prevalence and genotypes of HR-HPV playing role in the etiology of cervical cancer will be guiding for measures on prevention of cervical cancer and research on preventive vaccines.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

BackgroundThe diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.MethodsTwo multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC.ResultsMultiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.ConclusionsOur multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.Graphical

Development and Application of a Multiplex Real-Time Polymerase Chain Reaction Assay for the Simultaneous Detection of Bacterial Aetiologic Agents Associated With Equine Venereal Diseases

Venereal diseases caused by bacteria are important to the equine industry due to economic losses caused by decline of conception rate in breeding horses. Therefore, identification of infected animals as well as the implementation of appropriate managerial procedures based on accurate diagnosis is critical. In this study, two types of multiplex real-time polymerase chain reaction with high sensitivity and specificity were developed for the simultaneous detection and differentiation of five commonly associated bacterial pathogens of venereal diseases in horses, consisting of Taylorella equigenitalis, Taylorella asinigenitalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Streptococcus zooepidemicus. The assay was applied to samples collected as part of the surveillance of T.equigenitalis infection in South Korea. Swab samples collected from horses in 2015 were tested. T. equigenitalis and K. pneumoniae was detected in 21 (21.0%) and two (2.0%) samples, respectively. No samples were positive for T. asinigenitalis, P. aeruginosa, and S. zooepidemicus. Application of this assay to an existing surveillance program has allowed for an enhanced surveillance for a wider range of venereal diseases of equine to be implemented in South Korea.

Influenza A as a Common Viral Cause of Complex Febrile Seizures

Abstract Objective The most common childhood convulsive disorder happens to be febrile seizure (FS), which is an important health problem leading to economic burden and parental anxiety. Further investigation into the etiological causes of FS will guide us for appropriate measures during the follow-up period. The aim of study was to identify the percentage of viral and bacterial pathogens in the etiological causes of children with FS, and also if there is any difference between simple and complex FSs. Methods This prospective study randomly enrolled 100 pediatric patients with FS between January 2017 and July 2017. Nasopharyngeal swabs were obtained from all children at presentation. The respiratory panel was performed with a multiplex real-time polymerase chain reaction method to detect the 21 most common viruses. A complete blood count, absolute neutrophil count, absolute lymphocyte count, C-reactive protein, erythrocyte sedimentation rate, procalcitonin, blood culture, throat culture, urine analyses, urinary culture, and stool tests analysis were performed in all the patients. Results During the study period, at least one virus was detected in 87% of patients. Bacterial agents were detected in only 13% of patients. Coinfections of the viruses and bacterial pathogens were found in 24% of patients. The most frequently detected virus was influenza A (Inf A) (18%), followed by rhinovirus (12%). Coinfections of the viruses and bacterial pathogens, mixed viral infections, and Inf A were common in children who experienced complex FS. Inf A was detected in 16% of patients with simple FSs and 30% of patients with complex FSs and a significant difference between them (p &lt; 0.01). Conclusion The results of this study showed that respiratory viral and bacterial pathogens are important in the etiology of FS in children. It is considered that complex FSs may be triggered by Inf A. The fact is viral pathogens are very common; therefore, antibiotics must be carefully prescribed. These results also draw attention to the use of the quadrivalent influenza vaccine in the prevention of FS related to the flu.

Multiple Human Papilloma Virus (HPV) Infections Are Associated with HSIL and Persistent HPV Infection Status in Korean Patients.

Infections with multiple human papilloma virus (HPV) types have been reported, but their role in cervical carcinogenesis has not been fully elucidated. In this study, 236 cases with multiple HPV infection were examined and compared to 180 cases with single HPV infection. HPV genotyping was performed with cervico-vaginal swab specimens using multiplex (real-time) polymerase chain reaction (PCR). In multiple HPV infection, the most prevalent HPV genotype was HPV 53, followed by HPV 16, 58, 52, and 68. HPV 33, 35, 39, 51, 52, 53, 58, and 68 were high-risk-HPV (HR-HPV) genotypes that were more frequently detected in multiple HPV infection compared to that in single HPV infection. The association between multiple HPV infection and high-grade SIL (HSIL) was significantly stronger compared to that of single HPV infection and HSIL (p = 0.002). Patients with multiple HPV infection displayed persistent and longer duration of the HPV infection compared to patients with single HPV infection. Multiple HPV infections have distinct clinicopathologic characteristics. Since it is associated with persistent HPV infection, HSIL, and different HR-HPV strains in contrast to single HPV infection, the presence of multiple HPV infection should be reported; close follow up is warranted.

Avian Mycoplasma gallisepticum and Mycoplasma synoviae: Advances in diagnosis and control

Both of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) infections are the most common Mycoplasma infection in domestic poultry. The disease is associated with economic losses in poultry. MG and MS are commonly spread within chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo domesticus) flocks; however, they are frequently isolated from quails (Coturnix coturnix) and several avian species. Diagnosis of MG or MS infections is confirmed by isolating the organism in a cell-free medium or directly detecting its DNA in infected tissues or swab samples. Serological tests are also widely used for diagnosis. However, advances in molecular biology represented a rapid and sensitive alternative to the traditional culture methods requiring specialized techniques and sophisticated reagents. Several Mycoplasma molecular diagnostic tests are implemented: including polymerase chain reaction (PCR), Random Amplified Polymorphic DNA (RAPD), arbitrary primed polymerase chain reactions (AP‐PCR), and Multiplex real-time polymerase chain reaction (Multiplex MGMS). Current control practices against Mycoplasma infection include intense biosecurity, biosurveillance, medication, and vaccination. However, the egg-borne nature of avian Mycoplasma infection complicates controlling the infection. This review focuses on the advances in diagnosis and control of avian Mycoplasma infection, especially MG and MS infections.

Respiratory viruses in the healthy middle ear and middle ear with otitis media with effusion.

To investigate the presence of respiratory viruses in the middle ear cavity of the individuals with a healthy middle ear and the children with otitis media with effusion (OME). A total of 72 middle ear samples were collected from 25 children with OME (Group 1) and 47 individuals with no middle ear disease (Group 2). Multiplex real-time polymerase chain reaction was used to investigate the presence of 20 different respiratory viruses. Virus results were compared with bacteriomes of the same populations. At least one respiratory virus was detected in 56% of the patients in Group 1 and 12.8% of the individuals in Group 2. The viral co-infection rate for Group 1 and 2 was 8% and 2.1%, respectively. In Group 1, adenovirus was the most frequently detected virus with a rate of 24%, either alone (16%) or concurrent with other viruses (8%), followed by influenza B (12%), rhinovirus, and bocavirus (8%) each. Parainfluenza 4, coronavirus OC43, and RSV A/B were detected in 4% of the sample each. In Group 2, rhinovirus was detected in two samples (4.3%) followed by adenovirus, coronavirus OC43, coronavirus E299, and coronavirus NL63 with a rate of 2.1% each. The detection rate of respiratory viruses was significantly higher in children aged 6 to 11 years. There was no positive association between virus and bacteria found in the middle ear cavity. The current study has provided comprehensive data indicating the presence of diverse respiratory viruses in the healthy middle ear cavity. Our results also suggest that respiratory viruses might have a contribution to OME pathogenesis.

Detection of Crimean-Congo hemorrhagic fever virus in blood-fed Hyalomma ticks collected from Mauritanian livestock

BackgroundCrimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus (Nairovididae) and is a (re)emerging tick-borne pathogen. It is endemic in most parts of Africa, Asia and southern Europe, and can cause severe hemorrhagic symptoms in humans, with high fatality rates (5–30%).MethodsHyalomma ticks were collected from four different livestock herds (cattle and camels) in Mauritania in 2018. The tick species were determined morphologically and confirmed molecularly by using the cytochrome oxidase 1 gene marker. For the detection of CCHFV, ticks were tested individually by one-step multiplex real-time reverse-transcriptase quantitative polymerase chain reaction. The small segment of all positive samples was sequenced to determine the CCHFV genotype.ResultsIn total, 39 of the 1523 ticks (2.56%) collected from 63 cattles and 28 camels tested positive for CCHFV. Three Hyalomma species were identified. Hyalomma rufipes had the largest proportion of positivity (5.67%; 16/282), followed by Hyalomma dromedarii (1.89%; 23/1214). No Hyalomma impeltatum tested positive (0%; 0/21). Positive ticks were found in only six out of 91 host animals. Viral sequence analysis revealed the presence of two different CCHFV lineages (Africa I and Africa III).ConclusionsIn this study, 2.56% of Hyalomma ticks collected from camels and cattle in Mauritania tested positive for CCHFV. However, the true prevalence of CCHFV in unfed ticks may be lower, as a considerable number of ticks may have been passively infected during blood-feeding by co-feeding ticks or due to viremia of the host. The results indicate the need to track the actual area of circulation of this virus.Graphical

Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

Epidemiology and clinical features of intestinal protozoan infections detected by Real-time PCR in non-native children within an Italian tertiary care children's hospital: A cross-sectional study

BackgroundEnteric parasite infections are underestimated due to the limited sensitivity and specificity of microscopy, which remains the diagnostic gold standard in routine clinical practice. This could be a major problem in high-income countries, where the burden of parasitic diseases is low. In recent years, Multiplex Real-Time polymerase chain reaction (RT-PCR) based methods have been implemented. Therefore, the aim of this study was to evaluate the prevalence of four enteric protozoan species detected by RT-PCR in non-native children in Italy, and to describe their clinical characteristics. MethodsAdopted and immigrant children, evaluated for migration health assessment between 2017 and 2020 in a tertiary care children's hospital in Italy, were enrolled. Molecular analysis for Giardia lamblia, Dientamoeba fragilis, Blastocystis hominis, and Entamoeba histolytica, was conducted by in-house RT-PCR. ResultsOverall, 209 children were enrolled and 70% of them resulted positive by RT-PCR for at least one enteric parasite. B. hominis (47.8%) was the most commonly identified protozoa, followed by D. fragilis (44.5%). Co-infections with multiple pathogens were detected in 35.4% of the samples. Almost 80% of parasite-positive children were asymptomatic and the most common symptom was flatulence (60.7% of symptomatic children). Eosinophils were significantly increased in RT-PCR positive children compared to the negative ones and children with D. fragilis presented the highest eosinophils count. ConclusionsThe In-house Multiplex RT-PCR assay provides a valid molecular detection system for selected enteric parasites. This novel and accurate diagnostic method can help in increasing the detection rate of parasite infection, especially in high-risk population.

The African Network for Improved Diagnostics, Epidemiology and Management of common infectious Agents

BackgroundIn sub-Saharan Africa, acute respiratory infections (ARI), acute gastrointestinal infections (GI) and acute febrile disease of unknown cause (AFDUC) have a large disease burden, especially among children, while respective aetiologies often remain unresolved. The need for robust infectious disease surveillance to detect emerging pathogens along with common human pathogens has been highlighted by the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. The African Network for Improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA) is a sentinel surveillance study on the aetiology and clinical characteristics of ARI, GI and AFDUC in sub-Saharan Africa.MethodsANDEMIA includes 12 urban and rural health care facilities in four African countries (Côte d’Ivoire, Burkina Faso, Democratic Republic of the Congo and Republic of South Africa). It was piloted in 2018 in Côte d’Ivoire and the initial phase will run from 2019 to 2021. Case definitions for ARI, GI and AFDUC were established, as well as syndrome-specific sampling algorithms including the collection of blood, naso- and oropharyngeal swabs and stool. Samples are tested using comprehensive diagnostic protocols, ranging from classic bacteriology and antimicrobial resistance screening to multiplex real-time polymerase chain reaction (PCR) systems and High Throughput Sequencing. In March 2020, PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and analysis of full genomic information was included in the study. Standardised questionnaires collect relevant clinical, demographic, socio-economic and behavioural data for epidemiologic analyses. Controls are enrolled over a 12-month period for a nested case-control study. Data will be assessed descriptively and aetiologies will be evaluated using a latent class analysis among cases. Among cases and controls, an integrated analytic approach using logistic regression and Bayesian estimation will be employed to improve the assessment of aetiology and associated risk factors.DiscussionANDEMIA aims to expand our understanding of ARI, GI and AFDUC aetiologies in sub-Saharan Africa using a comprehensive laboratory diagnostics strategy. It will foster early detection of emerging threats and continued monitoring of important common pathogens. The network collaboration will be strengthened and site diagnostic capacities will be reinforced to improve quality management and patient care.

3' Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System.

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.

Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Taqman® probe based multiplex RT-PCR for simultaneous detection of Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli in foods

Three foodborne pathogenic bacteria, Listeria monocytogenes, Salmonella spp. and Shiga toxin-producing Escherichia coli are major contaminants in various foods and often cause food safety problems in public health. Therefore, a simple Taqman® probe based multiplex real-time polymerase chain reaction (mRT-PCR) for fast and reliable presence test to detect those three bacterial targets in different types of foods were developed. In order to improve the effectiveness of the developed method, a culturing step in Simultaneous Enrichment Broth at 37 °C for 18 h, followed by extraction of DNA using a boiling method was included. The developed Taqman® mRT-PCR platform gave all 100% of relative sensitivity, relative specificity and relative accuracy as compared to the conventional method (Bacteriological Analytical Manual) and iQ-check® RT-PCR detection kits. The results showed the developed method could detect as low as 1 CFU of each bacterial target in 25 g of artificially contaminated food samples. For practical food testing, the detection results of the bacterial targets naturally contaminated in 92 food samples by both the developed Taqman® probe mRT-PCR and the conventional method were essentially identical. The developed method is greatly specific, sensitive and effective to detect the three bacteria in foods simultaneously.

Multiplex RT-PCR provides improved diagnosis of skin and nail dermatophyte infections compared to microscopy and culture: a laboratory study and review of the literature

Dermatophytes are the most common cause of superficial mycosis, estimated to affect 20% to 25% of the general population. We assessed the performance of a novel real-time polymerase chain reaction (RT-PCR) multiplex assay for diagnosis of dermatophytosis.To evaluate sensitivity and specificity, 10 known bacteria and 10 known fungi commonly found on skin, as well as 105 samples with culture confirmed dermatophytosis were tested using Dermatophyte and Fungi assay (AusDiagnostics, Sydney, Australia), a novel multiplex assay for diagnosis of dermatophytosis in skin and nail. This was followed by prospective evaluation of 195 clinical samples for dermatophytosis by both conventional methods and RT-PCR.RT-PCR showed almost two-fold higher sensitivity and high specificity in the diagnosis of skin and nail dermatophytosis compared to traditional microscopy and culture. In addition, RT-PCR demonstrated markedly reduced turnaround time from 4 to 6 weeks to 4 to 6 hours and ability for high throughput.

#33: A Tale of Two Pneumos: the Impact of HIV Exposure or Infection Status on Pediatric Carriage of Streptococcus pneumoniae and Pneumocystis jirovecii, a Nested Case–control Analysis from the Pneumonia Etiology Research in Child Health (PERCH) study in Zambia

Abstract Background Most pediatric HIV cases in Africa reflect maternal to child transmission (MTCT). The persistently high seroprevalence of HIV in pregnant women (16.6%) and the low rates of mother to child HIV transmission (~2%) have resulted in a high number of children who are HIV exposed but uninfected (HEU). HIV exposed but uninfected (HEU) children have increased rates of morbidity and mortality vs. HIV unexposed and uninfected (HUU). Methods To explore the mechanisms behind this phenomenon, each case and control was sampled using a nasopharyngeal NP swab and an oropharyngeal (OP). A multiplex real-time polymerase chain reaction (PCR) assay was used to determine the presence of Streptococcus pneumoniae and Pneumocystis jirovecii in the NP/OP specimens. We compared nasopharyngeal carriage rates of S. pneumoniae and P. jirovecii between HIV-infected, HEU, or HUU case (pneumonia) and control (without pneumonia) children. In bivariate analyses, using carriage as a dichotomous outcome, proportions we compared using chi-square or odds ratios with 95% confidence intervals. In multivariate models, we created logistic and linear regression models adjusting for nutritional status as possible mediators of carriage status. Results SP carriage rates were similar between cases and controls. However, density was increased among HIV-infected children vs. HEU or HUU children (15.8 vs. 4.7 vs. 3.6 × 105 copies/mL, respectively). Among cases, PJ carriage among HIV positive, HEU and HUU children was 31%, 15% and 10%,, respectively, (P &amp;lt; 0.05) and carriage density was 63.9, 20.9, and 4.8 × 103 copies/mL, respectively (P &amp;lt; 0.05). In adjusted logistic regression models, HIV-infected cases were slightly more likely to be an S. pneumoniae carrier compared with HUU cases (aOR = 1.1; 95% CI: 0.57–2.14). In contrast, all other case/exposure combinations were less likely to be S. pneumoniae carriers with the lowest adjusted odds among HIV-infected controls children (aOR = 0.34; 95% CI: 0.17–0.71). The odds of being a PJ carrier was almost 6 times higher in HIV-infected cases than in HIV unexposed cases (aOR = 6.63; 95% CI: 3.24–13.54). Conclusions We demonstrate that HIV infection and HIV exposure without infection each have an impact on carriage rates and density for S. pneumoniae and P. jirovecii, though the magnitude and nature of these effects differs substantially between the two pathogens. This may be explained in part by differences in immune responses to these two different pathogens. Our analysis provides further evidence of fundamental differences in immune function among HIV exposed but uninfected infants compared with HIV unexposed, uninfected infants.

Comparison of the clinical characteristics and mortality of adults infected with human coronaviruses 229E and OC43

The purpose of the study was to compare clinical characteristics and mortality among adults infected with human coronaviruses (HCoV) 229E and OC43. We conducted a retrospective cohort study of adults (≥ 18 years) admitted to the ward of a university teaching hospital for suspected viral infection from October 2012 to December 2017. Multiplex real-time polymerase chain reaction (PCR) was used to test for respiratory viruses. Multivariate logistic regression was used to compare mortality among patients with HCoV 229E and HCoV OC43 infections. The main outcome was 30-day all-cause mortality. Of 8071 patients tested, 1689 were found to have a respiratory virus infection. Of these patients, 133 had HCoV infection, including 12 mixed infections, 44 HCoV 229E infections, and 77 HCoV OC43 infections. HCoV 229E infections peaked in January and February, while HCoV OC43 infections occurred throughout the year. The 30-day all-cause mortality was 25.0% among patients with HCoV 229E infection, and 9.1% among patients with HCoV OC43 infection (adjusted odds ratio: 3.58, 95% confidence interval: 1.19–10.75). Infections with HCoVs 229E and OC43 appear to have different seasonal patterns, and HCoV 229E might be more virulent than HCoV OC43.

Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis

The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.

Development of a Multiplex Real-Time PCR Assay for Predicting Macrolide and Tetracycline Resistance Associated with Bacterial Pathogens of Bovine Respiratory Disease.

Antimicrobial resistance (AMR) in bovine respiratory disease (BRD) is an emerging concern that may threaten both animal and public health. Rapid and accurate detection of AMR is essential for prudent drug therapy selection during BRD outbreaks. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction assay (qPCR) to provide culture-independent information regarding the phenotypic AMR status of BRD cases and an alternative to the gold-standard, culture-dependent test. Bovine clinical samples (297 lung and 111 nasal) collected in Nebraska were subjected to qPCR quantification of macrolide (MAC) and tetracycline (TET) resistance genes and gold-standard determinations of AMR of BRD pathogens. Receiver operating characteristic curve analysis was used to classify AMR based on the qPCR results. For lung tissues, the qPCR method showed good agreement with the gold-standard test for both MACs and TETs, with a sensitivity of 67–81% and a specificity higher than 80%. For nasal swabs, qPCR results passed validation criteria only for TET resistance detection, with a sensitivity of 88%, a specificity of 80% and moderate agreement. The culture-independent assay developed here provides the potential for more rapid AMR characterization of BRD cases directly from clinical samples at equivalent accuracy and higher time efficiency compared with the gold-standard, culture-based test.