Investigation of etiology of community-acquired pneumonia in hospitalized patients in a tertiary hospital of São Paulo City, Brazil

Abstract Background Community-Acquired Pneumonia (CAP) is the primary cause of hospitalization in the United States and the third leading cause of death in Brazil. The gold standard for diagnosing the etiology of CAP includes blood culture, Gram-stained sputum, and sputum culture. However, these methods have low sensitivity. No studies investigating the etiology of CAP have been conducted in Brazil in the last 20-years, and the empirical choice of antimicrobials is mainly based on the IDSA guidelines. This is the first national study with this aim, and as a result, there's potential for the Brazilian consensus to be impacted and possibly modify its guidelines rather than adhering strictly to the IDSA's recommendations. Methods The aim of this study is to identify the main microorganisms implicated in CAP by employing a multiplex Polymerase Chain Reaction (mPCR) at the foremost public hospital in Brazil. All patients who were admitted to the emergency department and diagnosed with severe CAP underwent an mPCR panel using nasopharyngeal and oropharyngeal swabs, with the aim of detecting 13 bacterial and 21 viral pathogens. Results A total of 169 patients were enrolled in the study. The mPCR panel identified an etiological agent in 61.5% of patients, with viruses being the most common (42.01%), led by Rhinovirus, followed by Influenza and Coronavirus (non-SARS-CoV-2). Bacterial agents were identified in 34.91% of patients, with S. pneumoniae being the most common, followed by H. influenzae, M. catarrhalis, and S. aureus. Additionally, we found that the prescription for 92.3% of patients could be modified, with most changes involving de-escalation of antibiotics and antiviral therapy. Conclusion Our study revealed different etiological causes of CAP than those suggested by the Brazilian guidelines. Using molecular diagnostic tests, we were able to optimize treatment by using fewer antibiotics.

Identificación molecular de Entamoeba histolytica, Entamoeba dispar y Entamoeba moshkovskii en niños con diarrea en Maracaibo, Venezuela / Molecular identification of Entamoeba histolytica, E. dispar, and E. moshkovskii in children with diarrhea from Maracaibo, Venezuela

Resumen | Introducción. Las amebas no patógenas Entamoeba dispar, Entamoeba moshkovskii y Entamoeba bangladeshi son morfológicamente idénticas a Entamoeba histolytica, parásito responsable de la amebiasis, por lo cual se necesitan técnicas moleculares para diferenciarlas. Objetivo. Determinar la frecuencia de las diferentes especies de Entamoeba mediante reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) en muestras fecales de niños menores de cinco años con diarrea, provenientes de Maracaibo (Venezuela). Materiales y métodos. Se recolectó una muestra fecal por individuo en 75 niños con diarrea (grupo de casos) y en 25 niños sin diarrea (grupo control). Las heces se evaluaron mediante examen microscópico, método de concentración de formól-éter y PCR múltiple anidada en una sola ronda para identificar E. histolytica, E. dispar y E. moshkovskii. Además, se hizo una encuesta en la que se recopilaron los datos demográficos, signos, manifestaciones clínicas y estrato socioeconómico de los niños. Resultados. El 48 % de los participantes (38 del grupo de casos y 10 del grupo de control) tenían enteroparásitos. Solo en las muestras de cuatro de los niños, se encontraron quistes del complejo Entamoeba (tres en el grupo de casos y uno en el de control). Mediante PCR se amplificaron nueve muestras (9 %) para la detección de las amebas estudiadas. En el grupo de casos se registraron tres (28,13 %) de E. histolytica, cuatro (30,50 %) de E. dispar y una (9,37 %) de E. moshkovskii, en tanto que solo una (25 %) muestra amplificó para E. dispar en el grupo de control. Conclusión. En general, predominó E. dispar; sin embargo, todos los infectados con E. histolytica se detectaron en el grupo de niños con diarrea y se detectó el primer caso de E. moshkovskii en la región.

Abstract | Introduction: Entamoeba histolytica is an amebiasis-producing parasite. However, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba bangladeshi are non-pathogenic amoebae morphologically identical to it and, therefore, molecular techniques are required for their differentiation. Objective: To determine the frequency of Entamoeba species by polymerase chain reaction (PCR) in fecal samples from children under five years with diarrhea from Maracaibo (Venezuela). Materials and methods: A fecal sample per individual was collected from 75 children with diarrhea (case group) and 25 children without diarrhea (control group). Stools were evaluated by microscopic examination, formol-ether concentration method, and nested-multiplex PCR in a single round for the identification of E. histolytica, E. dispar, and E. moshkovskii. In addition, a survey was conducted in which demographic data, signs, clinical manifestations, and socioeconomic status were registered. Results: In total, 48% of the children (38 from the case group and 10 from the control group) had intestinal parasites. Only four children presented cysts of the Entamoeba complex in their samples (three from the case group and one from the control group). By means of PCR, nine samples (9%) amplified for the studied amoebae. In the case group, three (28.13%) amplified for E. histolytica, four (30.50%) for E. dispar, and one (9.37%) for E. moshkovskii while only one (25%) sample amplified for E. dispar in the control group. Conclusion: In general, E. dispar predominated. Nevertheless, all those infected with E. histolytica were detected within the group of children with diarrhea and we reported the first case of E. moshkovskii in the region.

Polymorphisms of the GSTT1 and GSTM1 genes in polycystic ovary syndrome

SUMMARY BACKGROUND: This study aimed to investigate the deletion polymorphisms of the genes of the glutathione S-transferase family GSTT1 and GSTM1 in patients with Polycystic Ovarian Syndrome (PCOS), comparing them with a control population. METHODS: Blood was collected from 219 women (110 with PCOS and 109 controls) and genomic DNA was extracted. For the analysis of polymorphisms, the technique used was multiplex PCR. In the statistical analysis, the chi-square test and multiple logistic regression were used. RESULTS: There is no association between the GSTM1 null and GSTT1 null genotypes with PCOS when analyzed separately (P = 0.616 and P = 0.188). The analysis of the combined genotypes showed differences between the groups (P < 0.05), evidencing that the genotypic combination GSTT1 positive and GSTM1 negative is more frequent among patients. In the multivariate analysis, smoking was more frequent in the control group (OR = 0.22; 95% CI - 0.87-0.57; P = 0.002) while the presence of a family history of PCOS (OR = 2, 96; 95% CI - 1.54-5.68; P = 0.001) was more frequent in women with PCOS. CONCLUSIONS: In the studied sample, the deletion polymorphisms of the GSTT1 and GSTM1 genes isolated are not associated with PCOS, but in combination, they may be implicated in the etiology of the condition.

RESUMO OBJETIVO: Este estudo teve como objetivo investigar os polimorfismos de deleção dos genes da família glutationa S-transferase GSTT1 e GSTM1 em pacientes com síndrome dos ovários policísticos (SOP), comparando-as com uma população controle. MÉTODOS: Foi colhido sangue de 219 mulheres (110 com SOP e 109 controles) e extraído o DNA genômico. Para análise dos polimorfismos, a técnica empregada foi PCR multiplex. Na análise estatística foi utilizado o teste do qui-quadrado e regressão logística múltipla. RESULTADOS: Não há associação dos genótipos GSTM1 nulo e GSTT1 nulo com SOP quando analisados isoladamente (p=0,616 e p=0,188). A análise dos genótipos combinados mostrou diferenças entre os grupos (p<0,05), evidenciando que a combinação genotípica GSTT1 positivo e GSTM1 negativo é mais frequente entre as pacientes. Na análise multivariada, o hábito tabagista foi mais frequente no grupo controle (OR=0,22; IC 95% - 0,87-0,57; p=0,002), enquanto que a presença do histórico de SOP familiar (OR=2,96; IC 95% - 1,54-5,68; p=0,001) foi mais frequente nas mulheres com SOP. CONCLUSÕES: Na casuística estudada, os polimorfismos de deleção dos genes GSTT1 e GSTM1 isolados não estão associados a SOP, mas em combinação podem estar implicados na etiologia da condição.

Infecciones respiratorias virales en episodios de fiebre en niños con trasplante de precursores hematopoyéticos / Respiratory viral infections during episodes of fever in children undergoing hematopoietic stem cell transplantation

Resumen Introducción: Los niños que reciben trasplante de precursores hematopoyéticos (TPH) pueden presentar infecciones respiratorias virales (IRV) durante episodios febriles. Los datos sobre su evolución clínica son escasos, así como la comparación de ellos con infecciones bacterianas (IB). Objetivo: Caracterizar la evolución clínica de pacientes con IRV, en comparación con IB en niños con TPH, cursando un episodio febril. Método: Estudio prospectivo en pacientes ≤ 18 años con cáncer y TPH ingresados por fiebre en el Hospital Luis Calvo Mackenna (2016-2019). Se realizó evaluación clínica y de laboratorio: hemocultivos, RPC para patógenos respiratorios (Filmarray®), cuantificación viral y medición de citoquinas en muestra nasal (Luminex®, 38 citoquinas). Se compararon los grupos IRV, IB y los de etiología no precisada (ENP) en relación con: infección respiratoria aguda (IRA), citoquinas nasales, ingreso a UCI, necesidad de ventilación mecánica, mortalidad y suspensión de antimicrobianos. Resultados: De 56 episodios febriles, 35 fueron IRV, 12 IB y 9 de ENP. Mediana de edad fue 8,5 años, 62% masculino. Un 94% de los casos IRV presentó IRA sintomática, versus 33% en los grupos IB y ENP (p < 0,001), con IRA baja en 69% de las IRV (p < 0,001). Rinovirus (54%) y coronavirus (15%) fueron las etiologías más frecuentemente detectadas. No hubo diferencias en citoquinas nasales entre los grupos IRV e IB. Ingreso a UCI: 11% del grupo IRV, 17% de IB y 11% de ENP (p = 0,88). Requirieron ventilación mecánica sólo 2 pacientes (p = 0,37) sin fallecimiento. Tras la detección viral respiratoria por RPC, se suspendió antimicrobianos en 26% de los casos con IRV (p = 0,04). Conclusión: Las IRV son frecuentes en niños con TPH y episodios febriles. La detección viral podría optimizar y racionalizar el uso de antimicrobianos en esta población.

Abstract Background: Children undergoing hematopoietic stem cell transplant (HSCT) can develop respiratory viral infections (RVI) during fever episodes. There are few data about clinical outcomes in RVI and compared to bacterial infections (BI) in this population. Aim: To determine clinical outcome of RVI, compared to BI in children with HSCT. Methods: Prospective study, patients ≤ 18 years with cancer and HSCT admitted with fever at a National Bone Marrow Transplant Center (Hospital Calvo Mackenna), Chile, (April-2016 to May-2019). Clinical assessment, laboratory tests, blood cultures, nasopharyngeal sample for multiplex-PCR (Filmarray®), viral loads by PCR and cytokine panel (Luminex®, 38 cytokines) were performed. The following outcomes were evaluated: upper/lower respiratory tract disease (RTD), admission to ICU, mechanical ventilation, mortality and antimicrobial withdrawal. Results: Of 56 febrile episodes, 35 (63%) were RVI, 12 (21%) BI and 9 (16%) with unknown etiology (UE). Median of age was 8.5 years, 62% male gender. Rhinovirus (54%) and coronavirus (15%) were the more frequent detected viruses. No significant differences in cytokine levels were observed between RVI and BI. 94% of RVI patients had symptomatic RTD, versus 33% in BI and 33% in UE group (p < 0.001), with lower-RTD in 69% of RVI group (p < 0,001). Admission to ICU was 11% in RVI, 17% in BI and 11% in UE group (p = 0.88); only 2 patients required mechanical ventilation (p = 0.37) and no mortality was reported. After an RVI was detected by PCR, antimicrobials were withdrawal in 26% of patients with RVI (p: 0.04). Conclusion: RVI are frequent etiologic agents in febrile episodes of patients with HSCT. Viral detection might help to rationalize the use of antimicrobials in this population.

Identification of Panax ginseng and Panax quinquefolius by Mitochondrial Cox Ⅱ Intron / 中国实验方剂学杂志

Objective:To develop a simple and accurate method for molecular authentication of Panax ginseng and P. quinquefolius.Method:The mitochondrial cox Ⅱ sequences of P. ginseng and P. quinquefolius were amplified by polymerase chain reaction（PCR）with universal primers. PCR products of the two species were sequenced in both directions, and sequence alignments were conducted for intron length polymorphisms exploitation. Multiplex PCR was established for the identification of P. ginseng and P. quinquefolius with their specific primers，which were designed respectively based on their insertion sequences. And the limit of detection of the multiplex PCR was also determined.Result:The insertion/deletion sequences were exploited in mitochondrial cox Ⅱ. Under the established multiplex PCR assay，P. ginseng generated a 729 bp specific band, while P. quinquefolius yielded a 141 bp specific amplicon，and the mixture of the two species yielded both 729 bp and 141 bp fragments. The established multiplex PCR assay could detect 0.1% of intentional adulteration of P. quinquefolius into P. ginseng, with down to 0.001 ng of genomic DNA.Conclusion:The established multiplex PCR assay can accurately identify P. ginseng and P. quinquefolius from different sources, without the optimization of reaction system and the introduction of additional mismatches，so as to provide a new molecular marker method for identifying botanical origin of P. ginseng and P. quinquefolius.

Detección de mutaciones causantes de distrofia muscular de Duchenne/Becker: reacción en cadena de la polimerasa Multiplex vs. Amplificación múltiple dependiente de ligación por sondas / Detection of mutations causing Duchenne and Becker muscular dystrophies: multiplex polymerase chain reaction vs. Multiplex ligation dependent probe amplification

RESUMEN Las distrofias musculares de Duchenne/Becker son enfermedades raras que reciben poca atención en nuestro medio. El objetivo del presente estudio fue implementar la técnica de amplificación múltiple dependiente de ligación por sondas (MLPA) y demostrar que tiene ventajas sobre la técnica de reacción en cadena de la polimerasa multiplex (PCR-multiplex). Se analizaron muestras de 40 individuos con diagnóstico presuntivo de distrofia muscular de Duchenne/Becker, primero por PCR-multiplex y luego por MLPA. Con la PCR-multiplex se detectaron 15 individuos con deleciones causales y con la técnica MLPA se logró diagnosticar a 21 individuos, cuatro duplicaciones y 17 deleciones. En conclusión, la técnica MLPA logra detectar mutaciones de tipo deleción y duplicación de exones, consiguiendo un mayor número de diagnósticos moleculares por alteraciones en el gen DMD.

ABSTRACT Duchenne and Becker muscular dystrophies are rare diseases that receive limited attention in our field. The objective of this study was to implement the Multiplex Ligation-dependent Probe Amplification technique (MLPA) and to demonstrate that it has advantages over the Multiplex Polymerase Chain Reaction (Multiplex PCR) technique. Samples from 40 individuals with a presumptive diagnosis of Duchenne and Becker muscular dystrophies were analyzed: first by Multiplex PCR and then by MLPA. Fifteen individuals with causal deletions were detected with Multiplex PCR, while the MLPA technique was able to diagnose 21 individuals, four duplications, and 17 deletions. In conclusion, the MLPA technique can detect mutations of the exon deletion and duplication type, yielding a larger number of molecular diagnoses due to alterations in the DMD gene.

Evaluation of loop-mediated isothermal amplification assay for detection and typing of human papilloma virus 16 and 18 from endocervical samples

Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.

Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria

Background & objectives: The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings. Methods: In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo ?-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined. Results: Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM(63%) was the most prevalent gene followed by blaVIM(18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin. Interpretation & conclusions: Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.

Multiplex polymerase chain reaction-based reverse line blot hybridization to detect pathogens causing neonatal bacterial meningitis and relevant drug resistance genes / 中华围产医学杂志

Objective@#To evaluate the performance of multiplex polymerase chain reaction-based reverse line blot hybridization (mPCR/RLB) in the detection of pathogens causing neonatal bacterial meningitis and associated drug resistance genes.@*Methods@#Clinical data and cerebrospinal fluid (CSF) samples were collected retrospectively from 80 cases diagnosed with neonatal bacterial meningitis in Beijing Children's Hospital from January 1, 2012 to December 31, 2018. A total of 100 CSF samples were obtained including 80 samples collected after admission (12 before and 68 after antibiotic treatment) and 20 recollected at follow-up. All CSF samples were analyzed by conventional culture, susceptibility test and mPCR/RLB. Differences in the detection of pathogens and drug resistance genes were analyzed by Chi-square test.@*Results@#(1) Among the 80 first-collected CSF samples, mPCR/RLB revealed significantly higher positive rate than conventional culture [26.3% (21/80) vs 7.5% (6/80), χ2=10.025, P=0.002]. No significant difference was showed between the two methods in analyzing the 12 samples collected before antibiotic therapy (9/12 vs 5/12, χ2=1.543, P=0.214), while the positive rate in 68 samples collected after antibiotic intervention detected by mPCR/RLB was obviously higher than that by conventional culture [17.6% (12/68) vs 1.5% (1/68), χ2=13.176, P<0.001]. (2) Conventional culture results of the 20 samples collected during follow-up were all negative, but four were positive using mPCR/RLB, which were also positive previously. Furthermore, the results of both methods in previous detections were identical. (3) According to the conventional culture results, the pathogens were Escherichia coli (three cases), Group B Streptococcus (two cases) and Listeria monocytogenes (one case), while mPCR/RLB detected Escherichia coli (four cases), Group B Streptococcus (five cases), Listeria monocytogenes (four cases), Neisseria meningitidis (four cases), Haemophilus influenzae b (one case), Gram-negative bacteria (one case), Gram-positive bacteria (one case), and Listeria monocytogenes and Haemophilus influenzae b coinfection (one case) in 80 first-collected CSF samples. (4) Antibiotic susceptibility test showed that one Escherichia coli strain produced extended spectrum beta-lactamases. Drug resistance gene detection by mPCR/RLB showed that acrA, acrB, CTX-M (consistent with antibiotic susseptibility test) and TetM genes were positive in three, two, one and one case, respectively.@*Conclusions@#mPCR/RLB is of great clinical value due to its higher detection rate and better accuracy compared with bacterial culture and can also detect drug resistance genes.

Evaluation of FilmArray respiratory panel for the rapid and multi￣pathogen detection in children with severe pneumonia / 中国小儿急救医学

Objective To evaluate the value of nested polymerase chain reaction (PCR) for the rap-id detection of pathogens in children with severe pneumonia. Methods We prospectively enrolled the pa-tients with severe community-acquired pneumonia admitted to pediatric intensive care unit (PICU) in Shang-hai Children′s Hospital from January 2017 to June 2018. The sputum for PCR were collected within 24 h after PICU admission. Both nested PCR and routine microbiological methods were performed. Respiratory Panel (R-Panel) based on nested PCR could detect 17 kinds of respiratory pathogen at the same time. Results A total of 65 patients were enrolled in this study and the samples were detected using both R-Panel and routine microbiological method. (1) A total of 15 patients (23. 08 %) showed positive routine microbiological de-tection including 13 cases with virus-positive and 2 cases with mycoplasma-positive; (2) A total of 38 pa-tients (58. 46%) showed positive results using R-Panel within 2 h including 46 cases with virus-positive and 5 cases with mycoplasma-positive. The mainly primary infection was human rhinovirus and enterovirus in 15 cases (23. 08%),followed by adenovirus in 10 cases (15. 38%); and the positive rate of 2 or more patho-gens was 18. 46% (12/65);(3) The rate of adenovirus-positive using R-Panel was significantly higher than that using routine microbiological methods (15. 38 % vs. 4. 62%,χ2 ＝4. 188,P＝0. 041); the sensitivity of R-Panel for detection of adenovirus,respiratory syncytial virus,and parainfluenza virus was significantly high-er than those of routine microbiological methods (100% vs. 30%,χ2 ＝107. 692;50. 00% vs. 16. 67%,χ2 ＝ 24. 442;100% vs. 80%,χ2 ＝22. 222;100% vs. 40%,χ2 ＝85. 714; all P <0. 001). Conclusion R-Panel using nested PCR is a rapid,sensitive,and specific method for the detection of pathogens in children with severe community acquired pneumonia,which is valuable for targeted therapy in time.

Birth of a healthy baby after preimplantation genetic diagnosis in a carrier of mucopolysaccharidosis type II: The first case in Korea / 대한생식의학회지

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive lysosomal storage disease caused by mutation of the iduronate-2-sulfatase gene. The mutation results in iduronate-2-sulfatase deficiency, which causes the progressive accumulation of heparan sulfate and dermatan sulfate in cellular lysosomes. The phenotype, age of onset, and symptoms of MPS II vary; accordingly, the disease can be classified into either the early-onset type or the late-onset type, depending on the age of onset and the severity of the symptoms. In patients with severe MPS II, symptoms typically first appear between 2 and 5 years of age. Patients with severe MPS II usually die in the second decade of life although some patients with less severe disease have survived into their fifth or sixth decade. Here, we report the establishment of a preimplantation genetic diagnosis (PGD) strategy using multiplex nested polymerase chain reaction, direct sequencing, and linkage analysis. Unaffected embryos were selected via the diagnosis of a single blastomere, and a healthy boy was delivered by a female carrier of MPS II. This is the first successful application of PGD in a patient with MPS II in Korea

Comparison of Multiplex Real-Time Polymerase Chain Reaction Assays for Detection of Respiratory Viruses in Nasopharyngeal Specimens / 대한임상미생물학회지

BACKGROUND: Respiratory tract infections are major public health threats, and the identification of their causative microbes helps clinicians to initiate timely and appropriate antimicrobial therapy and prevent the secondary spread of infection. The main goal of this study was to compare two multiplex real-time polymerase chain reaction (PCR) assays used to detect respiratory viral pathogens in nasopharyngeal swab specimens. METHODS: Between September and October 2017, a total of 84 nasopharyngeal specimens were obtained consecutively from patients in a tertiary hospital using a flocked swab with 3 mL universal transport medium (COPAN Diagnostics, USA). A total of 64 positive and 20 negative sample results from the LG AdvanSure RV real-time RT-PCR kit (LG Life Sciences, Korea) were further retested using a new AdvanSure RV-plus a real-time RT-PCR kit to compare their performance. RESULTS: Statistical analysis of positive and negative agreement between the two different kits was conducted between the newly introduced AdvanSure RV-plus real-time RT-PCR kit and the AdvanSure RV real-time RT-PCR. The overall agreement was 96.4%, with positive agreement of 98.4% and negative agreement of 90%. The evaluated sensitivity and specificity of AdvanSure RV-plus real-time RT-PCR were 96.9% and 94.7%, respectively, with a kappa value of 0.9 (P<0.001). CONCLUSION: The performances of LG AdvanSure RV real-time RT-PCR and the new AdvanSure RV-plus real-time RT-PCR kit showed strong overall agreement. AdvanSure RV-plus real-time RT-PCR had a better detection rate and could detect coronavirus 229E and enterovirus, especially with a high detection rate in coinfection. AdvanSure RV-plus real-time RT-PCR can be considered a useful tool for respiratory virus diagnosis in clinical laboratories.

Single or Dual Infection with Respiratory Syncytial Virus and Human Rhinovirus: Epidemiology and Clinical Characteristics in Hospitalized Children in a Rural Area of South Korea

PURPOSE: Respiratory syncytial virus (RSV) and human rhinovirus (hRV) are the most common causes of child respiratory viral infections. We aimed to investigate epidemiological and clinical characteristics of RSV and hRV single infections and coinfections. METHODS: Nasopharyngeal aspirates of hospitalized children aged <5 years were tested using multiplex reverse transcription polymerase chain reaction (RT-PCR) from October 2014 to April 2017. Their medical records were retrospectively reviewed. RESULTS: RSV or hRV was detected in 384 patients who divided into 3 groups: patients with RSV (R group, n=258); patients with hRV (H group, n=99); and patients with both (RH group, n=27). The R group (median age, 6 months) consisted of 248 (96.1%) patients with lower respiratory tract infection (LRTI), and 14 (5.4%) needed oxygen inhalation. Infants aged <12 months (63.2%) had respiratory difficulty and were supplied oxygen more often. The H group (median age, 16 months) consisted of 56 (56.6%) patients with LRTI, 4 (4%) required oxygen inhalation, and 1 (1.0%) required mechanical ventilation. Infants (40.4%) showed longer hospitalization compared to patients aged ≥12 months (5 vs. 4 days, P<0.05). The RH group consisted of 24 (88.9%) patients with LRTI, and 2 (7.4%) needed oxygen inhalation. Hospitalization days and oxygen inhalation and mechanical ventilation rates did not differ between single infections (R and H groups) and coinfections (RH group). CONCLUSIONS: RSV was detected more often in younger patients and showed higher LRTI rates compared to hRV. Single infections and coinfections of RSV and hRV showed no difference in severity.

Genetic analysis and prenatal diagnosis of Duchenne or Becker muscular dystrophy / 中华妇产科杂志

Objective To explore the mutation characteristics of DMD gene in patients with Duchenne or Becker muscular dystrophy and female carriers, to provide effective prenatal diagnosis. Methods Samples were collected from 94 male patients clinically diagnosed with Duchenne or Becker muscular dystrophy and 121 corresponding female relatives from Qingdao Women and Children′s Hospital from June 2011 to October 2018. Multiplex ligation-dependent probe amplification (MLPA) was used to detect their DMD gene, and 23 high risk pregnants were performed prenatal diagnosis. Any candidate of DMD gene single-exon deletion was validated by further PCR amplification. The sample with whole DMD gene deletion was confirmed by chromosomal microarray analysis (CMA) to detect copy number variations and break site. Results Among 94 clinical Duchenne or Becker muscular dystrophy patients, 66(70.2%, 66/94) were detected gene mutation; 56 cases were exon deletion mutation and 10 cases were duplication mutation. In 121 female relatives, 48 cases (39.7%, 48/121) were diagnosed as carriers. The mutation carrying rate, was 64.5% (40/62) identified in 62 mothers of Duchenne or Becker muscular dystrophy patients. Five Duchenne or Becker muscular dystrophy fetuses and 5 carrier fetuses were prenatally diagnosed in 23 high risk pregnants. Two children with the entire DMD gene deletion were identified more deletions at Xp21, with deletions of 6.66 Mb and 10.64 Mb respectively. Conclusions MLPA may be an important method to detect DMD gene mutation of deletion and duplication. Therefore, the diagnosis of probands, female carriers and making an effective prenatal diagnosis are essential to reduce the birth of children with Duchenne or Becker muscular dystrophy.

Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India

The objective of this study was to detect the association of biofilm formation with IS256 among clinical and carrier isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). A total of 71 MRSE isolates were included in this study. Phenotypic detection of biofilm formation was done by Congo red agar method. Detection of genes associated with biofilm formation (icaAD, aap and atlE) and insertion sequence IS256 was done by polymerase chain reaction. Of the 71 MRSE isolates,19/40 (47.5%) clinical isolates from hospital settings and 11/31 (35.5%) carrier isolates from community settings respectively were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes. Nearly 80% of clinical isolates were found to harbour IS256, whereas only 13% of community isolates harboured IS256.

Estandarización de una prueba múltiple de reacción en cadena de la polimerasa en tiempo real para la identificación de Angiostrongylus cantonensis, A. costaricensis y A. vasorum / Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Resumen Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específica para el manejo de las angiostrongilosis humana y la animal. Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus. Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®. Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano. Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %). Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Abstract Introduction: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. Objective: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. Materials and methods: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. Results: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). Conclusions: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China / 中华医学杂志(英文版)

<p><b>Background</b>Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China.</p><p><b>Methods</b>We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA.</p><p><b>Results</b>We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions.</p><p><b>Conclusions</b>MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.</p>

Epidemiology and Clinical Severity of the Hospitalized Children with Viral Croup

PURPOSE: In this study, the clinical and epidemiological characteristics of patients admitted for viral croup were analyzed to evaluate disease severity based on the organism that caused the infection. METHODS: We retrospectively reviewed the medical records of 302 patients who were admitted to the Department of Pediatrics at the Wonju Severance Hospital between May 2013 and December 2016 for viral croup. Patients who showed positive results on multiplex polymerase chain reaction were subsequently diagnosed with respiratory virus infection. The Westley scoring system was used to evaluate the severity of viral croup. RESULTS: Of the 302 patients, 149 were admitted due to severe viral croup, including 88 boys and 61 girls, with a boy-to-girl ratio of 1.44:1. About 110 cases of parainfluenza virus infection have been reported, which accounted for almost half of the total cases. The other identified viruses included influenza virus, human rhinovirus, and respiratory syncytial virus. Analysis of the association between severe viral croup and causative pathogen revealed that only parainfluenza type 2 virus showed a significantly high risk. Parainfluenza type 2 virus did not show an age-based difference in frequency but showed relatively a higher frequency of infections during the summer and fall. CONCLUSIONS: In this study, parainfluenza virus type 2 was the only virus associated with severe viral croup. To facilitate proper preventive management, treatment, and prognosis evaluation of viral croup, prospective and multicenter studies should assess the additional variables and the severity of the virus. Additionally, further studies should be conducted to assess age-dependent influences, as well as the regional and seasonal incidence of viral infection.

HLA-B*15:02 screening in epileptic patients using a high resolution melting-real time PCR (HRM-QPCR) method

Background: The HLA-B*15:02 polymorphism in epileptic patients is known to be associated with carbamazepine-induced Stevens-Johnson syndrome (SJS). The prevalence of HLA-B*15:02 polymorphism seemed to be ethnic-specific with a higher frequency of HLA-B*15:02 in Asian compared to the Europeans. This study was performed to determine the frequency of the HLA-B*15:02 polymorphism in epileptic patients at the Chancellor Tuanku Muhriz Hospital-UKM Medical Centre (HCTM-UKMMC) using high resolution melting-real time PCR (HRM-QPCR) method. Methods: We performed a fast and effective in-house high resolution melting-real time polymerase chain reaction method and compared it with the conventional multiplex-PCR method. The specificity and sensitivity of each test were also determined using DNA from saliva. Results: Using the conventional multiplex-PCR approach for screening, 25 out of 64 (39.1%) epileptic patients were positive for HLA-B*15:02. However, using the HRM-QPCR technique, 24/64 (37.5%) of the patients were positive. The one patient who tested positive by the multiplex-PCR but negative using the HRM-QPCR turned out to be negative by DNA sequencing. The HRM-QPCR and DNA sequencing showed 100% sensitivity and specificity. The multiplex-PCR showed 100% sensitivity and 98.4% specificity compared to both HRM-QPCR and DNA sequencing. The HRM-QPCR is also more cost-effective (<$16.40 USD/test) and less time-consuming when compared to the multiplex-PCR ($25.15 USD/test).Conclusion: Our result suggested that multiplex PCR, HRM-QPCR and Sanger sequencing can be used for detection of HLA-B*15:02. However, a qualitative method such as multiplex PCR should be confirmed with other quantitative methods such as HRM-QPCR and Sanger sequencing.

No association between glutathione S-transferase M1 and T1 gene polymorphisms and susceptibility to endometriosis / Ausência de associação entre polimorfismos nos genes da glutationa-S transferase M1 e T1 e suscetibilidade à endometriose

ABSTRACT Introduction: Endometriosis is a common gynecologic disorder influenced by genetic and environmental factors. The glutathione S-transferase family is associated with endometriosis because its main function is cellular detoxification, so the absence of those enzymes may be a factor for the development of the disorder. Objective: Investigate the relationship between polymorphisms of GSTM1 and GSTT1 genes and endometriosis, in order to gain a better understanding of the association between detoxification genes and the susceptibility to endometriosis. Material and methods: Case-control study in 132 women (49 with endometriosis and 83 of the control group). The genotype was determined using multiplex polymerase chain reaction (PCR), observed in 10% polyacrylamide gel electrophoresis stained with silver nitrate, and statistical analysis was performed. Results: There was not a significant difference between the GSTM1 and GSTT1 null genotype in the endometriosis group and the control group (p = 0.9956). The same result was observed with the combined genotype (p = 0.8129). Conclusion: In the present study, the GSTM1 and GSTT1 polymorphisms are not associated with a higher risk of endometriosis.

RESUMO Introdução: A endometriose é uma doença ginecológica comum influenciada por fatores genéticos e ambientais. A família de glutationa-S-transferase está associada à endometriose porque tem como principal função a detoxificação celular, de modo que a ausência dessas enzimas pode ser um fator no desenvolvimento da doença. Objetivo: Investigar a relação entre polimorfismos nos genes GSTM1 e GSTT1 e endometriose, a fim de melhor compreender a associação entre os genes de detoxificação e a suscetibilidade à endometriose. Material e métodos: Estudo caso-controle em 132 mulheres (49 com endometriose e 83 do grupo- controle). O genótipo foi determinado utilizando reação em cadeia da polimerase (PCR) multiplex, observado em eletroforese em gel de poliacrilamida 10% corado com nitrato de prata, e a análise estatística foi empregada. Resultados: Não houve diferença significativa entre o genótipo nulo GSTM1 e GSTT1 no grupo endometriose e no controle (p = 0,9956). O mesmo resultado foi observado para o genótipo combinado (p = 0,8129). Conclusão: No presente estudo, os polimorfismos GSTM1 e GSTT1 não estão associados a maior risco de endometriose.