Optimizing a Conventional Multiplex PCR for Simultaneous Detection of Granulomatous Skin Infection Agents: Leishmania aethiopica, Mycobacterium leprae, and Mycobacterium tuberculosis.

An atypical presentation of tuberculous gumma heralding a diagnosis of lymph node tuberculosis: Hindsight is 20/20

Objective To develop a multiple polymerase chain reaction (PCR) technique based assay for rapid detection of vanA, vanB, vanD and vanM in high-level vancomycin-resistant enterococci. Methods After analyzing the uncleotide sequence divergence among D-Ala∶D-Lac ligase genes, an multiplex PCR assay for vanA, vanB, vanD and vanM genes in high-level vancomycin-resistant enterococci were designed. By using recombination plasmids containing vanA, vanB, vanD and vanM genes as positive control, and non-vancomycin resistant enterococci (non-VRE) common pathogenic bacterial DNA as negative control, the sensitivity and specificity of the assay were evaluated. Fifty vancomycin-resistant enterococci (VRE) isolates were detected by the assay. Fifty clinical strains of VRE were isolated from 9 hospitals in Shanghai from January 2006 to December 2014. The results were compared with the conventional PCR and sequencing methods. Results The identity of the D-Ala∶D-Lac ligase genes were 60.8%—71.3% of vanA, vanB, vanD and vanM genes. The multiplex PCR assay could identify the genotypes of the positive control samples accurately. No false positive results were found in negative control samples. Among fifty VRE strains detected by the assay, 18 were vanA genotype and 32 were vanM genotype. Comparison of the multiplex PCR assay and sequencing methods revealed sensitivity and specificity of 100%. The detection limit of the assay was 2×10 copies/PCR reaction. The experiment could be done within 3.5 h. Conclusions A multiplex PCR assay is developed to rapid identify the genotype of the high-level vancomycin-resistant enterococci, which can be used for the molecular epidemiology research and detection of VRE. Key words: Enterococcus; Vancomycin resistance; Genes; Genotype

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

Leishmaniasis is a neglected tropical disease caused by infected female sandflies (Phlebotomus and Lutzomyia), which are vector-borne protozoan parasites belonging to the genus Leishmania. The diagnosis of cutaneous leishmaniasis (CL) can be challenging and delayed, particularly in areas where leishmaniasis is not endemic. CL is known as “the great imitator” because it can mimic many skin disorders due to its various clinical manifestations. Cutaneous sarcoidosis (CS) and cutaneous tuberculosis (CTB), which are also known as “the great imitators,” should be differentiated from CL due to the common clinical, dermoscopic, and histopathologic features. In this article, we aim to help clinicians differentiate CL by listing its clinical manifestations those are similar to CS and CTB and highlighting common and uncommon dermoscopic and histopathologic findings. We have also created a brief approach to diagnose CL, CS, and CTB, which is presented as a diagram. A search was performed on PubMed and Google Scholar using the keywords CL, CTB, CS, and granulomatous disease for all articles, with no restrictions. Updated articles on leishmaniasis, tuberculosis, and sarcoidosis, including some new concepts in clinical presentations, dermoscopy, and histopathology, were reviewed.

Multiple ulcerated lesions in a patient from Amazon, Brazil

Development and Clinical Validation of a Multiplex Real-time PCR Assay for Herpes Simplex and Varicella Zoster Virus

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

What do positive and negative Cutibacterium culture results in periprosthetic shoulder infection mean? A multi-institutional control study.

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

Introduction: The identification of the pathogens involved in pleural empyema has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercial multiplex pneumonia PCR assay to identify the pathogens involved in pleural empyema. Methods: Prospective, monocentric, observational study. Pleural fluid samples collected from 194 consecutive patients were analysed at the time of hospitalisation using conventional microbiology. Commercial multiplex pneumonia bacterial PCR was performed retrospectively. Results: There was a suspicion of empyema based on ultrasound findings in 18.5% of the cases, of these, 85.7% were diagnosed with empyema. When only taking into account the pathogens included in the multiplex PCR assay more pathogens were identified using the multiplex PCR assay than with conventional methods (23.3% vs. 6.7%, p=0.008). In those cases without suspicion of empyema, 9.7% had a final diagnosis of empyema. Using the same criteria, conventional methods identified pathogens in 0% of the cases and the multiplex PCR-based assay identified pathogens in 6.7% of the cases. The most common microorganisms identified in the cases diagnosed with empyema, were anaerobes (31) followed by gram-positive cocci (10) and gram-negative rods (4). Conclusion: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

Identification and verification of porcine DNA in commercial gelatin and gelatin containing processed foods

BACKGROUND AND OBJECTIVES:There are few reports of cutaneous tuberculosis with immunosuppressed states such as HIV, use of immunosuppressants or malignancy. Diagnosis is thus difficult and despite scientific advances such as polymerase chain reaction, it is frequently missed. Although rare, given its worldwide prevalence and the rising incidence of HIV, it is important for clinicians to recognize the variants and promptly treat the patient.DESIGN AND SETTING:Retrospective study of all cases of cutaneous tuberculosis diagnosed from October 2007 to November 2009 at an outpatient clinic of a tertiary-care hospital in northern India.METHODS:We collected information on the clinical form of disease, histopathology and HIV concurrence rates and looked for differences in presentation between mmunocompetent and immunocompromised states. We also looked for differences and HIV concurrence between immunocompetent and immunocomprised patients. Diagnosis was based on clinical, histopathological and microbiological tests for tuberculosis and a test for HIV.RESULTS:The overall incidence of cutaneous tuberculosis was 0.7% (131 of 18720 outpatients). HIV concurrence was 9.1% (12 cases) of all cutaneous tuberculosis cases. Most common variants seen were scrofuloderma (36.5%), lupus vulgaris (31%), tuberculosis verruca cutis (12.9%), lichen scrofulosorum (11.4%), papulonecrotic tuberculids (3.8%), erythema nodosum (2.2%) and erythema induratum of Bazin (1.5%).CONCLUSIONS:Cutaneous tuberculosis rates were slightly higher in our study than in other studies from India. HIV co-infection rates were similar to those in other studies. Many atypical morphological forms and presentations were observed in HIV co-infected patients. Due to the varied clinical presentations, physician awareness and a high index of suspicion are necessary to diagnose cutaneous forms of tuberculosis.

Leprosy is not always an easy disease to diagnose, and patients can remain undiagnosed for longtime, not only at the peripheral clinics but also even at places with higher medical facilities, so, there is an urgent need for rapid and definitive modalities for leprosy diagnosis. This prospective study evaluates the ability of Fite-Faraco staining (FF staining) and multiplex polymerase chain reaction (PCR) over hematoxylin and eosin staining (H and E staining) and Ziehl-Neelsen staining (ZN staining). The aim of this perspective study is to evaluate the effectiveness of FF staining in combination with multiplex PCR for the early and rapid diagnosis of leprosy than any other coexisting diagnosis tool. Patients with new skin patches or nodules with or without evidence of nerve damage were selected for the study. Punch biopsy was collected according to standard procedures. Each biopsy sample was divided into two equal parts, one half was fixed in 4% (v/v) buffered neutral formalin and then accordingly embedded in paraffin. Sections were stained by three different methods: H and E staining for histopathological examination, ZN staining, and FF staining for detection of acid-fast bacilli (AFB). And the other part was subjected for DNA extraction and PCR was carried out by the obtained DNA sample. H and E staining, ZN staining, FF staining, and PCR yield 58.2%, 50.9%, 60%, and 67.7% successful diagnosis of leprosy. The true diagnostic performances for these techniques were as follows: H and E staining - sensitivity 70.6%, positive predictive value (PPV) 81.9%, negative predictive value (NPV) 53.6%. For ZN staining - sensitivity 59.9%, PPV 69%, NPV 45.7%. For FF staining - sensitivity 74.6%, PPV 85.9%, NPV 56.7%, and for PCR - sensitivity 87.8%, PPV 95.6%, NPV 71.2%. The combination of FF staining and PCR was shown to provide a rapid and definitive diagnosis in the majority of leprosy suspected cases with a higher positive likelihood ratio (+LR) of 7.76 and 2.716, respectively, than H and E staining of 2.244 and ZN staining of 1.378.

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.

A multiplex polymerase chain reaction (mPCR) assay was developed for detection and characterization of pathogenic Escherichia coli that cause diarrhea and edema disease in swine. The mPCR assay was designed as a single reaction for detecting 5 different adhesins (K88, K99, 987P, F41, and F18), 3 enterotoxins (LT, STaP, and STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the mPCR assay was evaluated by comparison with results from previous analysis of 100 porcine isolates characterized by colony blot hybridization with DNA probes for the 5 adhesins and 4 toxin genes. There was complete agreement between the 2 methods. The mPCR assay for E. coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. It was found that the mPCR assays targeting genes encoding for K88 and F18 amplified products with the appropriate sizes from strains containing genes for different K88 and F18 antigenic subtypes; mPCR assays targeting the gene encoding for STaP amplified product from only STaP-positive but not STaH-positive isolates; and mPCR assays targeting the gene encoding for the Stx2 amplified products from only Stx2-positive and not Stx1-positive isolates. Similarly, mPCR assays targeting the gene encoding for LTI did not produce the appropriate product from strains containing genes for LTII. The mPCR assays are simple to perform, and they should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E. coli isolates from pigs with diarrhea or edema disease.