Multiplex Tandem PCR Research Articles

Comparison of MT-PCR with Quantitative PCR for Human Bocavirus in Respiratory Samples with Multiple Respiratory Viruses Detection

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

Detection of bacterial co-infection in patients with severe influenza

Viral infections engender bacterial colonization in the host by causing alteration in the innate immune response to common bacterial pathogens. These alterations may lead to severe bacterial pneumonia during influenza. Bacterial co-infection is common in influenza pneumonia and increases morbidity and mortality. With the onset of the COVID-19 pandemic worldwide, research in viral diseases has intensified. With this study, it can be thought that bacterial colonization may also support viral activity in the human body. This study sought to investigate bacterial co-infection in respiratory specimens using samples from the Australian influenza epidemic of 2009 as a model for analysis. Furthermore, it was thought that this study could shed light on bacterial co-infection studies in patients with COVID-19, which caused the current pandemic. 200 clinical specimens were extracted for isolation of genomic DNA and directly tested in a multiplexed quantitative PCR. Multiplex-tandem PCR (MT-PCR) assay based on 16S rRNA consensus sequence of a family (Enterobacteriaceae species) and four genera (Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Pseudomonas spp.) was performed. According to MT-PCR results, respiratory tract samples showed a very high rate of bacterial identification (163/200-81.5%), and Enterococci were commonly found as the dominant genus identified in influenza patients. In a total of 200 putative co-infection samples, Enterococcus spp. was apparently present in 139 samples (69.5%), being the dominant signal (any other signal was ≤10%) from 106 samples. Streptococcus spp. was next most commonly indicated with 137 positive results (68.5%), but was the dominant signal in 19 of 137. It was determined that bacterial co-infection was high in the samples taken from patients with severe influenza, and Enterococcus was the dominant bacteria in the upper respiratory tract of patients treated for severe pneumonia, while Streptococcus was more dominant in the lower respiratory tracts than the other species.

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput

During the COVID-19 pandemic, sample pooling has proven an effective strategy to overcome the limitations of reagent shortages and expand laboratory testing capacity. The inclusion of influenza and respiratory syncytial virus (RSV) in a multiplex tandem PCR platform with SARS-CoV-2 provides useful diagnostic and infection control information. This study aimed to evaluate the performance of the influenza and RSV targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay, including the effect of pooling samples on target detection.RSV target detection in clinical samples was compared to the Cepheid Xpert Xpress Flu/RSV assay as a reference standard. Samples were then tested in pools of four and detection rates were compared. Owing to the unavailability of clinical samples for influenza, only the effect of sample pooling on simulated samples was evaluated for these targets.RSV was detected in neat clinical samples with a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.5% compared to the reference standard, demonstrating 99.7% agreement. This study demonstrates that sample pooling by four increases the average Ct value by 2.24, 2.29, 2.20 and 1.91 cycles for the target's influenza A, influenza A typing, influenza B and RSV, respectively. The commercial AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay was able to detect influenza and RSV at an intermediate concentration within the limit of detection of the assay. Further studies to explore the applicability of sample pooling at the lower limit of detection of the assay is needed. Nevertheless, sample pooling has shown to be a viable strategy to increase testing throughput and reduce reagent usage. In addition, the multiplexed platform targeting various respiratory viruses assists with public health and infection control responses, clinical care, and patient management.

Molecular Survey of Vector-Borne Pathogens of Dogs and Cats in Two Regions of Saudi Arabia.

Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood samples were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive samples were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.

A high-throughput multiplex tandem PCR assay for the screening of genetically modified maize

Genetically modified organisms (GMO) are increasingly gaining acceptance, which has led to an increasing demand for high-throughput methods for their detection. This study describes development of a high-throughput and low-cost multiplex tandem PCR (MT-PCR) assay for the screening of transgenic maize of 14 targets, containing 7 genetically modified (GM) common screening elements, 6 transgenic maize events and a maize reference gene, using SYBR Green I for quantification. The 14-target high-throughput multiplex tandem PCR assay successfully amplified products from single-event samples with a GM maize DNA amount less to 0.001 ng, and displayed high specificity. This assay can efficiently be used for screening GM maize in foods and feeds.

An automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study

Detecting the genera and species of gastrointestinal (GI) nematode infections in faecal samples obtained from cattle requires the incubation of faeces (‘larval culture’) followed by identification of the third-stage larvae that are harvested after 10–14days. Substantial research in the development of PCR-based methods for the rapid and specific identification GI nematodes has been conducted for small ruminants, whilst only few such assays have been developed for cattle. In the present paper we describe the development of an automated, robotic PCR platform for the detection and genus and/or species-specific identification of GI nematodes from bovine faecal samples. This test was then validated using samples from different regions of three countries (Australia, Belgium and Scotland). The PCR platform was found to be highly sensitive and specific for the identification of the important GI nematodes in naturally infected cattle (both estimates >90%). The PCR platform can also estimate the percentage of genera or species present in a mixed-species infection, and was found superior to larval culture in terms of speed (1–2days versus 1–2 weeks for culture), sensitivity and specificity. The PCR was simple to use and the operator requires no knowledge or experience to identify the nematodes present, compared to larval culture where even experienced operators can make substantial errors due to considerable overlap in the published characteristics of key species.

Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections.

Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods.

A multiplex tandem real-time PCR assay for the detection of enteric viruses

Noroviruses (NoVs) are important enteric pathogens that cause gastroenteritis worldwide. The first documented NoV outbreaks in South Africa (SA) were described in 1993. The current NoV prevalence and circulating genotypes are unknown. SA lacks NoV outbreak reporting systems and therefore the number and impact of NoV infections is underestimated.This study aimed to determine the prevalence and genetic diversity of NoV infections in hospitalised paediatric patients with gastroenteritis in SA during 2008.Stool specimens referred for virological analysis from hospitalised children ≤13 years, with gastroenteritis, were screened for rotavirus, human adenovirus and human astrovirus by enzyme immunoassay and for NoV genogroup I (GI), II (GII) and sapovirus by real-time RT-PCR. NoV strains were genotyped, and variants identified, based on sequence and phylogenetic analyses of the 5′ end or the full length of the capsid gene, respectively.Rotavirus was the most prevalent virus detected in 24.2% (61/252) of specimens, followed by NoV in 14.3% (35/245) and adenovirus, astrovirus and sapovirus in 9.6%, 6.7% and 4% of specimens, respectively. NoVs were only detected in children ≤2 years. The GII NoVs (89%) predominated and eight types were identified with GII.4 (43%) detected most frequently. The emerging 2008 GII.4 variant represented 80% of the GII.4 strains.A diverse range of NoV genotypes were identified in hospitalised children with gastroenteritis. The 2008 GII.4 variant was the most frequently detected strain in the study. This is the first report of NoV GII.4 viruses in SA.

Comparison of Direct Microscopy, Culture, ELISA and Molecular Methods for the Diagnosis of Entamoeba histolytica

Amebiasis, a parasitic infection caused by Entamoeba histolytica, is one of the most common parasitic infections worldwide. Since it is still an important public health problem in developing countries, rapid differential diagnosis of amebiasis is crucial in terms of treatment. The most frequently used method for laboratory diagnosis is direct microscopy, however more reliable and specific methods are needed in order to differentiate the apathogenic Entamoeba dispar under the microscope. This study was conducted to compare the results of different methods namely, direct microscopy, culture, ELISA and PCR for the detection of E.histolytica in stool samples and to evaluate the performances of those methods. A total of 1049 stool samples collected from pediatric and adult patients who were admitted to hospital with diarrhea complaint between January 2011-March 2013, and randomly selected samples from primary school children, were included in the study. Direct microscopic examination was performed by native-lugol, physiological saline, modified formol-ethyl acetate sedimentation and trichrome staining methods. The stool samples were also inoculated into TYI-S-33 media for axenic cultivation of amoeba. The presence of amebic antigens in the samples were screened by a commercial ELISA kit (TechLab, E.histolytica II, USA). For the molecular diagnosis, a multiplex tandem real-time PCR (MT-PCR) kit (AusDiagnostics Pty Ltd, Australia) was used, after the extraction of DNAs with QIAamp DNA Stool Mini Kit (Qiagen, USA). A total of 354 samples which could be evaluated by all of the methods, were included in the study. Of the 354 stool samples, 84 (23.7%) were found E.histolytica/E.dispar positive by direct microscopy, 61 (17.2%) by trichrome staining, 46 (12.9%) by culture, 31 (8.7%) by ELISA and 9 (2.5%) by MT-PCR. Of direct microscopy positive samples 54.7% (46/84) were also positive with trichrome staining, 39.3% (33/84) with culture, 15.5% (13/84) with ELISA and 7.1% (6/84) with MT-PCR methods. On the other hand, of the nine MT-PCR positive samples, six were positive with direct microscopy, four with trichrome staining and culture, and one with ELISA. It was remarkable that only one (0.3%) sample yielded positive results with all of the diagnostic methods used. When MT-PCR was considered as the reference method, the sensitivity and specificity values of direct microscopy, trichrome staining, culture and ELISA methods were estimated as; 66.7% and 77.4%, 44.4% and 83.5%, 44.4% and 87.8%, 11.1% and 91.3%, respectively. In conclusion, if the circumstances allow, the use of all methods in combination and evaluation together with the clinical symptoms seems to be the best approaches for the laboratory diagnosis of patients with amebiasis.

Molecular-based diagnostics in the clinical mycology laboratory: Storm in a teacup?

The rapidity of modern genomic/proteomic technologies has opened up new possibilities for the diagnosis of invasive fungal infections (IFIs). Diagnosis of yeast infections requires direct detection in blood cultures but ideally even earlier detection in blood (whole blood, serum) and from other clinical samples. A number of commercial systems employing multiplex real time PCR, multiplex tandem PCR, and PCR-magnetic bead technology, have been trialled or are being trialled but thus far their clinical value remains uncertain and their applicability in different populations has not yet been systematically evaluated. PCR assays have been developed to detect both Candida and Cryptococcus species. Direct detection of moulds in clinical samples is also problematic with the possible exception of detection of the pathogen in tissue biopsy specimens; the sensitivity, and positive predictive values of various molecular assays have been suboptimal. Screening for invasive aspergillosis by detection of Aspergillus DNA in blood has utility and prevents unnecessary empiric antifungal therapy, in early diagnosis in high risk haematology patients, particularly if they are not receiving mould active prophylaxis.

Prevalence of Shiga toxin-producing Escherichia coli (STEC) in Tasmania, Australia

(1) To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and the incidence of STEC associated enteritis within Tasmania and thereby evaluate the need for routine testing for this organism, and (2) compare detection method(s) for this purpose. 282 selected faecal samples were tested for STEC using selective culture media and multiplex tandem PCR (MT-PCR). The PCR used detected the virulence markers stx1, stx2 and eaeA. STEC were isolated in six of 282 faecal samples. This represents a STEC notification rate for Tasmania of 2.3 cases per 100 000 population. CT-SMAC medium detected one, CHROMagar STEC medium detected three and MT-PCR detected six STEC isolates. CHROMagar STEC medium displayed 50% sensitivity and 93% specificity compared to MT-PCR. The incidence of STEC associated enteritis in Tasmania was almost six times greater than the previously reported Australian average. STEC Screening Easy-Plex MT-PCR was more sensitive than both CHROMagar STEC and CT-SMAC media for the detection of STEC from pre-enriched faecal samples.

Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms

Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of ≥97.7% for each assay.

5. The utility of HPV PCR and p16 immunohistochemistry in the differential diagnosis of second branchial cleft cysts and level 2 cystic metastatic squamous cell carcinomas by fine needle aspiration cytology

Background Branchial cleft cysts (brCC) can resemble level 2 nodal cystic metastatic squamous cell carcinomas (mSCC). mSCC in this location often arise from an HPV-associated oropharyngeal primary. P16 immunohistochemistry (IHC) is used as a surrogate marker for infection by oncogenic HPV in this setting and can be combined with HPV PCR to identify this subset of mSCC. Aim (1) Evaluate the usefulness of p16 IHC and HPV PCR in the separation of brCC and mSCC. (2) Assess how different PCR techniques perform in this setting. Methods 54 brCC and 35 mSCC with cell blocks were assessed. 15/35 (42.8%) mSCC were oropharyngeal in origin. P16 IHC was scored by three pathologists. HPV detection was carried out by multiplex nested PCR (nPCR) and a newer multiplex tandem PCR (tPCR) technique. Results No brCC had ≥20% p16 positive cells in contrast to 17/35 (48.6%) mSCC; 12/17 (70.6%) were oropharyngeal in origin. tPCR identified HPV DNA in 20/54 (37%) brCC and 24/35 (68.6%) mSCC; 15/24 (62.5%) were oropharyngeal in origin. nPCR identified HPV DNA in 1/54 (1.8%) brCC and 14/27 (51.8%) mSCC; 10/14 (71.4%) were oropharyngeal in origin. No HPV PCR+ brCC were p16+ (≥20% cells staining). P16+ was present in 17/24 (70.8%) tPCR HPV+ mSCC and 13/14 (92.8%) nPCR HPV+ mSCC. 10/13 (76.9%) p16+/nPCR HPV+ mSCC and 12/17 (70.6%) p16+/tPCR HPV+ mSCC were of oropharyngeal origin. Conclusions (1) tPCR in isolation is not useful in separating brCC and mSCC; (2) dual p16/nPCR HPV DNA+ has the highest predictive value for mSCC from the oropharynx; (3) more experience is required with tPCR in this clinical setting given the high rate of HPV DNA detection in brCC.

Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay

The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7 %) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6 % (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100 % (172/172) of samples. MT-PCR identification for 94.7 % (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result.

Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis , Entamoeba histolytica , and Giardia intestinalis in Clinical Stool Samples

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis

BackgroundThe high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR.ResultsHere, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker.ConclusionsA pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness

BackgroundInfluenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak.MethodsWe developed a multiplexed PCR (MT-PCR) assay for the simultaneous diagnosis of respiratory viruses causing influenza-like illness, including the specific recognition of influenza A haemagglutinin subtypes H1, H3, and H5. We tested several hundred clinical specimens in two diagnostic reference laboratories and compared the results with standard techniques.ResultsThe sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens. The MT-PCR assays provided differential diagnoses as well as potentially useful quantitation of virus in clinical samples.ConclusionsMT-PCR is a potentially powerful tool for the differential diagnosis of influenza-like illness in the clinical diagnostic laboratory.

Comparison of Whole Blood, Serum, and Plasma for Early Detection of Candidemia by Multiplex-Tandem PCR

We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with Candida bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days). Candida DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) whole-blood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at -20 degrees C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide Candida species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction - a model for cross-modulation.

BackgroundA feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.MethodsPMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.ResultsWhen PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.ConclusionST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Colony Multiplex-Tandem PCR for Rapid, Accurate Identification of Fungal Cultures

We developed a multiplex tandem PCR (MT-PCR) assay for the rapid identification of 16 fungi directly from culture. MT-PCR results were concordant with phenotypic identification for all cultures studied (n = 183). The colony MT-PCR assay was rapid (<2 h), sensitive, and specific in identifying fungal pathogens directly from primary isolation plates.