Sensitive Molecular Assays Research Articles

Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks

Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

Abstract 503: Molecular characterization of in vivo isolated EpCAM-positive circulating tumor cells in breast cancer

Abstract Introduction: In the early stages of cancer, the chance to detect rare CTCs is increasing by increasing the sample volume. The aim of our study was to evaluate the diagnostic sensitivity of a novel clinical device for the in-vivo isolation of EpCAM-positive CTCs (CellCollectorTM, GILUPI, GmBH), by using highly sensitive RT-qPCR molecular assays. Patients and methods: 29 breast cancer patients without overt metastases before the beginning of adjuvant chemotherapy (M0), 26 breast cancer patients with overt metastases before starting of therapy (M1) and 12/26 of them before the second cycle of therapy (M2), as well as 18 healthy donors participated in the study. After in-vivo isolation, total RNA was extracted from captured cells, lysed in Trizol, followed by cDNA synthesis. RT-qPCR was used for the molecular characterization of captured cells, for: CK-19, HER-2, TWIST1, VEGF, ER, PR, EGFR, CD44, CD24, and ALDH1, while B2M was used as a reference gene. Peripheral blood was also collected for CTC analysis by the FDA cleared CellSearchTM system. In addition, immunofluorescence staining of cytospins was performed and screened for CTCs using the ARIOL system, using ER, HER2, CK (8, 18, 19) and CD45 for CTC identification. Results: Results are shown in Table 1. At least one gene was expressed in 10(34.5%) of M0, 15(57.7%) of M1 and 4(33.3%) of M2 patient groups, but in none of healthy donors 0/18(0%). CellSearchTM gave positive results in 5(17.2%) of M0, 10(38.5%) of M1 and 0(0%) of M2. Immunofluorescence (Ariol system) was positive for ER, HER2, CK (8, 18, 19) in 5/15(33.3%) M0, in 4/12(33.3%) M1 and in 1/7(14.3%) M2 groups. Table 1.Gene expression in CTCHealthy N = 18M0 N = 29M1 N = 26M2 N = 12CK-190 (0%)6(20.7%)6 (23.1%)2 (16.7%)HER20 (0%)2 (6.9%)0 (0%)0 (0%)ER0 (0%)2 (6.9%)0 (0%)0 (0%)PR0 (0%)0 (0%)0 (0%)0 (0%)EGFR0 (0%)0 (0%)0 (0%)0 (0%)TWIST10 (0%)1 (3.4%)0 (0%)2 (16.7%)VEGF0 (0%)3 (10.3%)5 (19.2%)1 (8.3%)CD44+/CD24−,0 (0%)4 (13.8%)3 (11.5%)1 (8.3%)ALDH1high/CD24−,0 (0%)2 (6.9%)8 (30.8%)1(8.3%) Conclusions: In-vivo isolation of CTC is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems promising. Comparison studies with the CellSearch and immunofluorescence have shown poor agreement. These results should be validated in large patient cohorts, and in respect to the clinical outcome. Citation Format: Areti D. Strati, Martha Zavridou, Galateia Kallergi, Eleni Politaki, Tobias Gorges, Andra Kuske, Anna-Lena Bohnen, George Koutsodontis, Amanda Psyrri, Klaus Lucke, Vasilis Georgoulias, Klaus Pantel, Evi Lianidou. Molecular characterization of in vivo isolated EpCAM-positive circulating tumor cells in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 503.

Abstract 1380: Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring

Abstract We have developed a new molecular assay that utilizes digital PCR to detect and quantify cancer mutations within poor quality and limited quantity samples such as archival tissue DNA and circulating tumor DNA (ctDNA). This digital PCR assay is highly sensitive and is capable of detecting the targeted mutant fraction at an individual DNA molecule resolution. Thus, this assay is ideal for applications where DNA is in low abundance. An example is where DNA is shed from tumors that can be extracted from the plasma fraction of routine blood draws. The resulting circulating DNA (ctDNA) is extremely low in concentration, which makes it a prime candidate for our highly sensitive molecular assay. Our assay incorporates small amplicon PCR primers and can be configured for nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. Mutation quantitation relies on two DNA primer sets that are identical with the exception of the mutant or wild type specific base at the 3’ end of the “detecting” primer sets, to amplify the genomic region of interest. Through the addition of artificial 5’ non-complementary tails to our mutant and wild type specific “detection” primers, we are able to consistently differentiate between droplets that contain the mutant or wild type alleles based upon their differential amplicon lengths. The synthetic amplicon extension tails minimize bias within the PCR reaction by allowing our primers to target an identical region of genomic DNA, with the exception of the single nucleotide variant specific base at the 3’ end of the detecting primer. The dPCR technology allows the standard PCR reaction to be partitioned into 20,000 independent wells; we can then assay each individual droplet to assess whether the individual DNA molecule partitioned into the droplet is “wild-type” or the target “mutant”. The resulting data provides an absolute count of mutant and wild-type templates in a given patient sample. We have optimized this new molecular assay technology for quantitatively measuring clinically actionable mutations including BRAF V600E, KRAS G12D among others. We have successfully validated the sensitivity, specificity and reproducibility of our assays through controlled cell line DNA mixed dilution samples ranging from 66% mutant to 0.1% mutant in each of our optimized primer sets. Our assay improves the limit of detection to seven DNA molecules containing a mutation among a total number of 7,000 genome equivalents. Because our assay is sensitive down to the single DNA molecule resolution, we have also been able to reduce the amount of clinical sample DNA required to determine the presence of clinically actionable mutations such as those occurring in KRAS or BRAF. We are testing this extremely low cost, highly sensitive diagnostic technology for detecting nearly any cancer mutation from longitudinal samples of ctDNA. Citation Format: Christina M. Wood, Billy Lau, Laura Miotke, Hanlee P. Ji, Stephanie Greer. Detecting cancer mutations at the resolution of individual DNA molecules for longitudinal monitoring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1380.

The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States.

Development of real-time PCR assays for evaluation of immune response and parasite load in golden hamster (Mesocricetus auratus) infected by Leishmania (Viannia) braziliensis.

BackgroundCutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions.MethodsHamsters were intradermally infected in the footpad with 105 promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-ɣ, IL-10, TGF-β TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology.ResultsA relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-ɣ, TGF-β, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-β to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-β to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-ɣ, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7–1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant.ConclusionsIn this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1647-6) contains supplementary material, which is available to authorized users.

Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

Ultra-Specific Isolation of Circulating Tumor Cells Enables Rare-Cell RNA Profiling.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Development of a sensitive molecular detection assay for mango malformation disease caused by Fusarium mangiferae.

To develop a sensitive and specific molecular assay for detection of mango malformation disease (MMD), which is caused primarily by Fusarium mangiferae. We screened 100 ISSR primers and identified one (UBC888) that directed the stable amplification of a specific gene fragment of 479bp (GenBank accession number KJ526382). Based on the DNA sequence of this fragment, a pair of SCAR primers (W342 and W1772) were designed to amplify another gene fragment of 1376bp (GenBank accession number KJ526383), demonstrating the successful conversion of an ISSR marker to a SCAR marker. An effective and simple detection assay for MMD was established based on this pair of PCR primers, with a high level of specificity and sensitivity to the DNA of F. mangiferae and other species of Fusarium both in vitro and in vivo. It can detect as little as 10pg fungal DNA from the DNA of mango's tissues. Our assay provides a practical method for the early diagnosis so that proper prevention of the mango malformation disease can be developed.

Updating the management of sexually transmitted infections.

The control of sexually transmitted infections relies on case-finding and treatment of sexual contacts to prevent further transmission. Screening for infections should be tailored to the demographic and sexual risk of the individual. For most sexually transmitted infections, screening is performed on self-collected, non-invasive samples using highly sensitive molecular assays. These are quick and inexpensive. Shorter courses of antivirals for genital herpes are now recommended. New chemoprophylactic strategies for preventing HIV transmission have emerged, including treatment to prevent transmission and the use of antiretrovirals for pre-exposure prophylaxis.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

SummaryAccurate, timely diagnosis is essential for the control, monitoring and eradication of foot‐and‐mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple‐to‐use technologies, including molecular‐based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)‐specific reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) and real‐time RT‐PCR (rRT‐PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory‐based rRT‐PCR. However, the lack of robust ‘ready‐to‐use kits’ that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT‐PCR and RT‐LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real‐time, and for the RT‐LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.

Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.

Rapid, specific and sensitive molecular detection assay for Alternaria helianthi that causes leaf blight disease in sunflower

The leaf blight disease of sunflower (Helianthus annuus) caused by Alternaria helianthi is a serious threat to its cultivation worldwide. Early and accurate detection of the pathogen is critical to efficient disease management and in avoiding further losses due to epidemics in sunflower. Conventional methods of detection and identification are time consuming, labour intensive, and lack specificity and sensitivity. A real-time PCR based TaqMan® probe assay was developed to target 156 bp Internal Transcribed Spacer (ITS) region of A. helianthi for its detection using fungal genomic DNA and infected plant tissues and seeds of sunflower. The specificity of the probe and primers was confirmed by testing their cross reactivity using genomic DNA of closely related Alternaria species isolated from 17 crop plants and 15 fungal species of other genera. No cross-reactivity could be detected with any of the other non-target fungal strains used in this study. The assay successfully detected as low as 1.0 pg fungal genomic DNA and up to 1 % infection in sunflower seed lots. To the best of our knowledge, this is the only probe based real-time PCR assay that enable high specificity and sensitivity for rapid detection of A. helianthi in infected seeds and plant tissues. The assay may also hold promise for application in effective bio-threat and risk mitigation program by early and accurate detection of the pathogen for effective management.

BOX-PCR- and ERIC-PCR-based genotyping and phylogenetic correlation among Fusarium oxysporum isolates associated with wilt disease in Psidium guajava L.

Abstract Fusarium oxysporum f. sp. psidii is a soilborne fungal pathogen that causes wilt disease in guava. There is an urgent need to establish a sensitive and specific molecular assay to diagnose the disease. A genetic fingerprint of the guava wilt pathogen also needs to be established. We isolated and identified 21 isolates of F. oxysporum f. sp. psidii from soil and infected roots of wilted guava, from different agro-climatic regions of India. We used BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR for generating a genetic fingerprint of F. oxysporum f. sp. psidii that ranged from 150 to 2500 bp in size. A dendrogram was constructed for the isolates on the basis of both the fingerprint patterns and the highest and lowest similarity values. The latter were observed for Fop123–Fop127 (at 94.0%) and Fop18–Fop24 (at 18.0%), respectively. Molecular data were derived from an analysis of the internal transcribed spacer sequences of 38 isolates belonging to 19 taxa within the species F. oxysporum. In this study, F. oxysporum f. sp. psidii was grouped in a single cluster that represented a different genetic structure. This study suggests that BOX-PCR and ERIC-PCR could be suitable, rapid, reproducible, and useful techniques for the discrimination of F. oxysporum f. sp. psidii, to better manage guava wilt disease.

The state of the art of diagnostic vitrectomy

AbstractThe emergence of increasingly sensitive molecular and biological assays has facilitated our diagnostic ability in uveitis. Together with the advances in surgical vitrectomy procedures, the risk to biopsy is much reduced. Combined therefore we are currently at an unprecedented state of ability to rapidly intervene and increase our diagnostic yield. The talk will highlight indications, simple techniques and evidence of how improved diagnostics have altered outcomes and discuss how diagnostic sampling should be employed in management of uveitis.

Accurate detection of BRAF p.V600E mutations in challenging melanoma specimens requires stringent immunohistochemistry scoring criteria or sensitive molecular assays.

Malignant melanoma patients require BRAF mutation testing prior to initiating BRAF inhibitor therapy. Molecular testing remains the diagnostic gold standard, but recent work suggests that BRAF immunohistochemistry (IHC) confers comparable results. Sample attributes and scoring criteria that may affect BRAF IHC interpretation, however, are poorly defined. We investigated formalin-fixed, paraffin-embedded samples with variable challenging interpretative attributes: metastases, core needle biopsies, sample tissues less than 60 mm(2), samples with greater than 50% necrosis, and/or samples with greater than 10% melanin pigmentation. Three pathologists independently scored 122 BRAF V600E IHC-labeled melanoma samples for percentage (0%-100%) of staining intensity (0-3+). Interscorer BRAF IHC discrepancies were resolved by consensus review. Lenient (≥1+, >0%) and stringent (≥2+, ≥10%) IHC scoring criteria were compared to BRAF V600 mutation (cobas) results (n = 118). Specimens with greater than 10% melanin pigmentation and metastatic samples produced the majority of interobserver IHC and IHC/cobas scoring discrepancies. Consensus review using stringent scoring criteria decreased the number of discrepant results, yielded very good interobserver reproducibility, and improved specificity and positive predictive value for BRAF p.V600E detection. BRAF p.V600K mutations accounted for 57.1% of false-negative IHC results when stringent, consensus criteria scoring were used. The cobas test detected 75.0% (8/12) of BRAF IHC-negative BRAF p.V600K mutations confirmed by next-generation sequencing. Molecular BRAF testing is the preferred screening test for BRAF inhibitor therapy eligibility because of superior sensitivity in challenging interpretative melanoma specimens. However, BRAF V600E IHC has excellent specificity and positive predictive value when stringent, consensus scoring criteria are implemented. To decrease IHC scoring discrepancies, pathologists should interpret metastatic and pigmented samples with caution.

Population- and Type-Specific Clustering of Multiple HPV Types Across Diverse Risk Populations in the Netherlands

In view of possible type replacement upon introduction of human papillomavirus (HPV) vaccination, we aimed to explore patterns of type-specific clustering across populations with various background infection risks. A total of 3,874 women from 3 cross-sectional studies in the Netherlands (in 2007-2009) provided vaginal self-samples, which were tested for 25 HPV genotypes by a sensitive molecular assay (SPF10 line probe assay, DDL Diagnostic Laboratory, Voorburg, the Netherlands). The number of concurrent HPV infections per woman was studied by Poisson regression. Associations between HPV types were investigated by generalized estimating equation analyses. The prevalence of any HPV type was 14% in a population-based study, 54% in a chlamydia screening intervention study, and 73% in a study among attendees of sexually transmitted infection clinics. Overall, multiple HPV infections were detected in 26% of the women. The number of concurrent HPV infections conformed to an overdispersed Poisson distribution, even after correction for known risk factors. Types differed significantly in their tendencies to be involved in coinfections, but no evidence for particular type-type interactions was found. Moreover, the strongest associations were observed in the lowest-risk population and vice versa.We found no indications of pairwise interactions, but our findings do suggest that clustering differs among HPV types and varies across risk groups.

An improved real-time PCR assay for the detection of Old World screwworm flies

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia's OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results.

Verrucous carcinoma of the head and neck – not a human papillomavirus‐related tumour?

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α-PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin-fixed, paraffin-embedded samples of 30 VC and 30 location-matched normal tissue samples were analysed, by using six different polymerase chain reaction-based methods targeting DNA of at least 87 HPV types from α-PV, β-PV, γ-PV and μ-PV genera, and immunohistochemistry against p16 protein. α-PV, γ-PV and μ-PV were not detected. β-PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV-19, -24 and -36 were identified in VC, and HPV-5, -9, -12, -23, -24, -38, -47, -49 and -96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α-PV, γ-PV and μ-PV are not associated with aetiopathogenesis of VC of the head and neck. β-PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation.

Molecular Assays for Determining Mycobacterium leprae Viability in Tissues of Experimentally Infected Mice

BackgroundThe inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues.Methodology/Principle FindingsTwo M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations.Conclusions hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Rapid Advances in Understanding Viral Gastroenteritis from Domestic Surveillance

“Winter vomiting disease” was the clinical moniker for viral acute gastroenteritis (AGE), including illnesses caused by norovirus and rotavirus, nearly 100 years ago (1). This nonspecific diagnosis represented a frequently observed illness, with the symptoms of vomiting and diarrhea that occurred particularly in the colder months. One hundred years later, diagnosing specific AGE pathogens in clinical settings continues to be an elusive task. Clinical treatment options are nonspecific as well—primarily rehydration and supportive therapies—and the identification of the viral pathogens is considered relatively time-consuming and costly. In truth, viral AGE has likely caused misery, illness, and death among human populations for thousands of years, since people first facilitated disease transmission by congregating in groups. But, it has been only during the lifetime of many current readers (and during the long career spans of several), beginning in the early 1970s, that norovirus, rotavirus, and an expanding collection of other viral AGE pathogens have been discovered. The advent of sensitive laboratory tools to detect and study the genetic evolution of these viruses has uncovered their critical role in the etiology of AGE. The flow of information is now so great that in each year since 2008, >800 scientific papers have been published on this topic as determined by a search of PubMed using the term acute gastroenteritis. The field of viral gastroenteritis is in the midst of an extraordinary period of rapid development and transition. Vaccines to prevent rotavirus, the leading cause of severe childhood AGE worldwide, are being rolled out globally and have already achieved remarkable success in reducing the burden of this pathogen in many countries, including the United States. In addition, the application of sensitive molecular assays is reaffirming the central etiologic role of noroviruses in both endemic and epidemic AGE, and vaccines against this pathogen are undergoing clinical testing. This issue of Emerging Infectious Diseases highlights recent developments in the field with a collection of timely findings from domestic viral gastroenteritis surveillance, which will further our understanding of disease effects, viral evolution and structure, implications of vaccination, and progress with other preventive measures. Figure Daniel Payne.