Evaluation of multiple sewage-associated bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for sewage pollution in environmental waters in Australia. The BacHum (i.e., 63% specificity) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims: Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies. Method: A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples. Conclusions: The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not. Significance and Impact of the Study: The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.

Closed-form design equations for decoupling networks of small arrays

Small element spacing in compact arrays results in strong mutual coupling between the array elements. A decoupling network consisting of reactive cross-coupling elements can alleviate problems associated with the coupling. Closed-form design equations for the decoupling networks of symmetrical arrays with two or three elements are presented.

Port decoupling for small arrays by means of an eigenmode feed network

An alternative approach to port decoupling and matching of arrays with tightly coupled elements is proposed. The method is based on the inherent decoupling effect obtained by feeding the orthogonal eigenmodes of the array. For this purpose, a modal feed network is connected to the array. The decoupled external ports of the feed network may then be matched independently by using conventional matching circuits. Such a system may be used in digital beam forming applications with good signal-to-noise performance. The theory is applicable to arrays with an arbitrary number of elements, but implementation is only practical for smaller arrays. The principle is illustrated by means of two examples.

Clustering of ad-hoc microphone arrays for robust blind beamforming

This paper proposes a clustered approach for blind beamfoming from ad-hoc microphone arrays. In such arrangements, microphone placement is arbitrary and the speaker may be close to one, all or a subset of microphones at a given time. Practical issues with such a configuration mean that some microphones might be better discarded due to poor input signal to noise ratio (SNR) or undesirable spatial aliasing effects from large inter-element spacings when beamforming. Large inter-microphone spacings may also lead to inaccuracies in delay estimation during blind beamforming. In such situations, using a cluster of microphones (ie, a sub-array), closely located both to each other and to the desired speech source, may provide more robust enhancement than the full array. This paper proposes a method for blind clustering of microphones based on the magnitude square coherence function, and evaluates the method on a database recorded using various ad-hoc microphone arrangements.

Single-layer decoupling networks for large circulant symmetric arrays

In this paper, the authors propose a new structure for the decoupling of circulant symmetric arrays of more than four elements. In this case, network element values are again obtained through a process of repeated eigenmode decoupling, here by solving sets of nonlinear equations. However, the resulting circuit is much simpler and can be implemented on a single layer. The corresponding circuit topology for the 6-element array is displayed in figure diagrams. The procedure will be illustrated by considering different examples.

Clustered blind beamforming from ad-hoc microphone arrays

Microphone arrays have been used in various applications to capture conversations, such as in meetings and teleconferences. In many cases, the microphone and likely source locations are known \emph{a priori}, and calculating beamforming filters is therefore straightforward. In ad-hoc situations, however, when the microphones have not been systematically positioned, this information is not available and beamforming must be achieved blindly. In achieving this, a commonly neglected issue is whether it is optimal to use all of the available microphones, or only an advantageous subset of these. This paper commences by reviewing different approaches to blind beamforming, characterising them by the way they estimate the signal propagation vector and the spatial coherence of noise in the absence of prior knowledge of microphone and speaker locations. Following this, a novel clustered approach to blind beamforming is motivated and developed. Without using any prior geometrical information, microphones are first grouped into localised clusters, which are then ranked according to their relative distance from a speaker. Beamforming is then performed using either the closest microphone cluster, or a weighted combination of clusters. The clustered algorithms are compared to the full set of microphones in experiments on a database recorded on different ad-hoc array geometries. These experiments evaluate the methods in terms of signal enhancement as well as performance on a large vocabulary speech recognition task.

Speech recognition using ad-hoc microphone arrays

While close talking microphones give the best signal quality and produce the highest accuracy from current Automatic Speech Recognition (ASR) systems, the speech signal enhanced by microphone array has been shown to be an effective alternative in a noisy environment. The use of microphone arrays in contrast to close talking microphones alleviates the feeling of discomfort and distraction to the user. For this reason, microphone arrays are popular and have been used in a wide range of applications such as teleconferencing, hearing aids, speaker tracking, and as the front-end to speech recognition systems. With advances in sensor and sensor network technology, there is considerable potential for applications that employ ad-hoc networks of microphone-equipped devices collaboratively as a virtual microphone array. By allowing such devices to be distributed throughout the users’ environment, the microphone positions are no longer constrained to traditional fixed geometrical arrangements. This flexibility in the means of data acquisition allows different audio scenes to be captured to give a complete picture of the working environment. In such ad-hoc deployment of microphone sensors, however, the lack of information about the location of devices and active speakers poses technical challenges for array signal processing algorithms which must be addressed to allow deployment in real-world applications. While not an ad-hoc sensor network, conditions approaching this have in effect been imposed in recent National Institute of Standards and Technology (NIST) ASR evaluations on distant microphone recordings of meetings. The NIST evaluation data comes from multiple sites, each with different and often loosely specified distant microphone configurations. This research investigates how microphone array methods can be applied for ad-hoc microphone arrays. A particular focus is on devising methods that are robust to unknown microphone placements in order to improve the overall speech quality and recognition performance provided by the beamforming algorithms. In ad-hoc situations, microphone positions and likely source locations are not known and beamforming must be achieved blindly. There are two general approaches that can be employed to blindly estimate the steering vector for beamforming. The first is direct estimation without regard to the microphone and source locations. An alternative approach is instead to first determine the unknown microphone positions through array calibration methods and then to use the traditional geometrical formulation for the steering vector. Following these two major approaches investigated in this thesis, a novel clustered approach which includes clustering the microphones and selecting the clusters based on their proximity to the speaker is proposed. Novel experiments are conducted to demonstrate that the proposed method to automatically select clusters of microphones (ie, a subarray), closely located both to each other and to the desired speech source, may in fact provide a more robust speech enhancement and recognition than the full array could.

Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh

This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh by using Quantitative PCR (qPCR) of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely, enterococci. The number of enterococci in lake water samples ranged from 1.1 x 104 to 1.9 x 105 CFU/100 ml of water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for the HF183 and CF128 markers, respectively. The numbers of the HF183 and CF128 markers in lake water samples were 3.9 x 104 to 6.3 × 107 and 9.3 x 103 to 6.3 x 105 genomic units (GU)/100 ml of water, respectively. The high numbers of enterococci and the HF183 markers indicate sewage pollution and potential health risks to those who use the lake water for non-potable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking (MST) markers in Dhaka, Bangladesh where diarrhoeal diseases is one of the major causes of childhood mortality. The molecular assay as used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimise potential health risks.

Highly discriminatory single-nucleotide polymorphism interrogation of Escherichia coli by use of allele-specific real-time PCR and eBURST analysis

In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.

Design of decoupling networks for circulant symmetric antenna arrays

Small element spacing in compact arrays results in strong mutual coupling between array elements. Performance degradation associated with the strong coupling can be avoided through the introduction of a decoupling network consisting of interconnected reactive elements. We present a systematic design procedure for decoupling networks of symmetrical arrays with more than three elements and characterized by circulant scattering parameter matrices. The elements of the decoupling network are obtained through repeated decoupling of the characteristic eigenmodes of the array, which allows the calculation of element values using closed-form expressions.