Comparison between a novel molecular tool and conventional methods for diagnostic classification of bacterial vaginosis: is integration of the two approaches necessary for a better evaluation?

The etiological cause of bacterial vaginosis (BV) is the change of the vaginal ecosystem characterized by a decrease of lactobacilli and an increase of other germs, such as Gardnerella vaginalis and Atopobium vaginae. Molecular tools have revolutionized the diagnosis of these conditions. The aim of this paper was to compare results obtained from 158 vaginal swabs collected from women aged between 18 and 59 years old and subjected to microscopic evaluation (Nugent Score), culture and to the multiparametric molecular assay Vaginitis and Vaginosis Multiplex-Tandem (MT) PCR (AU27117) - Nuclear Laser Medicine. In 50 samples we also used matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for bacterial microbiome identification. Our results showed a moderate concordance between traditional and molecular methods for diagnosis of candidiasis and a lower concordance for BV and normal flora. MALDI TOF MS allowed us to discriminate more than 10 species of lactobacilli with a greater abundance of Lactobacillus gasseri, Lactobacillus paracasei spp. paracasei, Lactobacillus pentosus and Lactobacillus crispatus in BV and altered flora. This work underlined how the integration of different assays and metagenomics studies can greatly expand our current understanding of vaginal microbial diversity, providing more reliable diagnostic criteria for BV and its intermediate condition diagnosis.

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or Ct-value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious.

An outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia.

This study reports an outbreak of oriental theileriosis in dairy cattle imported to Vietnam from Australia. Following clinical and pathological diagnoses, a total of 112 cattle blood samples were divided into three groups and tested using multiplexed tandem PCR. Group 1 were from aborted heifers in Vietnam; group 2 were from cattle before shipment from group 1 cattle and group 3 were from the same batch of cattle but transported to Taiwan. Theileria orientalis DNA was detected in 72·3% cattle. The prevalences of T. orientalis in groups 1, 2 and 3 were 77·6, 86·9 and 57·5%, respectively, and the difference in prevalence was significant between groups 1 and 3 (P < 0·0001). The infection intensities of genotypes chitose and ikeda of T. orientalis were higher in groups 1 (57 721 and 33 709, respectively) and 3 (5897 and 61 766, respectively) than those in group 2 (2071 and 6331, respectively). Phylogenetic analyses of the major piroplasm surface protein sequences revealed that genotypes chitose and ikeda determined herein were closely related to those previously reported from Australia. This first report of an outbreak of oriental theileriosis in imported cattle emphasizes improved measures for the export and import of cattle infected with T. orientalis.

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.

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.

Use of amplicon melt temperature to detect SARS-CoV-2 Lineage B.1.1.7 variant through the G28048T mutation in the ORF 8 gene.

A new variant of SARS-CoV-2 (Lineage B.1.1.7) was identified in the UK in December 2020 which was associated with higher transmissibility of COVID-19. The AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay is used at sixteen UK hospitals and detects part of the ORF8 gene (together with a segment from the ORF1a gene). The objective of this study was to determine if the recently identified mutation in ORF8 (G28048T) in the B.1.1.7. lineage could be used to identify the new variant quickly in clinical cases with PCR positive results. The melt data from SARS-CoV-2 positives from two hospitals (October through December 2020) were reviewed, and distribution over time and location was evaluated. A low melt variant of the ORF8 amplicon started to appear in samples from Guy's and St. Thomas' NHS Trust, London, at the start of November, and grew as a proportion of the total positives during the subsequent two months. These low melt variants were very rare during the same period at the Northern Care Alliance, Greater Manchester, North West of UK. It was confirmed that these carried the G28048T mutation. The geographic and temporal distribution of the low melt amplicons makes it very likely that these are lineage B.1.1.7 strains. The melt temperature of this amplicon could be used to discriminate between the original and new variants in advance of the full sequencing of the isolate. However, the appearance of other mutations in the same amplicon means that this approach would be of diminishing value over time.

Clinical evaluation of AusDiagnostics SARS-CoV-2 multiplex tandem PCR assay.

BACKGROUND: In the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays. AIMS: To evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2. METHODS: We reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory. RESULTS: Initial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results. CONCLUSION: The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.

Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle.

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance.

Comparison of multiplexed-tandem real-time PCR panel with reference real-time PCR molecular diagnostic assays for detection of Giardia intestinalis and Tritrichomonas foetus in cats.

Giardia intestinalis and Tritrichomonas foetus are frequent enteric protozoan parasites of the gastrointestinal track of domestic cats. Because of different treatment options for the parasites, confirmation of presence of one or both pathogens is necessary. The PCR based assays are suitable for differential diagnosis. We evaluated the performance of Small Animal Diarrhoea panel, a multiplexed-tandem real-time PCR (MT-PCR) assay, that detects DNA of both G. intestinalis and T. foetus. The sensitivity and specificity were compared to reference real-time PCR assays using 105 faecal samples, 39.05% (n = 41) positive for G. intestinalis and 30.48% (n = 32) were positive for T. foetus. The faecal samples positive for T. foetus had a high proportion of late amplifiers, determined by an arbitrary threshold of Ct-values > 35. On the other hand, only one G. intestinalis positive sample was considered a late amplifier. For G. intestinalis DNA, the MT-PCR assay had 95.1% sensitivity and 92.1% specificity. For T. foetus DNA, the MT-PCR assay had 41.9% sensitivity and 100.0% specificity. To evaluate the interlaboratory reproducibility of the MT-PCR assay, results were compared in two different laboratories and found to be in a very good agreement (Kappa = 0.9). Further analysis of the DNA using conventional PCR determined presence of G. intestinalis Assemblage F and T. foetus genotype 'feline'. In conclusion, the MT-PCR Small Animal Diarrhoea panel had a good and poor performance against reference assays for G. intestinalis and T. foetus, respectively. The assay is suitable for detection and differential diagnosis of G. intestinalis and moderate to high burdens of T. foetus in small animal clinical practice.

Molecular characterisation of Theileria orientalis in imported and native bovines from Pakistan.

The epidemiological aspects of Theileria orientalis in Pakistan are unknown; therefore, investigations using sensitive and precise molecular techniques are required. This study reports the first molecular characterisation of T. orientalis detected from imported (Bos taurus) and native cattle (Bos indicus×Bos taurus) and buffaloes (Bubalus bubalis) selected from four districts of Punjab, Pakistan. DNA samples from blood (n=246) were extracted and tested using conventional PCR utilising the major piroplasm surface protein (MPSP) gene and multiplexed tandem PCR (MT-PCR). Theileria orientalis DNA was detected (15%; 22/147) only in imported cattle by conventional PCR, whereas 24.5% (36/147), 6% (3/50) and 6.1% (3/49) of the imported cattle and native Pakistani cattle and buffaloes, respectively were test-positive for T. orientalis using MT-PCR. Using MT-PCR, the prevalence of T. orientalis was significantly higher (P<0.0001) in imported cattle compared to that of detected in native Pakistani bovines. The prevalence of T. orientalis and DNA copies of chitose and ikeda were significantly higher (P<0.05) in imported cattle than those detected in native Pakistani bovines. DNA sequencing of amplicons of the conventional PCR revealed the presence of buffeli, chitose and ikeda genotypes of T. orientalis. Phylogenetic analysis revealed that the MPSP sequences of buffeli, chitose and ikeda from imported cattle were closely related to those sequences reported previously from Australia and other regions. This study provides the first survey of T. orientalis infection in imported and native bovines in Pakistan, and highlights the need for future studies to understand the spread of transboundary animal diseases.

Multiplexed-tandem PCR for the specific diagnosis of gastrointestinal nematode infections in sheep: an European validation study.

BACKGROUND: Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories. RESULTS: MT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific. CONCLUSIONS: The MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.

Molecular characterization of Theileria orientalis from cattle in Ethiopia.

This study reports the first molecular characterization of Theileria orientalis in local breeds of cattle in Ethiopia. A conventional PCR utilizing major piroplasm surface protein (MPSP) gene and an established multiplexed tandem PCR (MT-PCR) were used to characterize T. orientalis and to assess the infection intensity, respectively. Of 232 blood samples tested, T. orientalis DNA was detected in only 2.2% of samples using conventional PCR; two genotypes buffeli (1.3%; 3/232) and type 5 (0.9%; 2/232) of T. orientalis were detected. Phylogenetic analysis revealed that the buffeli MPSP sequences from Ethiopia were closely related to those reported from Kenya, Sri Lanka and Myanmar, and type 5 sequences from Ethiopia grouped with those from Korea, Japan, Vietnam and Thailand. A higher number of samples (3.9%; 9/232) were test-positive by MT-PCR and four genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis were detected. The average intensity of infections with genotypes buffeli (DNA copy numbers 11,056) and type 5 (7508) were significantly higher (P<0.0001) than the pathogenic genotype ikeda (61 DNA copies). This first insight into T. orientalis from cattle in Ethiopia using MPSP gene provides a basis for future studies of T. orientalis in various agroclimatic zones and of the impact of oriental theilerosis on cattle in this and other countries of Africa.

Investigating the first outbreak of oriental theileriosis in cattle in South Australia using multiplexed tandem PCR (MT-PCR).

This study investigated the first outbreak of oriental theileriosis in a herd of beef cattle in South Australia using a newly established multiplexed tandem PCR (MT-PCR) to identify, differentiate and quantitate the four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis recognised to occur in Australasia. Following clinical diagnosis of oriental theileriosis (based on clinical signs, laboratory findings and post mortem examination), 155 blood samples were collected from individual cows (n = 85) and calves (n = 70), and tested by MT-PCR. In total, 117 (75.48%) cattle were shown to be test-positive for T. orientalis. All four genotypes were detected, and ikeda had the highest prevalence (90.6%; 106/117), followed by buffeli (83.8%; 98/117), chitose (18.8%; 22/117) and type 5 (5.1%; 6/117). Mixed infections with genotypes buffeli and ikeda had a higher prevalence (55.5%; 65/117) than any other combination of genotypes. The prevalences of buffeli and ikeda were significantly higher (P<0.005) than those of chitose and type 5. The average intensity of infection with genotype ikeda (329,775 DNA copies) was significantly higher (P<0.0001) than buffeli (212,843) and chitose (125,462). This study reinforces the utility of MT-PCR as a diagnostic tool for rapidly investigating oriental theileriosis outbreaks in cattle herds and as a pre-movement screening test for preventing the introduction of this disease into non-endemic regions.

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.