Standard Real-time Polymerase Chain Reaction Research Articles

Detecting SARS-CoV-2 Virus by Reverse Transcription-Loop-mediated Isothermal Amplification.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.

Performance Evaluation of Different RT-PCR Kits for the Direct Detection of SARS-CoV-2 in Preheated Specimens.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created high demand for molecular kits and consumables for mass screening of suspected individuals. Direct real-time polymerase chain reaction (RT-PCR) assay without nucleic acid extraction has several advantages in saving testing time and cost and helps in the rapid reporting of SARS-CoV-2. The present study evaluated the analytical performance of four SARS-CoV-2 RT-PCR for direct RT-PCR testing using preheated specimens. Methods A total of 100 clinical specimens were selected and divided into three different groups: (1) group I: 20 SARS-CoV-2 positive specimens with high viral load, viz., low Ct values (< 30 Ct), (2) group II: 50 SARS-CoV-2 positive specimens with low viral load, viz., high Ct values (> 30 Ct), and (3) group III: 30 SARS-CoV-2 negative specimens. Specimens were heat-inactivated at 70°C for 10 minutes and cooled down at 4°C and were evaluated for standard and direct RT-PCR method by using ViralDtect-II Multiplex Real-Time PCR kit, TaqPath COVID-19 Combo kit, COVIDsure Pro Multiplex RT-PCR kit, and Hi-PCR Coronavirus (COVID-19) Multiplex Probe PCR kit. Results Results showed that except ViralDtect-II kit, the other three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit were able to amplify all the SARS-CoV-2 genes in the direct RT-PCR method using preheated specimens. In group I specimens, 100% sensitivity was observed in all three RT-PCR kits. In group II specimens, COVIDsure Pro kit was found to be superior among other kits. Conclusion Direct RT-PCR method during pandemic situation is valuable and cost effective for the detection of SARS-CoV-2. All three TaqPath COVID-19 Combo kit, COVIDsure Pro kit, and Hi-PCR Coronavirus (COVID-19) RT-PCR kit can be used for direct RT-PCR method and COVIDsure Pro kit performance was found to be superior among all.

Perspectives on Rapid Antigen Tests for Downstream Validation and Development of Theranostics.

Point-of-care SARS-CoV-2 rapid antigen tests have proven to be useful over the years and have become more apparent to the public eye during COVID-19 pandemic due to their ease of use, rapid processing and result times, and low cost. Here, we have assessed the effectiveness and accuracy of rapid antigen tests in comparison to the standard real-time polymerase chain reaction analyses of the same samples.

Ligation-based assay for variant typing without sequencing: Application to SARS-CoV-2 variants of concern.

COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance. The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4× 104 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

No VTE Recurrence After 1-Year Follow-Up of Hospitalized Patients With COVID-19 and a VTE Event: A Prospective Study

No VTE Recurrence After 1-Year Follow-Up of Hospitalized Patients With COVID-19 and a VTE Event: A Prospective Study

Diagnostic performance of a colorimetric RT -LAMP for the identification of SARS-CoV-2: A multicenter prospective clinical evaluation in sub-Saharan Africa.

BackgroundManagement and control of the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus SARS-CoV-2 is critically dependent on quick and reliable identification of the virus in clinical specimens. Detection of viral RNA by a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a simple, reliable and cost-effective assay, deployable in resource-limited settings (RLS). Our objective was to evaluate the intrinsic and extrinsic performances of RT-LAMP in RLS.MethodsThis is a multicenter prospective observational study of diagnostic accuracy, conducted from October 2020 to February 2021 in four African Countries: Cameroon, Ethiopia, Kenya and Nigeria; and in Italy. We enroled 1657 individuals who were either COVID-19 suspect cases, or asymptomatic and presented for screening. RNA extracted from pharyngeal swabs was tested in parallel by a colorimetric RT-LAMP and by a standard real time polymerase chain reaction (RT-PCR).FindingsThe sensitivity and specificity of index RT LAMP compared to standard RT-PCR on 1657 prospective specimens from infected individuals was determined. For a subset of 1292 specimens, which underwent exactly the same procedures in different countries, we obtained very high specificity (98%) and positive predictive value (PPV = 99%), while the sensitivity was 87%, with a negative predictive value NPV = 70%, Stratification of RT-PCR data showed superior sensitivity achieved with an RT-PCR cycle threshold (Ct) below 35 (97%), which decreased to 60% above 35.InterpretationIn this field trial, RT-LAMP appears to be a reliable assay, comparable to RT-PCR, particularly with medium-high viral loads (Ct < 35). Hence, RT-LAMP can be deployed in RLS for timely management and prevention of COVID-19, without compromising the quality of output.FundingBill & Melinda Gates Foundation [Grant No. INV-022,816].

All‐in‐One DNA Extraction Tube for Facilitated Real‐Time Detection of Infectious Pathogens

An "all-in-one tube" platform is developed, where the genetic analysis involving DNA extraction, amplification, and detection can be performed in a single tube. The all-in-one tube consists of a polymerase chain reaction (PCR) tube in which the inner surface is conformally modified with a tertiary-amine-containing polymer to generate a strong electrostatic interaction with DNA. The all-in-one tube provides high DNA capture efficiency exceeding 80% from Escherichia coli O157: H7 pathogen at a wide range of DNA amount from 0.003 to 3ng. Indeed, the use of the surface-functionalized PCR tube enables direct amplification and detection of the surface-captured DNA without the modification of standard real-time PCR instrument. Besides, this platform has sensitivity, selectivity, and reliability enough for accurate detection at the minimal infective dose of both gram-positive and negative pathogens. The all-in-one tube enables the direct molecular diagnosis, substantially reducing the labor-intensive pathogen detection steps while providing high compatibility with the currently established real-time PCR instruments, and illustrates its on-site applicability with convenience expandable to various genetic analyses including food safety testing, forensic analysis, and clinical diagnosis.

Evaluation of SARS-CoV-2 antigen-based rapid diagnostic kits in Pakistan: formulation of COVID-19 national testing strategy

Rapid diagnosis of SARS-CoV-2 during pandemic enables timely treatment and prevention of COVID-19. Evaluating the accuracy and reliability of rapid diagnostic testing kits is crucial for surveillance and diagnosis of SARS-CoV-2 infections in general population, injection drug users, multi-transfused populations, healthcare workers, prisoners, barbers and other high risk populations. The aim of this study was to evaluate performance and effectiveness of nasopharyngeal swab (NSP) and saliva based rapid antigen detection testing kits in comparison with USFDA approved triple target gold standard real-time polymerase chain reaction. A cross-sectional study was conducted on 33,000 COVID-19 suspected patients. From RT-PCR positive patients, nasopharyngeal swab (NSP) and saliva samples were obtained for evaluation of rapid COVID-19 testing kits (RDT). 100/33,000 (0.3%) of specimens were RT-PCR positive for SARS-CoV-2. Among RT-PCR positive, 62% were males, 34% were females, and 4% were children. The NSP-RDT (Lepu Medical China) analysis revealed 53% reactivity among males, 58% reactivity among females, and 25% reactivity among children. However saliva based RDT (Lepu Medical China) analysis showed 21% reactivity among males and 23% among females, and no reactivity in children. False negative results were significantly more pronounced in saliva based RDT as compared to NSP-RDT. The sensitivity of these NSP-RDT and saliva based RDT were 52% and 21% respectively. The RDTs evaluated in this study showed limited sensitivities in comparison to gold standard RT-PCR, indicating that there is a dire need in Pakistan for development of suitable testing to improve accurate COVID-19 diagnosis in line with national demands.

The role of CT imaging for management of COVID-19 in epidemic area: early experience from a University Hospital

BackgroundTo evaluate the clinical value of the chest CT scan compared to the reference standard real-time polymerase chain reaction (RT-PCR) in COVID-19 patients.MethodsFrom March 29th to April 15th of 2020, a total of 240 patients with respiratory distress underwent both a low-dose chest CT scan and RT-PCR tests. The performance of chest CT in diagnosing COVID-19 was assessed with reference to the RT-PCR result. Two board-certified radiologists (mean 24 years of experience chest CT), blinded for the RT-PCR result, reviewed all scans and decided positive or negative chest CT findings by consensus.ResultsOut of 240 patients, 60% (144/240) had positive RT-PCR results and 89% (213/240) had a positive chest CT scans. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of chest CT in suggesting COVID-19 were 100% (95% CI: 97–100%, 144/240), 28% (95% CI: 19–38%, 27/240), 68% (95% CI: 65–70%) and 100%, respectively. The diagnostic accuracy of the chest CT suggesting COVID-19 was 71% (95% CI: 65–77%). Thirty-three patients with positive chest CT scan and negative RT-PCR test at baseline underwent repeat RT-PCR assay. In this subgroup, 21.2% (7/33) cases became RT-PCR positive.ConclusionChest CT imaging has high sensitivity and high NPV for diagnosing COVID-19 and can be considered as an alternative primary screening tool for COVID-19 in epidemic areas. In addition, a negative RT-PCR test, but positive CT findings can still be suggestive of COVID-19 infection.

Prevalence, Antibiotic-Resistance, and Virulence Characteristics of Vibrio parahaemolyticus in Restaurant Fish Tanks in Seoul, South Korea.

Vibrio parahaemolyticus is a marine bacterium that causes foodborne diarrhea. Many seafood restaurants keep live fish and shellfish in fish tanks for use in raw seafood dishes; thus, the present study aimed to investigate the prevalence, antibiotic-resistance, and virulence characteristics exhibited by V. parahaemolyticus detected in restaurant fish-tank water samples collected in Seoul, South Korea. Fish-tank water samples were collected from 69 restaurants in Seoul, and screened for the presence of V. parahaemolyticus via both a commercial detection kit, and a real-time polymerase chain reaction (RT-PCR) to detect the toxR gene. Antibiotic susceptibility and virulence determinants of V. parahaemolyticus isolates were evaluated and identified using standard disk-diffusion and RT-PCR methods, respectively. Thirty-five (50.7%) of the 69 analyzed water samples were found to be contaminated with V. parahaemolyticus. Those isolates were most often resistant to ampicillin (51.4% of isolates), followed by amikacin and tetracycline (11.4%), and ceftazidime (8.6%). Thirty (85.7%) out of the 35 isolates carried all four cytotoxicity-inducing type III secretion system 1 (T3SS1) genes [specifically, 34 (97.1%), 33 (94.3%), 35 (100%), and 32 (91.4%) isolates carried genes encoding the VP1670, VP1686, VP1689, and VP1694 T3SS1 proteins, respectively]. The type VI secretion systems (T6SS1 and T6SS2) genes were also detected in 11 (31.4%) and 27 (77.1%) isolates, respectively. However, virulence determinants such as the hemolysin (tdh and trh), urease (ureC), T3SS2α, or T3SS2β genes that are known to be associated with enterotoxicity were not detected in all isolates. Although some known major virulence genes were not detected in the V. parahaemolyticus isolates, the results of this study indicate that restaurant fish tanks are a potential source of antibiotic-resistant V. parahaemolyticus. The presented data support the need for strict guidelines to regulate the maintenance of restaurant fish tanks to prevent antibiotic-resistant foodborne vibriosis.

Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination.

A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspensions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin methylene blue and MacConkey detected only E. coli RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the background fluorescence was referred to as quantification cycle. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. Based upon standard curve analysis of B. cepacia DNA, the minimum DNA concentration that could be detected was 10 fg/uL with a correlation value of 0.98. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.LAY ABSTRACT: A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. B. cepacia is the number one reason for microbial contamination recalls of non-sterile drug products in the USA. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.

Pharmacogenetic testing revisited: 5' nuclease real-time polymerase chain reaction test panels for genotyping CYP2D6 and CYP2C19.

Due to their involvement in the metabolization of commonly prescribed psychopharmaceutical drugs, the cytochrome oxidase genes CYP2D6 and CYP2C19 are extensive targets for pharmacogenetic testing. The existence of common allelic variants allows the prediction of a metabolic phenotype based on a genotype result, hereby supplying a clinical tool for optimizing prescription and minimizing adverse effects. In this study, we present the development of two 5′ nuclease real-time polymerase chain reaction (PCR) test panels, capable of detecting eight of the most clinically relevant alleles of the CYP2D6 gene (*2, *3, *4, *6, *9, *10, 17, *41) and the three most common nonfunctional alleles of CYP2C19 (*2, *3, *4). The assays have been thoroughly validated using a large collection of reference samples, by parallel testing and by DNA sequencing. The reanalysis of reference samples provided the calculation of the frequency of the CYP2D6*4K allele in a population, not previously reported. Furthermore, original test results from CYP2D6*41, generated based on the presence of the 2850T and the lack of the −1584G single-nucleotide polymorphism (SNP), were compared with genotyping based on the current acknowledged founder SNP 2988G of this allele. These results indicate that up to 17.7% of the patients originally tested as carriers of the CYP2D6*41 allele may have had an incorrect phenotypic result assigned. The two 5′ nuclease real-time PCR test panels have subsequently been optimized for use in the clinical laboratory, using a standard real-time PCR instrument and software.

KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test

Background:Molecular techniques are relevant to modern cytopathology, but their implementation is difficult without molecular expertise and infrastructure. The assessment of KRAS mutational status on cytological preparations may be useful either to refine uncertain diagnoses on pancreatic aspirates or to yield predictive information to plan targeted treatment of metastatic colorectal cancer (mCRC). The novel test Idylla™ enables fully automated KRAS genotyping in approximately 2 h, even in less experienced hands.Materials and Methods:This study aims to validate this methodology to detect KRAS mutations on archival cytological preparations of pancreatic cancer (n = 9) and mCRC (n = 9) by comparing the Idylla™ performance to that of standard real-time polymerase chain reaction.Results:The same 11 mutations (n = 4: p.G12D; n = 2: p.G12V; n = 2: p.A59E/G/T; n = 1: p.G12R; n = 1: p.G13D; n = 1: p.Q61H) were detected by both techniques.Conclusion:Even in less experienced laboratories, a cytopathologist may easily integrate morphological diagnostic report with accurate KRAS mutation detection, which is relevant for diagnostic and treatment decisions.

An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT–PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL l-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT–PCR machines in a hydrophilic or hydrophobic environment.

Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae.

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

Evaluation of Systems for Nopaline Synthase Terminator in Fast and Standard Real-Time PCR to Screen Genetically Modified Organisms

The increasing number and diversity of genetically modified organisms (GMOs) developed and commercialised forces laboratories to apply many different methods of analysis. Matrix-based approach helps to minimize analytical effort reducing the number of identifications. However, the correctness of screening phase needs efficient methods. In this paper, 15 systems for the nopaline synthase terminator (T-nos) from Agrobacterium tumefaciens, a genetic element present in several genetically modified (GM) plants, were tested. The systems were obtained from three methods, and their primers and probes were combined and tested in real-time polymerase chain reaction (PCR) in fast and standard mode. The results have showed that the fast mode presented the lowest mean quantification cycle (Cq) and the highest ∆Rn, that is, the difference between normalized reporter and baseline. These parameters of system efficiency prove that the fast real-time PCR is a possible approach to obtain good data in less than half the time compared to standard mode. The proposed approach allows a practical way to evaluate the most efficient set of oligonucleotides (e.g., primers and probe) in fast and standard real-time PCR before validation. This article describes also in-house validation of the best set oligonucleotides.

Magnetoelastic Biosensors for Real-Time, Wireless Pathogen Detection on Surfaces

Recent outbreaks of Ebola in the United States have brought to the public’s attention the transfer of disease by contact with virus- and bacteria-contaminated surfaces ranging from hospital counters to door knobs to airplane seats. The need for a real-time, on-site technique by which pathogen-contaminated surfaces can be identified is immediate and real. Standard microbiological detection techniques, including enzyme-linked immunosorbent assay (ELSA), polymerase chain reaction (PCR) and plate count, are time-consuming and manpower-intensive. This paper describes research into wireless magnetoelastic (ME) biosensors combined with a surface-scanning coil that can be used to detect both viral and bacterial contamination of surfaces. This detection method takes less than 10 minutes and eliminates time-consuming sample preparation such as enrichment. This paper investigates the use of wireless ME biosensors for the surface-scanning detection of pathogens on surfaces. The ME biosensor consists of an ME resonator as the sensor platform and E2 phage as the bio-recognition element. The E2 phage is genetically engineered to specifically bind with Salmonella Typhimurium. The ME biosensor is actuated into mechanical resonance by an externally applied magnetic field. A microfabricated planar coil was used to measure the resonant frequency of multiple ME biosensors in real time. When this phage-coated ME biosensor comes into contact with Salmonella cells, the bacterial cells are captured and bound to the biosensor’s surface by the phage. The bound bacterial cells then cause the mass of the ME biosensor to increase, resulting in a decrease in the resonant frequency. The change of the resonant frequency is known to be directly proportional to the additional mass of the bacteria attached to the biosensor surface. As a model study, multiple E2 phage-coated biosensors (measurement sensors) were placed on surfaces spiked with Salmonella Typhimurium with various concentrations. Control sensors without phage were also used to compensate for environmental effects and non-specific binding. The limit of detection and repeatability of testing were found to be comparable to standard real-time PCR. In this manner, in-situ, continuous, real-time monitoring of pathogenic surface contamination can be performed on any surfaces.

Simple Method to Genotype the ACTN3 r577x Polymorphism.

The alpha-actinin-3 r577x polymorphism (rs1815739) is one of the most important polymorphisms associated with athletic performance. This single-nucleotide mutation leads to a premature stop codon, resulting in a nonfunctional protein product. The presence of the dominant R allele is associated with full power skeletal muscle contraction. Homozygosity for the X allele is correlated with more efficient energy disposure. Restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) are the standard methods used to genotype this polymorphism, but they are expensive and require special equipments. Here, we present a simple and cost-efficient method to genotype the ACTN3 r577x polymorphism by a single PCR. External primers yield a 690-bp product that indicates the template quality. Internal primers produce a 413-bp product if the R allele is present and a 318-bp product if the X allele is present. Our four-primer genotyping PCR was validated by the standard real-time PCR, generally used to genotype this single-nucleotide polymorphism, demonstrating the accuracy of this method. This protocol is perfect for small- or large-scale cohort genotyping of the ACTN3 r577x polymorphism.

Diagnosis and Management of Clostridium difficile Infection

Clostridium difficile infection (CDI) is increasing in frequency and severity in and out of the hospital, with a high probability of recurrence after treatment. The recent literature on CDI was reviewed using PubMed to include recent publications dealing with diagnosis and therapy. Real-time polymerase chain reaction is a sensitive and useful diagnostic test for CDI but there are growing concerns of false-positive test results if the rate of CDI is low in the patient population providing samples and/or if the population being studied commonly includes people with C difficile colonization. Recommended therapy of CDI includes oral metronidazole for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases, each given for 10 days. Colectomy is being performed more frequently in patients with fulminant CDI. For treatment of first recurrences the drug used in the first bout can be used again and for second recurrences longer courses of vancomycin often are given in a tapered dose or intermittently to allow gut flora reconstitution, or other treatments including fidaxomicin may be used. Bacteriotherapy with fecal transplantation is playing an increasing role in therapy of recurrent cases. Metagenomic studies of patients with CDI during successful therapy are needed to determine how best to protect the flora from assaults from antibacterial drugs and to develop optimal therapeutic approaches. Immunotherapy and immunoprophylaxis offer opportunities to prevent CDI, to speed up recovery from CDI, and to eliminate recurrent infection. Humanized monoclonal antitoxin antibodies and active immunization with vaccines against C difficile or its toxins are both in development and appear to be of potential value.

Prevalence of koala retrovirus in geographically diverse populations in Australia

To determine the prevalence of koala retrovirus (KoRV) in selected koala populations and to estimate proviral copy number in a subset of koalas. Blood or tissue samples from 708 koalas in Queensland, New South Wales, Victoria and South Australia were tested for KoRV pol provirus gene using standard polymerase chain reaction (PCR), nested PCR and real-time PCR (qPCR). Prevalence of KoRV provirus-positive koalas was 100% in four regions of Queensland and New South Wales, 72.2% in mainland Victoria, 26.6% on four Victorian islands and 14.8% on Kangaroo Island, South Australia. Estimated proviral copy number per cell in four groups of koalas from Queensland and Victoria showed marked variation, ranging from a mean of 165 copies per cell in the Queensland group to 1.29 × 10(-4) copies per cell in one group of Victorian koalas. The higher prevalence of KoRV-positive koalas in the north of Australia and high proviral loads in Queensland koalas may indicate KoRV entered and became endogenous in the north and is spreading southwards. It is also possible there are genetic differences between koalas in northern and southern Australia that affect susceptibility to KoRV infection or endogenisation, or that environmental factors affecting transmission in northern states are absent or uncommon in southern regions. Although further studies are required, the finding of proviral copy numbers orders of magnitude lower than what would be expected for the presence of a single copy in every cell for many Victorian animals suggests that KoRV is not endogenous in these animals and likely reflects ongoing exogenous infection.