Rapid Molecular Assay Research Articles

Evaluation of the Eazyplex®Candida ID LAMP Assay for the Rapid Diagnosis of Positive Blood Cultures.

Rapid molecular assays can be used to identify Candida pathogens directly from positive blood cultures (BCs) in a timely manner compared to standard methods using subcultures. In this study, the eazyplex®Candida ID assay, which is based on loop-mediated amplification (LAMP) and is currently for research use only, was evaluated for the identification of the most common fungal species. A total of 190 BCs were analysed. Sensitivity and specificity were 93.88% and 99.26% for C. albicans, 89.13% and 100% for Nakaseomyces glabratus (N. glabratus), 100% and 100% for Pichia kudravzevii (P. kudriavzevii), 100% and 100% for C. tropicalis, and 100% and 99.44% for C. parapsilosis. Sample preparation took approximately 11 min and positive amplification results were obtained between 8.5 and 19 min. The eazyplex®Candida ID LAMP assay is an easy-to-use diagnostic tool that can optimise the management of patients with candidemia.

Emerging public health strategies in malaria control: innovations and implications

Malaria remains a significant global health challenge, particularly in regions with limited resources and tropical climates. Despite extensive efforts, the disease continues to cause significant morbidity and mortality, with approximately 229 million cases and 409,000 deaths reported in 2020. However, recent years have seen promising advancements in public health strategies aimed at malaria control and elimination. Technological advancements have played a crucial role in improving malaria control efforts. Genomic surveillance techniques enable the monitoring of malaria parasite populations, aiding in the detection of drug resistance and informing targeted interventions. Additionally, innovative diagnostic technologies, such as rapid diagnostic tests (RDTs) and molecular assays, have enhanced the speed and accuracy of malaria diagnosis, facilitated prompt treatment and reduced transmission. These tools are instrumental in achieving the World Health Organization’s (WHO) goals of reducing malaria cases and deaths by at least 90% by 2030. Novel vector control methods offer innovative approaches to reduce malaria transmission. Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) remain foundational strategies, with advancements including the development of next-generation insecticides and long-lasting insecticidal nets (LLINs). Furthermore, genetic modification of mosquitoes, such as gene drive technology, holds promise for reducing mosquito populations and interrupting malaria transmission. These vector control innovations complement other strategies, contributing to comprehensive malaria control efforts aimed at achieving sustainable disease reduction and eventual elimination.

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.

A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes.

Recently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates. The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes. The standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/μL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method. The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.

Performance evaluation of Truenat MTB and Truenat MTB-RIF DX assays in comparison to gene XPERT MTB/RIF ultra for the diagnosis of pulmonary tuberculosis in Uganda

BackgroundThe World Health Organization endorsed Truenat MTB rapid molecular assay in 2020 and recommended additional in-country evaluation studies before uptake. We evaluated the accuracy and operational feasibility of Truenat MTB assay (Truenat) in comparison with GeneXpert Ultra and culture.MethodsIn a cross-sectional study of 250 presumptive TB patients, participants were requested to provide a sputum sample on the day of their visit to the clinic. The sputum sample was homogenized and a portion was tested using GeneXpert Ultra as per the routine standard procedure and the other portion was tested using Truenat assay at the clinic laboratory. The second sample portion was processed for Concentrated Fluorescent smear Microscopy (CFM), LJ, and MGIT cultures. Truenat sensitivity and specificity were compared to GeneXpert Ultra and culture. Test performance characteristics and operational feasibility assessment data through interview of the study laboratory staff were also collected and summarized as proportions and percentages.ResultsOf the 250 participants recruited in the study, the sensitivity and specificity of Truenat was n/N (%, 95%CI); 66/82 (80.5, 70.2–88.4) and 156/159 (98.1, 94.5–99.6) when compared with Ultra, 50/64 (89.3, 66.0-87.4) and 166/180 (92.2, 87.2–95.6) when compared with LJ, 58/71 (81.7,70.7–89.8) and 131/138 (94.9, 89.8–97.9) when compared to MGIT culture and 59/73 (80.8, 69.9–89.1) and 159/169 (94.1,89.3–97.1) when compared to LJ and/or MGIT culture. The sensitivity of Truenat was lower, 14/23 (60.9, 40.6–82.8) among smear-negative compared to 45/50 (90.0, 78.1–96.6) among smear-positive participants but not different by HIV status. There were no special training needs especially among laboratory personnel with previous GeneXpert /molecular test experience, 19/242 (7.8%) error/invalid, and 12 (17,4%) uninterpretable/indeterminate results mainly for rifampicin resistance determination. However, there were 3 (3.5%) of GeneXpert Ultra indeterminate results.ConclusionAmong presumptive TB patients in Uganda, the Truenat assay has high sensitivity and specificity. The Truenat assay has acceptable operational feasibility attributes when compared with the GeneXpert Assay.

Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease

BackgroundEnteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method.ResultsUsing our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines.ConclusionsOur study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry.

Discordance Between Phenotypic and WGS-Based Drug Susceptibility Testing Results for Some Anti-Tuberculosis Drugs: A Snapshot Study of Paired Mycobacterium tuberculosis Isolates with Small Genetic Distance.

Current tuberculosis treatment regimens primarily rely on phenotypic drug susceptibility testing and rapid molecular assays. Although whole-genome sequencing (WGS) offers a promising alternative, disagreements between phenotypic and molecular testing methods remain. In this retrospective study, we compared the phenotypic and WGS-predicted drug resistance profiles of paired Mycobacterium tuberculosis isolates with small genetic distances (≤10 single nucleotide variants) obtained from patients with longitudinal single-episode or recurrent tuberculosis. Additionally, we investigated the distribution of drug-resistance-conferring variants among the identified M. tuberculosis genotypes. Paired M. tuberculosis isolates from 46 patients with pulmonary tuberculosis (2002-2019) were analyzed. Spoligotyping was performed for all the isolates. WGS data were processed using TB-Profiler software to genotype the strains and detect variants in M. tuberculosis genes associated with drug resistance. The significance of these variants was evaluated using the M. tuberculosis variant catalog developed by the World Health Organization. Phenotypic drug susceptibility test results were obtained from patients' medical records. Among the 46 isolate pairs, 25 (54.3%) harbored drug-resistance-associated variants, with 20 demonstrating identical WGS-predicted drug resistance profiles. Drug-resistant isolate pairs belonged to Lineages 2 and 4, with the most common sub-lineages being 2.2.1 (SIT1 and SIT190 spoligotypes), and 4.3.3 (SIT42). Agreement between phenotypic and WGS-based drug susceptibility testing was highest (>90%) for rifampicin, isoniazid, ethambutol, fluoroquinolones, streptomycin, and amikacin when calculated for M. tuberculosis isolates or isolate pairs. In most discordant cases, isolate pairs harbored variants that could cause low- or moderate-level resistance or were previously associated with variable minimum inhibitory concentrations. Notably, such discrepancies mostly occurred in one isolate from the pair. In addition, differences in resistance-related variant distributions among M. tuberculosis genotypes were observed for most of the analyzed drugs. The simultaneous performance of phenotypic and WGS-based drug susceptibility testing creates the most accurate drug resistance profile for M. tuberculosis isolates and eliminates important limitations of each method.

Individualised treatment of sexually transmitted infections by rapid molecular assays

Individualised treatment of sexually transmitted infections by rapid molecular assays

Addressing patients' unmet needs related to multidrug-resistant tuberculosis (MDR-TB) care: A qualitative research study from Pune city, India.

We aimed to identify and describe the unmet needs of patients with multidrug-resistant tuberculosis (MDR-TB). As a part of larger cross-sectional mixed-methods (qualitative and quantitative data) study on pathways to MDR-TB care, here we present the qualitative component. We interviewed 128 (56 men and 72 women) individuals who had MDR-TB, aged > = 15 years, registered and treated under the National TB Elimination Program (NTEP) in Pune city of India. We carried out thematic analysis of participants' narratives. We found that delays in diagnosis, lack of counseling, late referral to the NTEP and unwarranted expenditure were the main barriers to care that study participants experienced in the private sector. Provider dismissal of symptoms, non-courteous behavior, lack of hygiene in the referral centers, forced stay with other patients and lack of support for psychological/psychiatric problems were identified as a few additional challenges that participants faced at the NTEP care centers. Using qualitative data from experiences of participants with MDR-TB, we identify patients' several unmet needs, attention to which can improve MDR-TB care. Educating private providers about MDR-TB risk and available rapid molecular assays can help the timely diagnosis of MDR-TB and reduce patients' out of pocket costs. At the RNTCP/NTEP, measures such as training health workers to build rapport with patients, maintaining hygienic environments in the health centers with adequate isolation of participants with MDR from other serious cases, referral of patients with psychiatric symptoms to mental health specialists and monitoring drug shortages can help in improving care delivery.

Discordance between genotypic and phenotypic methods for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis and the correlation with patient treatment outcomes

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) is essential for proper patient management. We investigated discordance between genotypic (Xpert MTB/RIF and MTBDRplus) and phenotypic (MGIT 960) methods for the detection of rifampicin (RIF) and isoniazid (INH) resistant MTB and its correlation with patient treatment outcomes in Jimma, Southwest Oromia, Ethiopia. MethodsA retrospective study was conducted on 57 stored MTB isolates with known Xpert RIF resistance status (45 RIF resistant and 12 RIF susceptible) at Jimma University Mycobacteriology Research Center from November 2, 2021, to December 28, 2022. We did MTBDRplus and phenotypic DST (using the Mycobacterial Growth Indicator Tube (MGIT) system). The Xpert and MTBDRplus results were compared using phenotypic DST as a reference standard method. The treatment outcome was determined as per national guideline. The discordance between the genotypic and phenotypic DST was calculated using GraphPad software. ResultsAmong the 57 MTB isolates, six (10.5 %) had discordant results between the two DST methods. Xpert yielded five discordant results for RIF when compared with phenotypic DST (kappa coefficient (κ) = 0.76, 95 % confidence interval 0.56–0.96). The MTBDRplus compared with phenotypic DST gave three discordant results for RIF (κ = 0.86, 95 % confidence interval 0.71–1.00) and three for INH (κ = 0.86, 95 % confidence interval 0.70–1.00). Compared with Xpert, MTBDRplus yielded lower discordance with phenotypic DST for RIF. Out of six patients with discordant results, three had unfavorable outcomes while the other three were cured. Of the three patients with unfavorable outcomes, only one patient has received an inappropriate treatment regimen. There was no correlation between unfavorable outcomes and incorrect treatment regimens due to discordant results (Χ2 = 0.404; P = 0.525). ConclusionsDiscordance between genotypic and phenotypic DST for RIF or INH occurred in 10.5 % of isolates. Only one patient with discordant results has received an inappropriate treatment regimen, resulting in an unfavorable outcome. The impact of parallel use of rapid molecular assay with phenotypic DST on patient treatment outcomes requires further study.

Laboratory diagnosis of CNS infections in children due to emerging and re-emerging neurotropic viruses.

Recent decades have witnessed the emergence and re-emergence of numerous medically important viruses that cause central nervous system (CNS) infections in children, e.g., Zika, West Nile, and enterovirus/parechovirus. Children with immature immune defenses and blood-brain barrier are more vulnerable to viral CNS infections and meningitis than adults. Viral invasion into the CNS causes meningitis, encephalitis, brain imaging abnormalities, and long-term neurodevelopmental sequelae. Rapid and accurate detection of neurotropic viral infections is essential for diagnosing CNS diseases and setting up an appropriate patient management plan. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis/encephalitis. However, the expansion of test menu has led to new challenges in selecting appropriate tests and making accurate interpretation of test results. There are unmet gaps in development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses. Herein we will discuss the advances and challenges in the laboratory diagnosis of viral CNS infections in children. This review not only sheds light on selection and interpretation of a suitable diagnostic test for emerging/re-emerging neurotropic viruses, but also calls for more research on development and clinical utility study of novel molecular assays. IMPACT: Children with immature immune defenses and blood-brain barrier, especially neonates and infants, are more vulnerable to viral central nervous system infections and meningitis than adults. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis and encephalitis. There are unmet gaps in the development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses.

Histopathological features in the clinical specimens with tuberculosis diagnosis by BACTEC MGIT 960 culture

Diagnosis of extrapulmonary tuberculosis remains challenging in lower-middle income countries with high burden of tuberculosis (TB). This study aims to describe the histological characteristics in biopsy samples from patients with confirmed TB. This is a retrospective study of clinical biopsy specimens with positive liquid medium culture for Mycobacterium tuberculosis and histopathological examination in the National Lung Hospital in Vietnam. Among 1045 biopsy specimens with mycobacteria culture, the overall rate of growth of Mycobacteria tuberculosis in culture was 20.7% (216/1045). The positivity rates of MIGT culture among surgical biopsy specimens were 75% in bone specimen, followed by vertebral specimens (51.3%), and joint specimens (26.4%). For specimens obtained by the fine needle aspiration, the positivity rates of MIGT culture were 26.3% in lymph node and 25.3% in pleural specimen. Among specimens with culture confirmation of TB, the most common histopathoglogical suggestive finding of TB was the presence of epithelioid cell (83.3%), Langhans giant cells (75.9%), and caseous necrosis (75.5%). The high proportion of histological features suggestive of TB among the TB culture confirmed biopsy samples support for further evaluation of histological examination and its combination with other recommended rapid molecular assays in specimens with suspicion of TB.

Implementation of large-scale pooled testing to increase rapid molecular diagnostic test coverage for tuberculosis: a retrospective evaluation

In 2021, only 6.4 million of the 10.6 million people with tuberculosis (TB) were diagnosed and treated for the disease. Although the World Health Organization recommends initial diagnostic testing using a rapid sensitive molecular assay, only 38% of people diagnosed with TB benefited from these, due to barriers including the high cost of available assays. Pooled testing has been used as an approach to increase testing efficiency in many resource-constrained situations, such as the COVID-19 pandemic, but it has not yet been widely adopted for TB diagnostic testing. Here we report a retrospective analysis of routine pooled testing of 10,117 sputum specimens using the Xpert MTB/RIF and Xpert MTB/RIF Ultra assays that was performed from July 2020 to February 2022. Pooled testing saved 48% of assays and enabled rapid molecular testing for 4156 additional people as compared to individual testing, with 6.6% of specimens positive for TB. From an in silico analysis, the positive percent agreement of pooled testing in pools of 3 as compared with individual testing for the Xpert MTB/RIF Ultra assay was estimated as 99.4% (95% CI, 96.6% to 100%). These results support the scale-up of pooled testing for efficient TB diagnosis.

Rapid genetic screening with high quality factor metasurfaces

Genetic analysis methods are foundational to advancing personalized medicine, accelerating disease diagnostics, and monitoring the health of organisms and ecosystems. Current nucleic acid technologies such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) rely on sample amplification and can suffer from inhibition. Here, we introduce a label-free genetic screening platform based on high quality (high-Q) factor silicon nanoantennas functionalized with nucleic acid fragments. Each high-Q nanoantenna exhibits average resonant quality factors of 2,200 in physiological buffer. We quantitatively detect two gene fragments, SARS-CoV-2 envelope (E) and open reading frame 1b (ORF1b), with high-specificity via DNA hybridization. We also demonstrate femtomolar sensitivity in buffer and nanomolar sensitivity in spiked nasopharyngeal eluates within 5 minutes. Nanoantennas are patterned at densities of 160,000 devices per cm2, enabling future work on highly-multiplexed detection. Combined with advances in complex sample processing, our work provides a foundation for rapid, compact, and amplification-free molecular assays.

Antimicrobial susceptibility assays for Neisseria gonorrhoeae: a proof-of-principle population-based retrospective analysis

Neisseria gonorrhoeae treatment guided by molecular antimicrobial susceptibility assays could improve treatment options and antimicrobial stewardship; however, few commercial assays are available. We aimed to investigate antimicrobial susceptibility of N gonorrhoeae isolates in New South Wales, Australia, and estimate the potential usefulness of hypothetical combinations of rapid molecular antimicrobial susceptibility assays. In this proof-of-principle, population-based, retrospective analysis, we assessed N gonorrhoeae susceptibility data for ceftriaxone, azithromycin, ciprofloxacin, and penicillin. Isolates were previously collected as part of the Australian Gonococcal Surveillance Programme between Jan 1, 2008, and Dec 31, 2019. All cultured N gonorrhoeae isolates with susceptibility data to all four antimicrobials were included. However, only one isolate was included if several isolates originated from the same individual within 13 days of the previous isolate originating from that individual, and there were less than two standard double-dilution minimum inhibitory concentrations between the isolates. We assessed the use of different combinations of hypothetical antimicrobial susceptibility assays and treatment combinations in terms of their ability to minimise overall ceftriaxone use, and use specifically in isolates with decreased susceptibility to ceftriaxone, compared with standard non-assay-guided empirical ceftriaxone treatment. We included 23 089 N gonorrhoeae isolates. The prevalence of antimicrobial sensitivity fluctuated significantly during the study. Isolates with decreased susceptibility to ceftriaxone were more likely to be resistant to one or more antimicrobials than isolates without decreased susceptibility (782 [98·6%] of 793 vs 10 661 [47·8%] of 22 296), particularly ciprofloxacin (p<0·0001) and penicillin (p<0·0001). Compared with empirical ceftriaxone treatment, we estimated that strategies based on the use of hypothetical antimicrobial susceptibility would reduce ceftriaxone use (p<0·0001). However, because of co-resistance, most assay-directed treatment strategies, including those involving use of assays for two antibiotics, would result in only moderate reductions in ceftriaxone use among isolates with decreased susceptibility to ceftriaxone. Individualised treatment guided by molecular antimicrobial susceptibility diagnostics could help to reduce overall ceftriaxone use in gonorrhoea. However, the use of these assays needs to be informed by the non-random nature of co-resistance among circulating N gonorrhoeae strains. Australian Government and Queensland Government.

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart’s hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

Rapid molecular assay for the evaluation of clove essential oil antifungal activity against wheat common bunt.

Common bunt of durum wheat (DW), Triticum turgidum L. ssp. durum (Desf.) Husn., is caused by the two closely related fungal species belonging to Tilletia genus (Tilletiales, Exobasidiomycetes, Ustilaginomycotina): Tilletia laevis Kühn (syn. T. foetida (Wallr.) Liro.) and T. caries (DC) Tul. (syn. T. tritici (Bjerk.) G. Winter). This is one of the most devastating diseases in wheat growing areas worldwide, causing considerable yield loss and reduction of wheat grains and flour quality. For these reasons, a fast, specific, sensitive, and cost-effective method for an early diagnosis of common bunt in wheat seedlings is urgent. Several molecular and serological methods were developed for diagnosis of common bunt in wheat seedlings but at late phenological stages (inflorescence) or based on conventional PCR amplification, with low sensitivity. In this study, a TaqMan Real Time PCR-based assay was developed for rapid diagnosis and quantification of T. laevis in young wheat seedlings, before tillering stage. This method, along with phenotypic analysis, was used to study conditions favoring pathogen infection and to evaluate the effectiveness of clove oil-based seed dressing in controlling the disease. The overall results showed that: i) the Real Time PCR assay was able to quantify T. laevis in young wheat seedlings after seed dressing by clove oil in different formulations, greatly reducing times of analysis. It showed high sensitivity, detecting up to 10 fg of pathogen DNA, specificity and robustness, allowing to directly analyze crude plant extracts and representing a useful tool to speed up the tests of genetic breeding for disease resistance; ii) temperature was a critical point for disease development when using wheat seeds contaminated by T. laevis spores; iii) at least one of the clove oil-based formulations tested was able to efficiently control wheat common bunt, suggesting that clove oil dressing could represent a promising tool for managing the disease, especially in sustainable farming.

Curtailing virus-induced inflammation in respiratory infections: emerging strategies for therapeutic interventions.

Acute respiratory viral infections (ARVI) are the most common illnesses worldwide. In some instances, mild cases of ARVI progress to hyperinflammatory responses, which are damaging to pulmonary tissue and requiring intensive care. Here we summarize available information on preclinical and clinical effects of XC221GI (1-[2-(1-methyl imidazole-4-yl)-ethyl]perhydroazin-2,6-dione), an oral drug with a favorable safety profile that has been tested in animal models of influenza, respiratory syncytial virus, highly pathogenic coronavirus strains and other acute viral upper respiratory infections. XC221GI is capable of controlling IFN-gamma-driven inflammation as it is evident from the suppression of the production of soluble cytokines and chemokines, including IL-6, IL-8, CXCL10, CXCL9 and CXCL11 as well as a decrease in migration of neutrophils into the pulmonary tissue. An excellent safety profile of XC221GI, which is not metabolized by the liver, and its significant anti-inflammatory effects indicate utility of this compound in abating conversion of ambulatory cases of respiratory infections into the cases with aggravated presentation that require hospitalization. This drug is especially useful when rapid molecular assays determining viral species are impractical, or when direct antiviral drugs are not available. Moreover, XC221GI may be combined with direct antiviral drugs to enhance their therapeutic effects.