From Epidemiology of Community-Onset Bloodstream Infections to the Development of Empirical Antimicrobial Treatment-Decision Algorithm in a Region with High Burden of Antimicrobial Resistance.

Antimicrobial-resistant (AMR) infections have increased in community settings. Our objectives were to study the epidemiology of community-onset bloodstream infections (BSIs), identify risk factors for AMR-BSI and mortality-related factors, and develop the empirical antimicrobial treatment-decision algorithm. All adult, positive blood cultures at the emergency room and outpatient clinics were evaluated from 08/2021 to 04/2022. AMR was defined as the resistance of organisms to an antimicrobial to which they were previously sensitive. A total of 1151 positive blood cultures were identified. There were 450 initial episodes of bacterial BSI, and 114 BSIs (25%) were AMR-BSI. Non-susceptibility to ceftriaxone was detected in 40.9% of 195 E. coli isolates and 16.4% among 67 K. pneumoniae isolates. A treatment-decision algorithm was developed using the independent risk factors for AMR-BSI: presence of multidrug-resistant organisms (MDROs) within 90 days (aOR 3.63), prior antimicrobial exposure within 90 days (aOR 1.94), and urinary source (aOR 1.79). The positive and negative predictive values were 53.3% and 83.2%, respectively. The C-statistic was 0.73. Factors significantly associated with 30-day all-cause mortality were Pitt bacteremia score (aHR 1.39), solid malignancy (aHR 2.61), and urinary source (aHR 0.30). In conclusion, one-fourth of community-onset BSI were antimicrobial-resistant, and one-third of Enterobacteriaceae were non-susceptible to ceftriaxone. Treatment-decision algorithms may reduce overly broad antimicrobial treatment.

ACORN (A Clinically-Oriented Antimicrobial Resistance Surveillance Network) II: protocol for case based antimicrobial resistance surveillance.

Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs). Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies. Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest.

In Vitro Activity of Cefiderocol, Cefepime-Zidebactam, and ß-Lactam Combinations Versus Other Antibiotic Classes Against Various Sequence Types of Clinically Isolated Carbapenemase-Producing Klebsiella pneumoniae.

Aim: This study aimed to establish the in vitro efficacy and susceptibility profiles of new ß-lactam antibiotics against clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) strains. Materials and Methods: A total of 117 nonduplicated CPKP isolates were tested against cefiderocol, cefepime-zidebactam, ceftazidime-avibactam, tigecycline, and other 20 antibiotics by broth microdilution. The carbapenemase genes were identified using PCR and sequencing, while multilocus sequence typing established the bacterial strains. Results: Three significant sequence types (STs), including ST147, ST16, and ST11, were shown to be the dominant STs, which occupied ∼90% of the tested population. Three carbapenemase genes, blaNDM-1, blaOXA-181, and blaOXA-232, were detected. The blaNDM-1 was found in ST147 and ST16 but not in ST11, while the blaOXA-232 was not detected in ST147. The majority of ST16 isolates contained both blaNDM-1 and blaOXA-232, which was not seen in other strains. Cefiderocol, cefepime-zidebactam, and tigecycline were the most active agents against CPKP. Both MIC50 and MIC90 of these three antibiotics remained within the susceptible categories, while nearly all other antibiotics were in the resistant levels. However, in ST11, which carried only blaOXA genes without blaNDM-1, ceftazidime-avibactam was effective with the MIC90 at 2 µg/mL. In addition, amikacin was shown to have good activity in ST11. In contrast, gentamicin was active in only ST16 and ST147. Conclusions: This study is the first report that demonstrates the prevalence of CPKP, distribution of strains, resistant genes, and antimicrobial susceptibility profiles in northern Thailand. These data would contribute to appropriate individual treatment and the selection of infection control strategies.

Genomic analysis of Enterococcus faecium strain RAOG174 associated with acute chorioamnionitis carried antibiotic resistance gene: is it time for precise microbiological identification for appropriate antibiotic use?

BACKGROUND: Preterm labor syndrome is associated with high perinatal morbidity and mortality, and intra-amniotic infection is a cause of preterm labor. The standard identification of causative microorganisms is based on the use of biochemical phenotypes, together with broth dilution-based antibiotic susceptibility from organisms grown in culture. However, such methods could not provide an accurate epidemiological aspect and a genetic basis of antimicrobial resistance leading to an inappropriate antibiotic administration. Hybrid genome assembly is a combination of short- and long-read sequencing, which provides better genomic resolution and completeness for genotypic identification and characterization. Herein, we performed a hybrid whole genome assembly sequencing of a pathogen associated with acute histologic chorioamnionitis in women presenting with PPROM. RESULTS: We identified Enterococcus faecium, namely E. faecium strain RAOG174, with several antibiotic resistance genes, including vancomycin and aminoglycoside. Virulence-associated genes and potential bacteriophage were also identified in this genome. CONCLUSION: We report herein the first study demonstrating the use of hybrid genome assembly and genomic analysis to identify E. faecium ST17 as a pathogen associated with acute histologic chorioamnionitis. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. The occurrence of E. faecium ST17 raised the awareness of the colonization of clinically relevant E. faecium and the carrying of antibiotic resistance. This finding has brought the advantages of genomic approach in the identification of the bacterial species and antibiotic resistance gene for E. faecium for appropriate antibiotic use to improve maternal and neonatal care.

Temporal shifts in the predominant carbapenemase gene types among carbapenemase-producing Klebsiella pneumoniae isolated in Bangkok, Thailand, during 2013-2016.

Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice.Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48-like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited.Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand.Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013-2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships.Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014-2015 was bla NDM-1, whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1. Other carbapenemase gene variants, such as bla NDM-4, bla NDM-5, bla OXA-48, bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181, emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years.Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.

Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity.

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42-3.59 µM. The most active compound 1 showed an IC50 value of 0.42 µM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 µM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products.

Platelet transfusions may lead to more significant risks of infection and septic transfusion reactions that can be fatal to the recipient. Platelet products should be screened to limit or detect bacterial contamination before application to patients to minimise any adverse reactions. This study aimed to develop a helicase-dependent amplification (HDA) technique targeting a universal highly conserved bacterial gene, 16S rRNA, in combination with naked-eye detection using SYBR Green I (HDA/SYBR Green I) to detect bacterial contamination in platelet products. Thirty positive samples were obtained from spiked platelet products by five transfusion-relevant bacterial strains and were screened for bacterial contamination by HDA/SYBR Green I. HDA/SYBR Green I showed an enhanced yield of bacterial contaminant detection when performed with medium to late shelf life, Day 2 of storage or later platelet products (98.67% sensitivity and 100% specificity compared to the BACT/ALERT culture system). The limit of detection of HDA/SYBR Green I was 1 ng, and there was no cross-reaction with other organisms that could likely contaminate platelet products. The developed HDA/SYBR Green I assay is rapid and simplistic and only requires an easy-to-find heat box, available in general blood bank laboratories, for the amplification step. This technique is suitable for further development as an alternative method to detect bacterial contamination in platelet products in the near future.

Poisoning from Ingestion of Fungus-Infected Cicada Nymphs: Characteristics and Clinical Outcomes of Patients in Thailand.

The current data regarding poisoning associated with ingestion of fungus-infected cicada nymphs are limited. We performed a retrospective cohort study of patients who ingested fungus-infected cicada nymphs and were referred to the Ramathibodi Poison Center for consultation from June 2010 to June 2022. Thirty-nine patients were included for analysis. Most were men (53.8%). Mean age was 40.2 ± 15.0 years. All nymphs were ingested as a health/food supplement. Thirty-one patients (79.5%) reported gastrointestinal symptoms. Median time from ingestion to symptom onset was 5 h. Twenty-nine patients (74.4%) reported neurological symptoms, including tremor, myoclonus, muscle rigidity, nystagmus/ocular clonus, drowsiness, dysarthria, seizure, and confusion. Some complained of dizziness, urinary retention, and jaw stiffness. Most patients (94.9%) were admitted to the hospital. Median hospital stay was 3 days. Ibotenic acid was detected in the blood and urine samples of one patient. All received supportive care. Four patients developed infectious complications. No deaths occurred. Consuming fungus-infected cicada nymphs may cause poisoning in humans. Gastrointestinal and neurological symptoms were common. Ibotenic acid might be the underlying cause. The main treatment is supportive care and appropriate management of complications. Education of the general public is advocated to prevent the incidence of this type of poisoning.

A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-ß-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae Clones and Spreading Plasmids.

Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome. We analyzed 371 Enterobacteriaceae isolates carrying blaNDM-1 and 114 Enterobacteriaceae isolates carrying blaNDM-5 obtained from clinical samples of 473 patients in 11 representative hospitals located in six provinces in Thailand between 2012 and 2017. The complete structures of plasmids carrying blaNDM and chromosomal phylogeny were determined by combining Southern blotting hybridization analysis and our previously performed whole-genome short-read sequencing data. Dissemination of the blaNDM-5 gene among the Enterobacteriaceae isolates in Thailand was mainly owing to the nationwide clonal spread of Escherichia coli ST410 and regional clonal spreads of Escherichia coli ST361 and ST405. Analysis of blaNDM-1-carrying isolates revealed nationwide dissemination of two specific plasmids and nationwide clonal dissemination of Klebsiella pneumoniae ST16 accompanied with regional disseminations of three distinctive K. pneumoniae clones (ST231, ST14, and ST147) with different plasmids. Dissemination of CRE carrying blaNDM in Thailand is mainly based on nationwide clonal expansions of E. coli ST410 carrying blaNDM-5 and K. pneumoniae ST16 carrying blaNDM-1, nationwide dissemination of two distinctive plasmids carrying blaNDM-1, and accumulation of clonal expansions in regional areas. Although the overuse of antibiotics can promote CRE dissemination, the limited variety of transmitters highlights the importance of preventing horizontal dissemination among patients.

Diagnostic Yield and Impact on Antimicrobial Management of 16S rRNA Testing of Clinical Specimens.

16S rRNA gene sequencing is increasingly used in clinical practice for bacterial identification of clinical specimens. However, studies on its applicability to direct clinical specimens are limited. Here, we studied the diagnostic yield and impact of 16S rRNA gene sequencing from direct clinical specimens on antimicrobial management. Adult inpatients whose attending physician requested 16S rRNA gene sequencing and corresponding bacterial culture from a direct clinical specimen between January and December 2021 in a university hospital were prospectively included in this study. A total of 434 specimens from 374 patients were requested. Of these, 253 (58.3%) specimens were collected from patients whose final diagnosis indicated a bacterial infection, whereas 181 (41.7%) specimens were from nonbacterial infections. Using the final diagnosis as a "gold standard," the sensitivity and specificity of 16S rRNA gene sequencing were 38.3% and 93.9%, respectively. Among the bacterial infection cases, the proportion of 16S rRNA gene sequencing-positive and culture-positive cases was 32.4%, and the proportion of sequencing-positive and culture-negative cases was 5.9%. The impact on antimicrobial management was evident in 10 (2.3%) specimens, which all resulted in the continuation of antibiotics. The impact on antimicrobial management was highest in skin and soft tissue infections, followed by bone and joint infections. In this study, the long turnaround time of 16S rRNA gene sequencing of clinical specimens was a limiting factor. In conclusion, the overall diagnostic yield of 16S rRNA gene sequencing in bacterial infection cases was fair, being useful in selected cases. Restrictions on test requests may improve test utilization in this setting. IMPORTANCE 16S rRNA gene sequencing has been increasingly used in clinical practice. Using the final diagnosis as a gold standard, the sensitivity of 16S rRNA gene sequencing was fair. In the setting with no 16S rRNA gene sequencing test ordering restrictions, only small percentages of the test results had an impact on antimicrobial management. Restrictions on test requests should be developed to maximize the benefit of the test.

Clonal dissemination of carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-232 in Thailand.

Background: Klebsiella pneumoniae ST258 and ST11 carrying bla KPC are among the most widespread carbapenem-resistant K. pneumoniae strains worldwide. Our carbapenem-resistant Enterobacteriaceae surveillance in Thailand revealed a nationwide dissemination of K. pneumoniae ST16 isolates carrying bla NDM-1 and bla OXA-232. Objectives: To analyse the genomic details of this nationwide dissemination by focusing on plasmids and virulence factors. Methods: Using WGS data of 119 K. pneumoniae ST16 isolates carrying bla NDM-1 obtained in our previous surveillance study, clonality of chromosomes and plasmids of the isolates with carriage of virulence factors was evaluated. Results: Of the 119 isolates, 111 carried plasmid pKP151_NDM1, and all 104 isolates harbouring bla OXA-232 carried plasmid pKP151_OXA232. These 104 K. pneumoniae ST16 isolates showing chromosomal clonality possessed both pKP151_NDM1 and pKP151_OXA232, demonstrating clonal dissemination of K. pneumoniae ST16 with these plasmids. The isolates had essentially similar virulence factors as those of K. pneumoniae ST16 clones carrying bla KPC, which were recently reported as highly invasive clones in Brazil. Conclusions: The potential global dissemination of these invasive clones with resistance to several antibiotics highlights the importance of appropriate monitoring and strict standard precautions.

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity.

Mycobacterium tuberculosis protein kinase B (PknB) is essential to mycobacterial growth and has received considerable attention as an attractive target for novel anti-tuberculosis drug development. Here, virtual screening, validated by biological assays, was applied to select candidate inhibitors of M. tuberculosis PknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitro biological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosis H37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 µg/mL, respectively. Two compounds, 2 and 10, inhibited M. tuberculosis PknB activity in vitro, with IC50 values of 14.4 and 12.1 µM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4 displayed anti-tuberculosis activity against M. tuberculosis H37Rv but showed no inhibition of PknB activity (IC50 > 128 µM). We hypothesize that hydrolysis of its ethyl ester to a carboxylate moiety generates an active species that inhibits other M. tuberculosis enzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10 bound to M. tuberculosis PknB indicated that compound 4 has a lower affinity for M. tuberculosis PknB than compounds 2 and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2 and 10 therefore represent candidate inhibitors of M. tuberculosis PknB that provide attractive starting templates for optimization as anti-tubercular agents.

Maternal septicemia caused by Streptococcus mitis: a possible link between intra-amniotic infection and periodontitis. Case report and literature review.

BACKGROUND: Intra-amniotic infection has a strong causal association with spontaneous preterm birth and preterm prelabor rupture of membranes (PPROM). The most common route of intra-amniotic infection is the ascending pathway in which microorganisms from the vagina gain access to the amniotic cavity. Distant microorganisms such as those from the oral cavity have been reported in intra-amniotic infection through hematogenous spreading. CASE PRESENTATION: A 31-year-old gravida 1, para 0 Thai woman at 33+6 weeks' gestation presented with leakage of vaginal fluid and irregular uterine contraction. She developed fever at 4 h after admission and was later diagnosed with acute chorioamnionitis. A Cesarean section was performed to terminate pregnancy. In addition to a blood culture, the cultures of amniotic fluid, vaginal and chorioamniotic membrane swabs were positive for Streptococcus mitis with identical susceptibility profiles. After the delivery and antibiotic prescription, oral examination showed dental caries and chronic periodontitis. CONCLUSIONS: This is the first case report demonstrating maternal septicemia and intra-amniotic infection caused by S. mitis which might be attributed to periodontitis in women presenting with preterm PROM. We highlighted the association of periodontal disease and preterm labor/PROM syndrome. Oral cavity examination should be included in the prenatal care to ensure good dental hygiene.

Identification of Potent DNA Gyrase Inhibitors Active against Mycobacterium tuberculosis.

Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 µg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 µM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Utility and Applicability of Rapid Diagnostic Testing in Antimicrobial Stewardship in the Asia-Pacific Region: A Delphi Consensus.

Rapid diagnostic tests (RDTs) facilitate fast and accurate identification of infectious disease microorganisms and are a valuable component of multimodal antimicrobial stewardship (AMS) programs but are currently underutilized in the Asia-Pacific region. An experienced group of infectious diseases clinicians, clinical microbiologists, and a clinical pharmacist used a modified Delphi consensus approach to construct 10 statements, aiming to optimize the utility and applicability of infection-related RDTs for AMS in the Asia-Pacific region. They provide guidance on definition, types, optimal deployment, measuring effectiveness, and overcoming key challenges. The Grading of Recommendations Assessment, Development, and Evaluation system was applied to indicate the strength of the recommendation and the quality of the underlying evidence. Given the diversity of the Asia-Pacific region, the trajectory of RDT development will vary widely; the collection of local data should be prioritized to allow realization and optimization of the full benefits of RDTs in AMS.

Rapid diagnostic testing for antimicrobial stewardship: Utility in Asia Pacific.

Rapid diagnostic testing (RDT) can provide prompt, accurate identification of infectious organisms and be a key component of antimicrobial stewardship (AMS) programs. However, their use is less widespread in Asia Pacific than western countries. Cost can be prohibitive, particularly in less resource-replete settings. A selective approach is required, possibly focusing on the initiation of antimicrobials, for differentiating bacterial versus viral infections and identifying locally relevant tropical diseases. Across Asia Pacific, more data are needed on RDT use within AMS, focusing on the impact on antimicrobial usage, patient morbidity and mortality, and cost effectiveness. Moreover, in the absence of formal guidelines, regional consensus statements to guide clinical practice are warranted. These will provide a regionally relevant definition for RDT; greater consensus on its role in managing infections; advice on implementation and overcoming barriers; and guidance on optimizing human resource capacity. By addressing these issues, the outcomes of AMS programs should improve.

Rapid detection of multidrug-resistant tuberculosis based on allele-specific recombinase polymerase amplification and colorimetric detection.

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.

Predictive Value of Urinalysis and Recent Antibiotic Exposure to Distinguish Between Bacteriuria, Candiduria, and No-Growth Urine.

PURPOSE: Urinary tract infections are diagnosed by clinical symptoms and detection of causative uropathogen. Antibiotics are usually not indicated in candiduria and no-growth urine. We aimed to develop a predictive score to distinguish bacteriuria, candiduria, and no-growth urine, and to describe the distribution of microorganisms in urine. PATIENTS AND METHODS: A single-center, retrospective cohort study was conducted between January 2017 and November 2017. Patients with concomitant urinalysis and urine culture were randomly sorted for a clinical prediction model. Multivariable regression analysis was performed to determine factors associated with bacteriuria, candiduria, and no-growth urine. A scoring system was constructed by rounding the regression coefficient for each predictor to integers. Accuracy of the score was measured by the concordance index (c-index). RESULTS: There were 8091 positive urine cultures: bacteria 85.6%, Candida 13.7%. Randomly selected cases were sorted into derivation and validation cohorts (448 cases and 272 cases, respectively). Numerous yeast on urinalysis predicted candiduria with complete accuracy; therefore, it was excluded from a score construction. We developed a NABY score based on: positive nitrite, 1 point; Antibiotic exposure within 30 days, -2 points; numerous Bacteria in urine, 2 points; few Yeast in urine, -2 points; moderate Yeast in urine, -5 points. The c-index was 0.85 (derivation) and 0.82 (validation). A score ≥0 predicted 76% and 54% of bacteriuria in the derivation and validation cohorts, respectively. A score ≤-3 predicted 96% of candiduria in both cohorts. CONCLUSION: Numerous yeast on urinalysis and the NABY score may help identify patients with a low risk of bacteriuria in whom empiric antibiotics for UTIs can be avoided.

Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice.

BACKGROUND: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. METHODS: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. RESULTS: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. CONCLUSIONS: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.