Genomic and Phenotypic Analysis of bla KPC-2 Associated Carbapenem Resistance in Klebsiella aerogenes: Insights into Clonal Spread and Resistance Mechanisms Across Hospital Departments in Beijing.

Purpose: This study conducted an phenotypic and whole-genome sequencing analysis with Klebsiella aerogenes to elucidate its clinical epidemiological characteristics, antimicrobial resistance (AMR) phenotype, biofilm formation ability and hemolytic activity testing, AMR genes and phylogenetic relationships, so as to provide a further understanding of the intra-hospital strain transmission. Methods: Samples were collected from a hospital in Beijing between 2020 and 2022. All strains underwent bacterial identification, antimicrobial susceptibility testing (AST) using the VITEK-2 compact system. Biofilm formation ability and hemolytic activity were tested. Second-generation sequencing was applied to all strains, with those carrying the bla KPC gene were selected for third-generation sequencing. Whole-genome analysis identified resistance genes, plasmid types, MLST typing, and phylogenetic relationships. Plasmids were assembled to detect plasmid structures and AMR gene location. Results: Among the 42 K. aerogenes isolates, 21 were carbapenem-resistant K. aerogenes (CRKA). All strains exhibited strong biofilm formation and no hemolytic activity. Most were sourced from sputum (83.3%). CRKA demonstrated extensive resistance to antibiotics, particularly ß-lactamase inhibitors and Cefotetan. This resistance pattern was closely associated with the presence of an IncFII(pHN7A8) plasmid, which carried multiple resistance genes, including bla KPC-2, bla CTX-M-65, bla TEM-1, rmtB and a large number of mobile elements. The majority of CRKA strains clustered within the same branch of the phylogenetic tree, exhibiting minimal single nucleotide polymorphism (0-13 SNPs) differences, and they shared the same sequence type (ST292), resistance genes, and plasmids, originating from different departments, suggesting clonal transmission among the hospital. Conclusion: Our research reveals that the clonal transmission of CRKA occurs across various departments within the hospital. The widespread resistance observed in CRKA, attributed to the presence of bla KPC and ESBLs genes, underscores the need for heightened vigilance to prevent the further dissemination of CRKA within the hospital and, potentially, throughout the wider community.

Genomic insights and antimicrobial resistance profiles of CRKP and non-CRKP isolates in a Beijing geriatric medical center: emphasizing the blaKPC-2 carrying high-risk clones and their spread.

Background: The escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably ß-lactams, constitutes a formidable challenge for healthcare and global public health management. Methods: This research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features. Results: The study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments. Conclusion: In summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.

Contamination dynamics of personal protective equipment (PPE) by SARS-CoV-2 RNA in a makeshift hospital with COVID-19 positive occupants.

Background: Personal protective equipment (PPE) helps protect healthcare workers (HCWs) from infection and prevents cross-contamination. Knowledge of the contamination dynamics of PPE during the management of COVID-19 patients in a makeshift hospital is limited. Aim: To describe the rate of SARS-CoV-2 contamination in PPE and to assess the change of contamination at different time points. Methods: HCWs were followed up for up to 4 hours with hourly collection of swab samples from PPE surfaces in a makeshift COVID-19 hospital setting. Swabs were tested using quantitative reverse transcription polymerase chain reaction (RT-qPCR) for SARS-CoV-2 RNA. Results: SARS-CoV-2 was detected on 50.9% of the 1620 swabbed samples from 9 different sites of full-body PPE worn by HCWs. The proportion of sites contaminated with SARS-CoV-2 RNA varied from 10.6% to 95.6%. Viral RNA was most frequently detected from the sole of the outer foot cover (95.6%) and least frequently on the face shield (10.6%). The median Ct values among positive samples were 34.20 (IQR, 32.61-35.22) and 34.05 (IQR, 32.20-35.39) for ORF1ab and N genes, respectively. The highest rate of contamination with SARS-CoV-2 RNA for the PPE swab samples was found after 3 hours of use. The positive rate of outer surface of HEPA filters from air supply device was 82.1% during the full capacity period of the makeshift hospital. Conclusion: A higher rate of contamination was identified at 3 hours after the entrance to the COVID-19 patient care area. Virus-containing aerosols were trapped in the HEPA filter of air supply equipment, representing a potential protective factor against infection to HCWs.

Monitoring Prevalence and Persistence of Environmental Contamination by SARS-CoV-2 RNA in a Makeshift Hospital for Asymptomatic and Very Mild COVID-19 Patients.

Objective: To investigate the details of environmental contamination status by SARS-CoV-2 in a makeshift COVID-19 hospital. Methods: Environmental samples were collected from a makeshift hospital. The extent of contamination was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for SARS-CoV-2 RNA from various samples. Results: There was a wide range of total collected samples contaminated with SARS-CoV-2 RNA, ranging from 8.47% to 100%. Results revealed that 70.00% of sewage from the bathroom and 48.19% of air samples were positive. The highest rate of contamination was found from the no-touch surfaces (73.07%) and the lowest from frequently touched surfaces (33.40%). The most contaminated objects were the top surfaces of patient cubic partitions (100%). The median Ct values among strongly positive samples were 33.38 (IQR, 31.69-35.07) and 33.24 (IQR, 31.33-34.34) for ORF1ab and N genes, respectively. SARS-CoV-2 relic RNA can be detected on indoor surfaces for up to 20 days. Conclusion: The findings show a higher prevalence and persistence in detecting the presence of SARS-CoV-2 in the makeshift COVID-19 hospital setting. The contamination mode of droplet deposition may be more common than contaminated touches.

Epidemiological Characteristics and Drug Resistance of Fungemia in General Hospitals from 2010 to 2019.

OBJECTIVE: This study intends to analyze the data of fungemia in a large tertiary hospital from 2010 to 2019, and is aimed at understanding its epidemic characteristics and drug resistance. METHODS: The "Hospital Infection Real-Time Monitoring System" was used to retrieve the case information of patients who were hospitalized for more than 48 hours from 2010 to 2019. The questionnaire was designed to collect patients' basic information, infection situation, drug resistance, and other related information. Statistical software was used for analysis. RESULTS: The fungi detection rate was in the range of 0.19%~0.75% in ten years, the average rate was 0.29%, and the rate 0.2%~0.3% since 2013, which was lower than that from 2010 to 2012. Non-Candida albicans was the main fungus, accounting for 62.50%. The drug resistance of non-C. albicans was higher than that of C. albicans, among which C. glabrata had the highest resistance rate. Data analysis showed that the patients with more serious basic diseases, combined with infection of other sites, surgery, long hospital stay, combination of antibiotics, and invasive catheterization, were more likely to occur fungemia. CONCLUSION: We should pay more attention to the patients with high-risk factors of fungemia and focus on the drug resistance of non-C. albicans, choose the right antifungal drugs, so as to improve the level of diagnosis and treatment.

Prospective investigation of carbapenem-resistant Klebsiella pneumonia transmission among the staff, environment and patients in five major intensive care units, Beijing.

BACKGROUND: Following the alarming outbreak of carbapenem-resistant Klebsiella pneumonia (CRKP) in five intensive care units (ICUs) of a tertiary care hospital in China, a prospective investigation of CRKP colonized/infected patients was conducted. AIM: To describe the diffusion and transmission of CRKP among epidemiologically linked ICU patients, staff and environment. METHODS: Enhanced CRKP infected/colonized case monitoring was performed by the real-time nosocomial infection surveillance system (RT-NISS). The immediate surroundings of each CRKP patient bed unit and the staff hands/gloves/gowns were sampled and then evaluated for the presence of CRKP. Antimicrobial susceptibility tests, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) were used to identify and to characterize these isolates. FINDINGS: Among 2750 patients monitored, 67 CRKP patients were newly labeled and 11 patients' CRKP isolates were available. A total of 31.34% (21/67) bed units were positive at one or more surrounding surfaces, 7.99% (49/613) environmental samples and 3.57% (4/112) ICU staff samples were CRKP positive. The selected CRKP isolates (N = 64) exhibited intermediate to high resistance levels to the antibiotics tested apart from colistin and tigecycline. RT-NISS data combined with MLST and PFGE revealed nine likely transmission clusters. WGS analysis of these CRKP isolates revealed extensive sharing of multiple antimicrobial resistance genes and plasmid replicons among these isolates. Two carbapenemase genes blaKPC-2 (62/64) and blaOXA-48 (2/64) were identified. These CRKP isolates carried one or more plasmid replicons. CONCLUSIONS: The contamination of ICU environment and staff's hands, gloves or gowns is frequent with CRKP patients. Our study also supports the hypothesis that an association between environmental contamination and transmission of CRKP bacteria in ICUs.

Complete genome analysis of three Acinetobacter baumannii clinical isolates in China for insight into the diversification of drug resistance elements.

BACKGROUND: The emergence and rapid spreading of multidrug-resistant Acinetobacter baumannii strains has become a major health threat worldwide. To better understand the genetic recombination related with the acquisition of drug-resistant elements during bacterial infection, we performed complete genome analysis on three newly isolated multidrug-resistant A. baumannii strains from Beijing using next-generation sequencing technology. METHODOLOGIES/PRINCIPAL FINDINGS: Whole genome comparison revealed that all 3 strains share some common drug resistant elements including carbapenem-resistant bla OXA-23 and tetracycline (tet) resistance islands, but the genome structures are diversified among strains. Various genomic islands intersperse on the genome with transposons and insertions, reflecting the recombination flexibility during the acquisition of the resistant elements. The blood-isolated BJAB07104 and ascites-isolated BJAB0868 exhibit high similarity on their genome structure with most of the global clone II strains, suggesting these two strains belong to the dominant outbreak strains prevalent worldwide. A large resistance island (RI) of about 121-kb, carrying a cluster of resistance-related genes, was inserted into the ATPase gene on BJAB07104 and BJAB0868 genomes. A 78-kb insertion element carrying tra-locus and bla OXA-23 island, can be either inserted into one of the tniB gene in the 121-kb RI on the chromosome, or transformed to conjugative plasmid in the two BJAB strains. The third strains of this study, BJAB0715, which was isolated from spinal fluid, exhibit much more divergence compared with above two strains. It harbors multiple drug-resistance elements including a truncated AbaR-22-like RI on its genome. One of the unique features of this strain is that it carries both bla OXA-23 and bla OXA-58 genes on its genome. Besides, an Acinetobacter lwoffii adeABC efflux element was found inserted into the ATPase position in BJAB0715. CONCLUSIONS: Our comparative analysis on currently completed Acinetobacter baumannii genomes revealed extensive and dynamic genome organizations, which may facilitate the bacteria to acquire drug-resistance elements into their genomes.

Clinical and microbiological characterization of Staphylococcus lugdunensis isolates obtained from clinical specimens in a hospital in China.

BACKGROUND: Several reports have associated Staphylococcus lugdunensis with the incidence of severe infection in humans; however, the frequency and prevalence of this microorganism and thus the propensity of its antimicrobial drug resistance is unknown in China. The objective of the current study was to determine the prevalence of Staphylococcus lugdunensis among six hundred and seventy non-replicate coagulase negative Staphylococcus (CoNS) isolates collected in a 12-month period from clinical specimens in the General Hospital of the People's Liberation Army in Beijing, China. RESULTS: Five (0.7%) of the 670 isolates of CoNS were identified as S. lugdunensis. Whereas three isolates were resistant to erythromycin, clindamycin, and penicillin and carried the ermC gene and a fourth one was resistant to cefoxitin and penicillin and carried the mecA gene, one isolate was not resistant to any of the tested antimicrobials. Pulse field gel electrophoretic analysis did not reveal widespread epidemiological diversity of the different isolates. CONCLUSION: Hence, even though S. lugdunensis may be yet unrecognized and undefined in China, it still might be the infrequent cause of infection and profound multi-drug resistance in the same population.

Nosocomial bloodstream infection in patients caused by Staphylococcus aureus: drug susceptibility, outcome, and risk factors for hospital mortality.

BACKGROUND: Previous studies have different viewpoints about the clinical impact of methicillin resistance on mortality of hospital-acquired bloodstream infection (BSI) patients with Staphylococcus aureus (S. aureus). The objective of this study was to investigate the mortality of hospital-acquired BSI with S. aureus in a military hospital and analyze the risk factors for the hospital mortality. METHODS: A retrospective cohort study was performed in patients admitted to the biggest military tertiary teaching hospital in China between January 2006 and May 2011. All included patients had clinically significant nosocomial BSI with S. aureus. Multivariate Logistic regression analysis was used to identify the risk factors for hospital mortality of patients with S. aureus BSI. RESULTS: One hundred and eighteen patients of more than one year old were identified as clinically and microbiologically confirmed nosocomial bacteraemia due to S. aureus, and 75 out of 118 patients were infected with methicillin-resistant S. aureus (MRSA). The overall mortality of nosocomial S. aureus BSI was 28.0%. Methicillin resistance in S. aureus bacteremia was associated with significant increase in the length of hospitalization and high proportion of inappropriate empirical antibiotic treatment. After Logistic regression analysis, the severity of clinical manifestations (APACHE II score) (odds ratio (OR) 1.22, 95% confidence interval (CI) 1.12 - 1.34) and inadequacy of empirical antimicrobial therapy (OR 0.25, 95%CI 0.09 - 0.69) remained as risk factors for hospital mortality. CONCLUSIONS: Nosocomial S. aureus BSI was associated with high in-hospital mortality. Methicillin resistance in S. aureus has no significant impact on the outcome of patients with staphylococcal bacteremia. Proper empirical antimicrobial therapy is very important to the prognosis.

Viral shedding in Chinese young adults with mild 2009 H1N1 influenza.

BACKGROUND: The duration of viral shedding and the transmission of 2009 H1N1 influenza among individuals, especially among the younger population with mild illness, are not well understood now. The aim of this study was to determine the viral shedding of the young adult patients with mild 2009 H1N1 influenza in China. METHODS: From September 2009 to January 2010, the clinical data and serial nasopharyngeal swabs of 67 patients with 2009 H1N1 influenza and 37 patients with seasonal influenza aged from 18 years to 35 years were collected. The nasopharyngeal swab samples were detected by real time RT-PCR to determine the viral shedding. All the patients did not receive the antiviral therapy but Chinese medicine for detoxicating. RESULTS: Among the patients with H1N1 virus infection, 82.1% (55/67) patients presented with fever symptom, while more patients with high fever (≥ 39°C) were found in seasonal influenza patients (P < 0.05). For the H1N1 patients, the median interval between the symptom onset and the undetectable RNA was six days (4 - 10 days). But viral shedding was still found in 31.3% patients after 7 days following illness onset. The median interval between disappearance of fever and an undetectable viral RNA level was three days (2 - 8 days), and 17.9% patients were found to be viral shedding 6 days later after normalization of body temperature. For the seasonal influenza patients, 94.6% patients were detected out viral RNA within 7 days. The median interval of seasonal influenza between the symptom onset and the undetectable RNA was four days (3 - 8 days). The median interval between disappearance of fever and an undetectable viral RNA level was three days (2 - 6 days). CONCLUSION: It suggests that 7 days isolation period from the illness onset or 24 hours after the resolution of fever and respiratory symptoms are not long enough to cut off the transmission among Chinese young adults with mild illness.

[Colloidal gold and dot-ELISA rapid tests for screening influenza A virus].

OBJECTIVE: To evaluate the diagnostic efficiency of colloidal gold and dot ELISA rapid tests in clinical screening of influenza A virus. METHODS: The pharyngeal swabs were collected from 297 outpatients suspected of influenza between June and October, 2009 for detection with colloid gold and dot ELISA rapid test, with real-time PCR as the golden methods. The discrepant results of colloid gold and dot ELISA methods were confirmed by sequencing, and the diagnostic efficiency of the two methods was evaluated. RESULTS: Among the 166 samples with influenza A virus infection as confirmed by real-time PCR and sequencing, the diagnostic sensitivity of dot ELISA and colloid gold methods was 54.82% (91/166) and 4.22% (7/166), respectively. The total concordance rate with PCR was 66.67% (Kappa value of 0.35). Among the 133 samples negative for influenza A virus, the specificity of dot ELISA and colloid gold methods was 81.68% (107/131) and 98.47% (129/131), respectively, with a total concordance rate with PCR of 45.79% (Kappa value 0.02). Of the 99 H1N1 influenza samples confirmed by real-time PCR, the detection rate of dot ELISA was 67.3%, whereas that of colloid gold was 5.1%. Out of the 107 dot ELISA-positive but colloid gold-negative samples, 84 were confirmed to be influenza A virus-positive by real-time PCR and sequencing. One sample negative for dot ELISA but positive for colloid gold test was confirmed to be influenza A virus-negative. The detection rate and diagnostic concordance rate for influenza A virus by dot ELISA were significantly higher than those of colloid gold (P<0.05). CONCLUSION: Dot ELISA is better than colloid gold in influenza A virus detection and shows great prospect in clinical screening.

[Genotyping on 47 Staphylococcus aureus strains associated with bloodstream infection.].

OBJECTIVE: To investigate the molecular epidemiological characteristics of Staphylococcus aureus associated with bloodstream infection in hospital. METHODS: 47 Staphylococcus aureus strains isolated from bloodstream in PLA General Hospital were collected from January 2006 to December 2008. Susceptibility of the strains to 11 antimicrobial agents was detected and DNA homology of them was analyzed with Rep-based DiversiLab(TM) Microbial Typing System. Panton-Valentine leukocidin (PVL) gene was determined by PCR. For methicillin-resistant Staphylococcus aureus (MRSA) strains, the genotypes of SCCmec were determined and ST239 clone was screened with multiplex PCR. Multilocus sequence typing (MLST) was used to determine the STs of the selected isolates. RESULTS: In the 47 Staphylococcus aureus isolated from blood, methicillin-resistant strains accounted for 51.1%, all belonged to SCCmec III type, with only 2 pvl gene positive strains identified. 12 different patterns (A-L) were found among 47 strains with Rep-PCR. All MRSA strains clustered in the A and B subtypes. CONCLUSION: Most MRSA strains isolated from blood in PLA General Hospital belonged to ST239-MRSA-SCCmec III clone. DiversiLab(TM) Microbial Typing System could provide a rapid and effective method to investigate the molecular epidemiological characteristics of Staphylococcus aureus in the hospital settings.

Susceptibility patterns and molecular epidemiology of multidrug-resistant Acinetobacter baumannii strains from three military hospitals in China.

To date, little has been reported on the susceptibility patterns and molecular characterisation of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates from different Chinese military hospitals. In this study, 49 MDRAB strains were collected from three military hospitals during 2007. The minimum inhibitory concentrations (MICs) of 13 antibiotics were determined for each strain. Genotyping and dendrogram analysis of MDRAB strains were performed using the repetitive sequence-based polymerase chain reaction (rep-PCR) DiversiLab Microbial Typing System. PCR screening was carried out to investigate the distribution of various genes contributing to each resistance phenotype in the main clonal types. The rates of resistance to the majority of antibiotics tested varied between 75.5% and 100%, with the exception of polymyxin B. Two DiversiLab rep-PCR clones (A and B) were widespread in three hospitals in different cities, one clone (D) existed only in two hospitals located in the same city (Beijing), and the other two clones (C and E) were present in only one hospital. In addition, this study shows a high distribution of intI1, ISAba1, bla(OXA-23), bla(ADC), adeB, adeJ, abeM and tet(B) genes, which mediate resistance to structurally unrelated antimicrobials in MDRAB isolates. These results suggest that all isolates were resistant to at least three classes of antibiotics. In addition, clonal dissemination among the three hospitals located in two different cities in China, previously documented in many regions of Europe and Asia-Pacific nations, emphasises the epidemic potential of these MDRAB isolates.

[Study on the molecular characteristics of multidrug-resistant Acinetobacter baumannii].

OBJECTIVE: To investigate antibiotic resistance, carbapenemase genotype and the molecular epidemiology of multidrug-resistant Acinetobacter baumannii (Aba) collected from 3 military hospitals in China. METHODS: The minimum inhibitory concentrations (MIC) were examined by ager dilution method. Genotypes of carbapenemases were amplified by multiplex PCR and its products were sequenced. PCR was used to detect per gene. Homology of the resistant isolates was analyzed by pulse-field gel electrophoresis (PFGE). RESULTS: Among the 64 MDRA strains, 78.1% (50) strains possessed bla(OXA-23) gene, 89.1% (57) carried Class 1 integrase gene, 39.1% (25) with bla(PER-1) gene, and 1 strain with bla(OXA-58-like) gene. PFGE showed that 13 (A, B, C, D, E genotype) different clones were identified in these strains. A, B, and U clones were the predominant clones in three hospitals, respectitively. CONCLUSION: Outbreaks of multidrug-resistant Aba occurred at 3 military hospitals with the most prevalent carbapenemase as OXA-23 enzyme. OXA-58 type of carbapenemase and per-1 in Aba were also isolated.

[Transmission and molecular characteristics of carbapenem-resistant Acinetobacter baumannii].

OBJECTIVE: To study the mode of transmission and molecular characteristics on carbapenem-resistant Acinetobacter baumannii strain. Strains were isolated from different parts of samples in various patients. METHODS: Clinical information of carbapenem-resistant Acinetobacter baumannii isolates were stored and analyzed by WHONET 5.4 software. The transmission and pathopoiesis of the strains were learned through case file review. Genotypes of isolates were identified by pulse-field gel electrophoresis (PFGE) and genes of carbapenemase were detected by multiple PCR, in order to find molecular characteristics and relatedness between strains. RESULTS: 29 stains of Acinetobacter baumannii resistant to carbapenem were isolated from 2 or more kinds of samples among 13 patients'. Two genotypes were identified by PFGE: genotype A was obtained from 22 isolates in 11 patients and genotype B was obtained from 7 isolates in 4 patients. PCR amplification showed that all strains possessed OXA-23 gene except 1, and all strains possessed Integrase gene I except 3. CONCLUSION: There were 2 different genotypes from 29 strains of carbapenem-resistant Acinetobacter baumannii with Genotype A as the main type. OXA-23 carbapenemase gene and integrase gene I were detected from most of the isolates. All the strains could be easily transmitted in the body of the patients and among patients, hence becoming the epidemic pathogen of iatrogenic infection.

Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor.

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V T and V C stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V T/V C is directly proportional to the number of Y. pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y. pestis above the detection limit, which was approximately 104 CFU/ml. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry.