Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection

Background Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005–2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method. Results Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04). Conclusions Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections.

Positive correlation between the number of bacteria in soybean tempeh with the bioactivity of its extract against enterotoxigenic Escherichia coli (ETEC) adhesion to eukaryotic cells

Bioactive oligosaccharides from soybean tempeh can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells, thus reducing the severity of ETEC-mediated diarrhea. Bacteria are also present in tempeh but there has yet been any report regarding their effect on the anti-adhesion bioactivity of tempeh. In this research, the bacterial number in tempeh was quantified and the anti-adhesion bioactivity of tempeh extract was determined using yeast agglutination assay. Statistical analysis showed a moderately (R = 0.69) significant positive correlation (P < 0.01) between the number of bacteria in tempeh and the anti-adhesion bioactivity of its extract. In conclusion, tempeh that contains more bacteria is more effective in inhibiting ETEC adhesion to eukaryotic cells. This could be due to a symbiosis between Rhizopus and bacteria in breaking down soy matrix polysaccharides to release bioactive oligosaccharides.

Erytra Blood Group Analyser and Kode Technology testing of SARS-CoV-2 antibodies among convalescent patients and vaccinated individuals

SummarySurveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires tests to monitor antibody formation and prevalence. We detected SARS-CoV-2 antibodies using red cells coated by Kode technology with short peptides derived from the SARS-CoV-2 spike protein. Such modified red cells, called C19-kodecytes, can be used as reagent cells in any manual or automated column agglutination assay. We investigated the presence of SARS-CoV-2 antibodies in 130 samples from COVID-19 convalescent plasma donors using standard manual technique, two FDA authorized ELISA assays and a virus neutralisation assay. The sensitivity of the C19-kodecyte assay was 88%, comparable to the anti-SP and anti-NCP ELISAs (86% and 83%) and the virus neutralisation assay (88%). The specificity of the C19-kodecyte assay was 90% (anti-SP 100% and anti-NCP 97%). Likewise, 231 samples from 73 vaccinated individuals were tested with an automated analyser and we monitored the appearance and persistence of SARS-CoV-2 antibodies. The C19-kodecyte assay is a robust tool for SARS-CoV-2 antibody detection. Automated blood group analyser use enables large-scale SARS-CoV-2 antibody testing for vaccination monitoring in population surveys.

Performance of a New Microfluidic Dengue NS1 Immuno-magnetic Agglutination Assay for the Rapid Diagnosis of Dengue Infection in Adults

Dengue (DENV) infections are a public health concern worldwide and thus early diagnosis is important to ensure appropriate clinical management. The rapid diagnostic test (RDT) targets nonstructural protein 1 (NS1) detection and is the main tool used for diagnostic purpose. In this study, we evaluated the performance of a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay or IMA (ViroTrack Dengue Acute, BluSense Diagnostics, Copenhagen, Denmark). We studied 233 subjects confirmed to have DENV infection (by a real-time reverse transcriptase polymerase chain reaction) and 200 control samples were taken from patients with confirmed diagnoses of other febrile illnesses, in Thailand. Samples were tested using the NS1 antigen (Ag) detection methods: in-house NS1 Ag ELISA (ELISA), SD BIOLINE Dengue NS1 Ag RDT (ICT), and ViroTrack Dengue Acute (IMA). Sensitivities of these tests were 86.3%, 78.9%, and 85.5%, respectively. All tests showed high specificity (100%, 99%, and 97% for ELISA, ICT, and IMA, respectively). The sensitivities of both RDTs were affected by the low sensitivity to DENV-2 and DENV-4. NS1 Ag was detected in every patient on day 1 and day 2 after onset of illness by ELISA and IMA with a decline in detection rates over time after day 6 of illness. NS1 detection rate using ICT decreased from 100% on day 1 of illness to 98.6% on day 2 after onset of illness. By day 6, the detection rate was 45.9%. Thus, IMA performed better than ICT for early and rapid diagnosis of DENV infections in endemic countries.

Development of a Bacteriophage Tail Fiber-Based Latex Agglutination Assay for Rapid Clinical Screening of Burkholderia pseudomallei

Melioidosis is a life-threatening disease in humans caused by the Gram- negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 hours, a rapid diagnosis of melioidosis is critical for ensuring an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and whole genome sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an endemic area of melioidosis over 11 months. The 94TF-LAA took less than 5 minutes to produce positive agglutination, demonstrating 98% (95% CI; 94.2%−99.59%) sensitivity and 83% (95% CI; 75.64%−88.35%) specificity when compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring optimal antibiotic courses are prescribed to patients, and thus warrants the development of cost-effective and easy-to-use tests for implementation in under-resourced areas such as Northeast Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust, easy to manufacture, and critically many tail fibers (such as 94TF investigated here) can target a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many endemic areas of meilodosis.

Quantitative particle agglutination assay for point-of-care testing using mobile holographic imaging and deep learning

Particle agglutination assays are widely adapted immunological tests that are based on antigen-antibody interactions. Antibody-coated microscopic particles are mixed with a test sample that potentially contains the target antigen, as...

Rapid and accurate point-of-care testing for SARS-CoV2 antibodies

ABSTRACTThe COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has grown into worst public health crisis since the 1918 influenza pandemic. As COVID-19 continues to spread around the world, there is urgent need for a rapid, yet accurate antibody test to identify infected individuals in populations to inform health decisions. We have developed a rapid, accurate and cost-effective serologic test based on antibody-dependent agglutination of antigen-coated latex particles, which uses ∼5 µl plasma and takes <5 min to complete with no instrument required. The simplicity of this test makes it ideal for point-of-care (POC) use at the community level. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid (N) protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin converting enzyme 2 (ACE2) in a surrogate neutralization assay, suggesting that the agglutination assay may be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of positive viral RNA test, suggesting that seroconversion may occur earlier than previously thought and that the agglutination antibody test may complement RNA testing for POC diagnosis of SARS-CoV-2 infection.