Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Rapid tests should be used with caution for HIV-1 primary infection screening.

Rapid tests allow outpatient, low cost, reliable, screening for chronic HIV infection. However, data regarding their sensitivity on primary infection remain scarce. The objective of this study was to assess sensitivity of nine HIV rapid tests for primary HIV-1 infection screening. Seventy-five serum samples from patients during HIV-1 primary infection were included. Primary infection was diagnosed by a positive 4th generation ELISA and HIV-1 RNA positivity confirmed by Western blot patterns associated with HIV-1 primary infection. Early seroconversion was defined as the absence of antibodies on HIV-1 Western blot associated with HIV-1 RNA and p24-antigen positivity. An identical sensitivity (95% CI) of 76.7% (65.2-84.2%) was observed for HIV 1/2 STAT-PAK® Assay (STAT-PAK), INSTI™ HIV-1/HIV-2 antibody Test (INSTI), SURE CHECK® HIV 1/2 (SURE CHECK) and MULTISURE HIV rapid test (MULTISURE) with visual reading. Sensitivity was 74.7% (63.8-83.1%) for MULTISURE (automatic reading), 77.0% (66.3-85.1%) for FIRST RESPONSE® Test VIH 1-2.O CARTE (FIRST RESPONSE), 83.8% (73.8-90.5%) for VIKIA HIV1/2® (VIKIA), 88.0% (78.7-93.6%) for Genie™ Fast HIV 1/2 (Genie Fast), 88.6% (79.0-94.1%) for Hexagon HIV (Hexagon), and 92.8% (83.6-96.3%) for Exacto® TEST HIV Pro (Exacto). However, rapid tests performed poorly for the early seroconversion subgroup (n = 14), with sensitivities ranging from 7% (1.3-31.5%) for STAT-PAK, INSTI, SURE CHECK, MULTISURE (automatic reading), to 29% (12-55%) for FIRST RESPONSE, 31% (13-58%) for VIKIA, 43% (21-67%) for Hexagon and 57.1% (32.6-78.6%) for Exacto and Genie Fast. Overall, despite significant discrepancies in sensitivity, HIV rapid tests should be used with caution in the context of a suspected primary infection.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Development of a new accurate lateral flow immunoassay for diagnosis of human leptospirosis.

PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

(Un)Reliable detection of menstrual blood in forensic casework - evaluation of the Seratec® PMB test with mock samples.

The identification of the type of body fluid in crime scene evidence may be crucial, so that the efforts are high to reduce the complexity of these analyses and to minimize time and costs. Reliable immunochromatographic rapid tests for specific and sensitive identification of blood, saliva, urine and sperm secretions are already routinely used in forensic genetics. The recently introduced Seratec® PMB test is said to detect not only hemoglobin, but also differentiate menstrual blood from other secretions containing blood (cells) by detecting D-dimers. In our experimental set-up, menstrual blood could be reliably detected in mock forensic samples. Here, the result was independent of sample age and extraction buffer volume. It was also successfully demonstrated that all secretions without blood cells were negative for both, hemoglobin (P) and D-dimer (M). However, several blood cell-containing secretions/tissues comprising blood (injury), nasal blood, postmortem blood and wound crust also demonstrated positive results for D-dimer (M) and were therefore false positives. For blood (injury) and nasal blood, this result was reproduced for different extraction buffer volumes. The results of this study clearly demonstrate that the Seratec® PMB test is neither useful nor suitable for use in forensic genetics because of the great risk of false positive results which can lead to false conclusions, especially in sexual offense or violent acts.

Rapid screening of positive blood cultures for extended-spectrum ß-lactamases and metallo-ß-lactamases using a drug susceptibility testing microfluidic method.

OBJECTIVES: An increasing number of drug-resistant bacteria have been identified recently. In particular, drug-resistant bacteria have been linked to unfavorable prognoses in patients with bacteremia, highlighting the need for rapid testing. Our previous studies have focused on the utility of a drug susceptibility testing microfluidic (DSTM) method using microfluidic channels. A system with this DSTM method for screening for ß-lactamases can rapidly detect extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs). In this study, we have evaluated the clinical utility of pre-treatment for screening positive blood cultures using the DSTM method. METHODS: A total of 178 positive blood cultures and five simulated samples of MBL-producing bacteria were prepared at Kochi University Hospital, Japan. The pretreatment consisted of a two-step centrifugation. The obtained sediments were screened with the DSTM method for the production of ß-lactamase based on morphological changes in the bacteria after 3 h of incubation. RESULTS: The pretreatment functioned properly for all samples. Of the 25 ESBL samples, 21 were positive for ESBLs. Four false-negative samples, all obtained from the same patient, contained CTX-M-2 enzyme-producing Proteus mirabilis and showed insusceptibility to an ESBL inhibitor. The simulated samples prepared for MBL screening were positive for MBLs. CONCLUSIONS: When combined with a method for rapidly identifying bacterial species, DSTM may enable patients with bloodstream infections to start receiving appropriate treatment within 4 h after positive blood cultures are screened.

Fish DNA Sensors for Authenticity Assessment-Application to Sardine Species Identification.

Food and fish adulteration is a major public concern worldwide. Apart from economic fraud, health issues are in the forefront mainly due to severe allergies. Sardines are one of the most vulnerable-to-adulteration fish species due to their high nutritional value. Adulteration comprises the substitution of one fish species with similar species of lower nutritional value and lower cost. The detection of adulteration, especially in processed fish products, is very challenging because the morphological characteristics of the tissues change, making identification by the naked eye very difficult. Therefore, new analytical methods and (bio)sensors that provide fast analysis with high specificity, especially between closely related fish species, are in high demand. DNA-based methods are considered as important analytical tools for food adulteration detection. In this context, we report the first DNA sensors for sardine species identification. The sensing principle involves species recognition, via short hybridization of PCR-amplified sequences with specific probes, capture in the test zone of the sensor, and detection by the naked eye using gold nanoparticles as reporters; thus, avoiding the need for expensive instruments. As low as 5% adulteration of Sardina pilchardus with Sardinella aurita was detected with high reproducibility in the processed mixtures simulating canned fish products.

Probable Transmission of SARS-CoV-2 from African Lion to Zoo Employees, Indiana, USA, 2021.

We describe animal-to-human transmission of SARS-CoV-2 in a zoo setting in Indiana, USA. A vaccinated African lion with physical limitations requiring hand feeding tested positive for SARS-CoV-2 after onset of respiratory signs. Zoo employees were screened, monitored prospectively for onset of symptoms, then rescreened as indicated; results were confirmed by using reverse transcription PCR and whole-genome virus sequencing when possible. Traceback investigation narrowed the source of infection to 1 of 6 persons. Three exposed employees subsequently had onset of symptoms, 2 with viral genomes identical to the lion's. Forward contact tracing investigation confirmed probable lion-to-human transmission. Close contact with large cats is a risk factor for bidirectional zoonotic SARS-CoV-2 transmission that should be considered when occupational health and biosecurity practices at zoos are designed and implemented. SARS-CoV-2 rapid testing and detection methods for big cats and other susceptible animals should be developed and validated to enable timely implementation of One Health investigations.

Rapid detection of cefiderocol susceptibility/resistance in Acinetobacter baumannii.

PURPOSE: Due to its ability to disseminate worldwide and its multiple resistance trait, Acinetobacter baumannii is becoming a threat for public health worldwide. Cefiderocol (FDC) is a promising broad-spectrum cephalosporin recently approved for treating Gram-negative infection. The aim of this study was to develop a rapid test, namely the rapid FDC Acinetobacter baumannii NP test, for the detection of FDC susceptibility/resistance in A. baumannii since the current FDC susceptibility tests are rather time-consuming (at least 24 h). MATERIALS AND METHODS: The rapid test is based on the reduction of resazurin to resorufin product by bacterial viable cells, thus detecting bacterial growth in the presence of FDC (38.4 mg/L). A color change from blue (resazurin) to violet or pink (resorufin) represents visual detection of bacterial growth. 95 randomly selected A. baumannii isolates were used to evaluate the performance of the rapid FDC Acinetobacter baumannii NP test. RESULTS: The test showed 95.5% (95% CI 78.2-99.2%) and 100.0% (95% CI 95.0-100.0%) of sensitivity and specificity, respectively. All the results were obtained within 4 h30-4 h45 incubation time at 35 °C ± 2 °C, saving virtually one day when compared with currently-used antimicrobial susceptibility tests. The test showed only a single very major error, an isolate with a MIC of 8 mg/L. CONCLUSIONS: The rapid FDC Acinetobacter baumannii NP test can be a valuable method which is easier and faster to interpret when compared with the gold standard broth microdilution method. The test showed remarkable performances; hence, it may be suitable for implementation in clinical microbiology routine laboratories.

Awareness of and willingness to use oral HIV self-test kits among Kenyan young adults living in informal urban settlements: a cross-sectional survey.

Self-administered HIV testing may be a promising strategy to improve testing in hard-to-reach young adults, provided they are aware of and willing to use oral HIV self-testing (HIVST). This study examined awareness of and willingness to use oral HIVST among 350 high-risk young adults, aged 18-22, living in Kenya's informal urban settlements. Bivariate and multivariate logistic regressions were used to examine differences in HIVST awareness and willingness by demographic and sexual risk factors. Findings showed that most participants were male (56%) and less than 20 years old (60%). Awareness of oral HIVST was low (19%). However, most participants (75%) were willing to use an oral HIV self-test in the future and ask their sex partner(s) to self-test before having sex (77%). Women (OR = 1.80, 95%CI:1.11, 2.92), older participants (aged 20+) (OR = 2.57, 95% CI:1.48, 4.46), and more educated participants (OR = 2.25, 95%CI:1.36, 3.70) were more willing to use HIVST as compared to men, teen-aged, and less educated participants, respectively. Young adults who reported recent engagement in high-risk sexual behaviors, such as unprotected sex, sex while high or drunk, or sex exchange, were significantly less likely to be willing to use an oral HIV self-test kit (OR = 0.34, 95%CI:0.13,0.86). Those with the highest monthly income (OR = 0.47, 95%CI: 0.25, 0.89) were also less willing to use HIVST. More community- and peer-based efforts are needed to highlight the range of benefits of HIVST (i.e., social, clinical, and structural) to appeal to various youth demographics, in addition to addressing concerns relating to HIVST.

An overview of the most important preanalytical factors influencing the clinical performance of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs).

Due to the many technical limitations of molecular biology, the possibility to sustain enormous volumes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic testing relies strongly on the use of antigen rapid diagnostic tests (Ag-RDTs). Besides a limited analytical sensitivity, the manually intensive test procedures needed for performing these tests, very often performed by unskilled personnel or by the patients themselves, may contribute to considerably impair their diagnostic accuracy. We provide here an updated overview on the leading preanalytical drawbacks that may impair SARS-CoV-2 Ag-RDT accuracy, and which encompass lower diagnostic sensitivity in certain age groups, in asymptomatic subjects and those with a longer time from symptoms onset, in vaccine recipients, in individuals not appropriately trained to their usage, in those recently using oral or nasal virucidal agents, in oropharyngeal swabs and saliva, as well as in circumstances when instructions provided by the manufacturers are unclear, incomplete or scarcely readable and intelligible. Acknowledging these important preanalytical limitations will lead the way to a better, more clinically efficient and even safer use of this important technology, which represents an extremely valuable resource for management of the ongoing pandemic.

SARS-CoV-2 antigen detection in deceased bodies: implications for infection prevention.

Background & objectives: High transmissibility of the SARS-CoV-2 has significant implications on healthcare workers' safety, preservation, handling, transportation and disposal of the deceased bodies. The objective of this study was to detect SARS-CoV-2 antigen in nasopharyngeal samples and its implications in handling and care of COVID-19 deceased bodies. Methods: A study was conducted at a dedicated COVID-19 centre on deceased individuals from April to December 2020. Rapid antigen test (RAT) and reverse transcription (RT)-PCR was compared on all the SARS-CoV-2 positive cadavers recruited in the study. Results: A total of 115 deceased individuals were included in the study. Of these, 79 (68.7%) were male and 36 (31.3%) were female and majority were in the age group of 51-60 yr [31 (27%)]. SARS-CoV-2 antigen test was positive in 32 (27.8%) and negative in 83 (72.1%) individuals. The mean time interval between deaths to the sample collection was 13.2 h with interquartile range of eight to 20 h. Reverse transcription (RT)-PCR was used as the reference test and 24 (20.9%) cases were true positive; 93.6 per cent [95% confidence interval (CI) 88.8-98.4%] sensitivity, 45.2 per cent (95% CI 35.5-55%) specificity, 60.2 per cent (95% CI 50.6-69.8%) positive predictive value and 88.8 per cent (95% CI 82.7-95%) negative predictive value of antigen test was computed. Interpretation & conclusions: SARS-CoV-2 antigen test was positive beyond 19 h in COVID-19 deceased individuals. Antigen test was found to be highly sensitive in the deceased. Patients, suspected of having died due to COVID-19, can be screened by this method. As infectiousness of the virus in the deceased bodies cannot be directly concluded from either the antigen or RT-PCR test, yet possible transmission cannot be completely ruled out. Strict infection control measures need to be followed during the handling and clearance of COVID-19 cadavers.

Combined antibodies against internalins A and B proteins have potential application in immunoassay for detection of Listeria monocytogenes.

Listeria monocytogenes is a food-borne bacterium that causes listeriosis upon the ingestion of contaminated food. Traditional methods to detect L. monocytogenes require pre-enrichment broths to increase its concentration. To improve the screening of contaminated food and prevent listeriosis outbreaks, rapid, specific and sensitive assays are needed to detect L. monocytogenes. This study developed a prototype lateral flow immunochromatographic assay (LFIA) employing antibodies against L. monocytogenes Internalin A (InlA) and Internalin B (InlB) proteins, that are involved in non-phagocytic cell invasion. The following antibodies were used to capture L. monocytogenes antigenic targets: mouse anti-Internalin A monoclonal antibody (MAb-2D12) conjugated to colloidal gold nanoparticles and a mouse anti-Internalin B polyclonal antibody. This test was able to detect pure L. monocytogenes from culture with a limit of detection (LOD) ranging from 5.9 × 103 to 1.5 × 104 CFU/mL. In milk artificially contaminated with L. monocytogenes, the LOD was 1 × 105 CFU/mL. This prototype test discriminated L. monocytogenes from other bacterial species (Listeria innocua, Enterobacter cloacae, Bacillus cereus). Results indicate that this LFIA developed using antibodies against L. monocytogenes InlA and InlB proteins is a sensitive and specific tool that can be potentially useful to rapidly detect L. monocytogenes in contaminated food. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05597-9.

Accuracy of a Rapid Multiplex Polymerase Chain Reaction Plus a Chromogenic Phenotypic Test Algorithm for Detection of Extended-Spectrum ß-Lactamase and Carbapenemase-Producing Gram-Negative Bacilli in Positive Blood Culture Bottles.

We studied the performance of an algorithm combining multiplex polymerase chain reaction with phenotypic detection of extended-spectrum ß-lactamases and carbapenemases directly from positive blood culture bottles in patients with gram-negative bacteremia and found good concordance with routine cultures. Such an algorithm may be a tool to improve time to optimal therapy in patients with gram-negative bacteremia.

Nomograms including the UBC® Rapid test to detect primary bladder cancer based on a multicentre dataset.

OBJECTIVES: To evaluate the clinical utility of the urinary bladder cancer antigen test UBC® Rapid for the diagnosis of bladder cancer (BC) and to develop and validate nomograms to identify patients at high risk of primary BC. PATIENTS AND METHODS: Data from 1787 patients from 13 participating centres, who were tested between 2012 and 2020, including 763 patients with BC, were analysed. Urine samples were analysed with the UBC® Rapid test. The nomograms were developed using data from 320 patients and externally validated using data from 274 patients. The diagnostic accuracy of the UBC® Rapid test was evaluated using receiver-operating characteristic curve analysis. Brier scores and calibration curves were chosen for the validation. Biopsy-proven BC was predicted using multivariate logistic regression. RESULTS: The sensitivity, specificity, and area under the curve for the UBC® Rapid test were 46.4%, 75.5% and 0.61 (95% confidence interval [CI] 0.58-0.64) for low-grade (LG) BC, and 70.5%, 75.5% and 0.73 (95% CI 0.70-0.76) for high-grade (HG) BC, respectively. Age, UBC® Rapid test results, smoking status and haematuria were identified as independent predictors of primary BC. After external validation, nomograms based on these predictors resulted in areas under the curve of 0.79 (95% CI 0.72-0.87) and 0.95 (95% CI: 0.92-0.98) for predicting LG-BC and HG-BC, respectively, showing excellent calibration associated with a higher net benefit than the UBC® Rapid test alone for low and medium risk levels in decision curve analysis. The R Shiny app allows the results to be explored interactively and can be accessed at www.blucab-index.net. CONCLUSION: The UBC® Rapid test alone has limited clinical utility for predicting the presence of BC. However, its combined use with BC risk factors including age, smoking status and haematuria provides a fast, highly accurate and non-invasive tool for screening patients for primary LG-BC and especially primary HG-BC.

Low-entry-barrier point-of-care testing of anti-SARS-CoV-2 IgG in the population of Upper Austria from December 2020 until April 2021-a feasible surveillance strategy for post-pandemic monitoring?

Already at the very beginning of the COVID-19 pandemic, an extensive PCR and antigen testing strategy was considered necessary and subsequently also proved successful in order to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on international and national levels. However, equally important will be the continuous monitoring of the seroprevalence status of populations from defined regions to detect-in a timely manner-any recurrence of infections or an eventual decline in antibody levels of vaccinated individuals, especially in the emerging post-pandemic situation. The aim of this study was to estimate the prevalence of SARS-CoV-2-specific immunoglobulin G antibodies in the federal state of Upper Austria (Austria) during the period of December 2020 until April 2021. To achieve this goal, we have analyzed anonymized data on the immune status of self-referral volunteers that have been determined at local pharmacies through a low-entry-barrier point-of-care analysis approach. The seroprevalence values for immunoglobulin type G antibodies against SARS-CoV-2 antigens obtained by rapid diagnostic testing on peripheral blood from volunteers reflect the current population-based estimates reported in the literature as well as the positivity rates detected by PCR-screening analyses. In conclusion, broad-based monitoring of IgG antibodies by means of a point-of-care testing network represents a valuable tool to assess the current immune situation within regionally defined populations.

Visceral leishmaniasis in a recent transmission region: 27.4% infectivity rate among seronegative dogs.

American visceral leishmaniasis (VL) is a parasitic disease whose main domestic reservoir in the urban environment is dog and is considered one of the most important zoonoses in the context of public health. Serological tests are typically used for the diagnostic screening of the disease. This study aimed to analyse the performance of different methodologies used in the diagnosis of VL in dogs sampled from a recent transmission area. The sample consisted of 52 dogs separated into groups based on the absence and presence of clinical signs of VL. The following serological techniques were carried out: the DPP® rapid test (RT), the ALERE® RT and an RT and immunoenzymatic assay with a recently developed protein (rKDDR-plus). In addition, molecular techniques were carried out with conjunctival swabs, and bone marrow aspirate samples and parasitological samples were obtained directly from bone marrow aspirates. It was concluded that 27.4% of seronegative dogs were infected, but the serological tests, used as screening tests, showed unsatisfactory sensitivity results (average: 51.2%) for dogs without clinical signs. It was suggested that polymerase chain reaction with conjunctival swabbing be used as a screening test for dogs without clinical signs, as this is a non-invasive collection technique with high-sensitivity values.

Novel approach for rapid detection of extended spectrum ß-lactamase and metalloid-ß-lactamase using drug susceptibility testing microfluidic device (DSTM).

BACKGROUND/PURPOSE: Rapid detection of ß-lactamases is important in a recent situation where resistant bacteria are increasing. By using the drug susceptibility testing microfluidic device (DSTM), rapid screening of extended spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) has become possible. METHODS: ß-lactams and ß-lactamase inhibitors were pre-fixed in the DSTM for use. A bacterial suspension in Mueller-Hinton broth (McF 0.25) was introduced into the device, and the effects of ß-lactamase inhibitor on morphological changes caused by ß-lactam were evaluated after 3 h incubation. RESULTS: Clinical isolates genetically confirmed to produce ß-lactamase were used. Of the 84 ESBL-producing strains, 80 strains (95%) turned to be ESBL positive, and five strains (6%) of them MBL were positive as well as ESBL. Four strains (5%) were negative for both ESBL and MBL. Of the 24 MBL-producing strains, 23 strains (96%) were positive for MBL. All the 43 AmpC-producing strains were negative for both ESBL and MBL. Of the 156 ESBL- and MBL-nonproducing strains, 155 strains (99%) were negative for both ESBL and MBL, and one strain was positive for ESBL. With this method, the detection sensitivity was 95% and the specificity was 100% for ESBL, whereas the detection sensitivity was 96% and the specificity was 98% for MBL. These results were not significantly different from the results of the disc diffusion method. CONCLUSION: The DSTM method allows rapid detection of ß-lactamases in 3 h and may be a useful replacement for the disc diffusion method.

Leptospirosis in central & eastern Uttar Pradesh, an underreported disease: A prospective cross-sectional study.

Background & objective: Leptospirosis is a zoonotic disease associated with potentially fatal consequences and a grossly underreported disease in Uttar Pradesh. However, only a few studies are available which report the prevalence of leptospirosis in this State. Hence, this study was undertaken to know the status of the disease in central and eastern Uttar Pradesh. Methods: A total of 143 serum and urine samples were collected from patients with acute febrile illness from July 2017 to March 2019. All the serum samples were tested for Leptospira by rapid IgM antibody card and IgM ELISA and urine samples were tested by real-time polymerase chain reaction (RT-PCR) to detect Leptospira DNA. All positive and 10 per cent negative sera from ELISA and RT-PCR (all rapid test positive were also ELISA positive) were sent to the ICMR-Regional Medical Research Centre, Port Blair for microscopic agglutination test (MAT). Results: Thirty eight (26.6%) out of 143 samples were positive for leptospirosis either by ELISA or RT-PCR. Positive results were eight (6%) by Rapid card, 32 (22%) by IgM ELISA, 10 (7%) by MAT, 10 (7%) by RT-PCR. In MAT, the most common serovar was Lai followed by Hebdomadis, Bangkinang and Pomona. Interpretation & conclusions: Leptospirosis was found to be one of the important causes for acute febrile illness in the central and eastern parts of Uttar Pradesh. The results of the present study suggest that it is necessary to increase diagnostic facility and awareness in clinicians for the screening of leptospirosis in acutely febrile patients to decrease morbidity and mortality associated with this disease.

Bayesian estimation of sensitivity and specificity of a rapid mastitis test kit, bacterial culture, and PCR for detection of Staphylococcus aureus, Streptococcus species, and coliforms in bovine milk samples.

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.