Development of an immunochromatographic lateral flow assay to rapidly detect OXA-23-, OXA-40-, OXA-58- and NDM-mediated carbapenem resistance determinants in Acinetobacter baumannii.

Introduction. Acinetobacter baumannii infections can be extremely challenging to treat owing to the worldwide prevalence of multidrug-resistant isolates, especially against carbapenems. Colonization with carbapenem-resistant A. baumannii (CRAb) requires rapid action from an infection control perspective because the organism is known for its propensity for epidemic spread. Hypothesis/Gap Statement. There is an unmet medical need to rapidly identify CRAb to enable appropriate antimicrobial treatment and to prevent transmission. Aim. Our aim was to expand the OXA-detection abilities of the rapid immunochromatographic test (ICT) OXA-23 K-SeT (Coris BioConcept) to include OXA-40- and OXA-58-like carbapenemases, which together confer carbapenem resistance to more than 94 % of CRAb isolates worldwide. Methodology. We used hybridoma technology to generate mAbs against OXA-40 and OXA-58 and selected them for productivity and specificity against recombinant and endogenous OXA-40 and OXA-58. Combinations of the resulting mAbs were analysed in ICT format for their ability to detect recombinant rOXA-40His6 or rOXA-58His6, respectively. Subsequently, selected antibody pairs were implemented into single-OXA-40 or single-OXA-58 prototypes and the final OXA-23/40/58/NDM ICT and were evaluated on clinical Acinetobacter spp. isolates with well-defined carbapenem resistance mechanisms. Results. Five anti-OXA-40 and anti-OXA-58 mAbs were selected. Competition ELISA with combinations of these antibodies revealed that the anti-OXA-40 antibodies bind to one of two binding clusters on OXA-40, while anti-OXA-58 antibodies bind to one of four binding clusters on OXA-58. Direct binding to the corresponding antigen in an ICT format has left only three antibodies against rOXA-40His6 and rOXA-58His6, respectively for the subsequent sandwich ICT selection procedure, which revealed that the anti-OXA-40 (#5) and anti-OXA-58 (#A8) mAbs in combination with the cross-reactive mAb #C8 performed best. They were implemented into single-OXA-40 and single-OXA-58 ICT prototypes and evaluated. These single ICT prototypes demonstrated 100 % specificity and sensitivity. Based on these results, an OXA-23/40/58/NDM-ICT was developed, complemented with OXA-23 and NDM-specific detection. An evaluation with selected carbapenem-resistant Acinetobacter spp. isolates (n=34) showed 100 % specificity. Conclusion. With this easy-to-use detection assay, one can save 12-48 h in diagnostics, which helps to treat patients earlier with appropriate antibiotics and allows immediate intervention to control transmission of CRAb.

Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. METHODOLOGY/PRINCIPAL FINDINGS: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. CONCLUSIONS/SIGNIFICANCE: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.

Visceral Leishmaniasis IgG1 Rapid Monitoring of Cure vs. Relapse, and Potential for Diagnosis of Post Kala-Azar Dermal Leishmaniasis.

Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti-Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment (p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference (p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11-30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes.

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care. METHODOLOGY/PRINCIPAL FINDINGS: All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001). CONCLUSIONS/SIGNIFICANCE: Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.