Diagnostic Characteristics of Lactate Dehydrogenase on a Multiplex Assay for Malaria Detection Including the Zoonotic Parasite Plasmodium knowlesi

Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07–5.16) and 4.56 (3.43–6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56–2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37–2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.

Download Full-text

Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i38b32115 ◽

2021 ◽

pp. 197-209

Author(s):

Rupam R. Nashine ◽

Amit R. Nayak ◽

Aliabbas Husain ◽

Gargi D. Mudey ◽

Hatim F. Daginawala ◽

...

Keyword(s):

Whole Blood ◽

Cross Reactivity ◽

Clinical Samples ◽

Interferon Gamma Release Assay ◽

Antigenic Peptides ◽

Whole Blood Assay ◽

Blood Assay ◽

Variable Sensitivity ◽

Latent Tb Infection

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major cross-reactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.

Download Full-text

OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution

Biomedicines ◽

10.3390/biomedicines9101314 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1314

Author(s):

Eduarda Carvalho-Correia ◽

Carla Calçada ◽

Fernando Branca ◽

Nuria Estévez-Gómez ◽

Loretta De Chiara ◽

...

Keyword(s):

Cross Reactivity ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Analytical Sensitivity ◽

S Gene ◽

Genomic Knowledge ◽

Gene Assay ◽

One Step

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide–sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Download Full-text

Comparative evaluation of a new immunoradiometric assay for corticotropin

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2006.113 ◽

2006 ◽

Vol 44 (5) ◽

Cited By ~ 11

Author(s):

Charles W. Wilkinson ◽

Hershel Raff

Keyword(s):

The United States ◽

Cross Reactivity ◽

Clinical Samples ◽

Immunoradiometric Assay ◽

Standard Curve ◽

United States Food ◽

In Vitro Diagnostic ◽

States Food ◽

Diagnostic Measurement

AbstractWe have characterized the performance of a commercial two-site immunoradiometric assay for manual in vitro diagnostic measurement of plasma corticotropin from Scantibodies Laboratory. We compared the results with those of a similar commonly used assay from Nichols Institute Diagnostics that has recently been withdrawn from production. The lower detection limit, range of the standard curve, cross-reactivity, and intra-assay and inter-assay imprecision of the two assays are very similar. Measurement of clinical samples and a series of samples from an experimental subject demonstrate high correlations between the two assays. These factors, together with recent clearance by the United States Food and Drug Administration for manual in vitro diagnostic measurement, make the Scantibodies corticotropin immunoradiometric assay an appropriate replacement for the Nichols assay.

Download Full-text

Evidence of IgE-Mediated Cross-Reactions between Anisakis simplex and Contracaecum osculatum Proteins

Pathogens ◽

10.3390/pathogens10080950 ◽

2021 ◽

Vol 10 (8) ◽

pp. 950

Author(s):

Foojan Mehrdana ◽

María Lavilla ◽

Per Walter Kania ◽

Miguel Ángel Pardo ◽

María Teresa Audicana ◽

...

Keyword(s):

Cross Reactivity ◽

Secretory Proteins ◽

Allergic Reactions ◽

Cross Reactions ◽

Contracaecum Osculatum ◽

Ige Mediated ◽

Skin Prick Tests ◽

Zoonotic Parasites

Fish consumers may develop allergic reactions following the ingestion of fish products containing nematode larvae within the genus Anisakis. Sensitized patients may cross-react with proteins from insects, mites and mollusks, leading to allergic reactions even in the absence of the offending food. Potential cross-reactivity in Anisakis-allergic patients with larval proteins from other zoonotic parasites present in freshwater and sea fish should be investigated due to an increasing occurrence in certain fish stocks, particularly Contracaecum osculatum. In this work, we evaluated IgE-cross reactions by in vivo (skin prick tests with parasites extracts) and in vitro methods (IgE-ELISA and IgE-immunoblot). In vivo skin prick tests (SPT) proved the reactivity of Anisakis-sensitized patients when exposed to C. osculatum antigens. Sera from Anisakis-sensitized patients confirmed the reaction with somatic antigens (SA) and excretory/secretory proteins (ES) from C. osculatum. Only anecdotal responses were obtained from other freshwater worm parasites. Consequently, it is suggested that Anisakis-sensitized humans, especially patients with high levels of specific anti-Anisakis antibodies, may react to C. osculatum proteins, possibly due to IgE-mediated cross-reactivity.

Download Full-text

Reliability of a Single β-Thromboglobulin Measurement in a Diabetic Population : Importance of PGE1 in Anticoagulant Mixture

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651094 ◽

1987 ◽

Vol 57 (02) ◽

pp. 201-204 ◽

Cited By ~ 2

Author(s):

P Y Scarabin ◽

L Strain ◽

C A Ludlam ◽

J Jones ◽

E M Kohner

Keyword(s):

In Vitro Release ◽

Mean Value ◽

Reliable Estimate ◽

Diabetic Patients ◽

Single Measurement ◽

Diabetic Population ◽

The Difference ◽

Vitro Release

SummaryDuring the collection of samples for plasma β-thromboglobulin (β-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single β-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE!. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p <0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artifically high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGEi decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.

Download Full-text

The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650659 ◽

1996 ◽

Vol 76 (05) ◽

pp. 774-779 ◽

Cited By ~ 14

Author(s):

John T Brandt ◽

Carmen J Julius ◽

Jeanne M Osborne ◽

Clark L Anderson

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Complement Activation ◽

Thromboembolic Disease ◽

Clinical Samples ◽

Hla Antigen ◽

Aggregation Response ◽

Immune Mediated ◽

Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

Download Full-text

Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01948-20

Author(s):

Dalin Rifat ◽

Si-Yang Li ◽

Thomas Ioerger ◽

Keshav Shah ◽

Jean-Philippe Lanoix ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Evidence ◽

Clinical Samples ◽

Loss Of Function ◽

Wild Type ◽

Content Type ◽

Solid Media ◽

High Level ◽

Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Download Full-text

Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae

Scientific Reports ◽

10.1038/s41598-021-83505-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aiyan Guan ◽

Inge Van Damme ◽

Frank Devlieghere ◽

Sarah Gabriël

Keyword(s):

Enzymatic Digestion ◽

Infected Fish ◽

Effect Of Temperature ◽

Video Recordings ◽

Different Temperatures ◽

Semi Solid ◽

Zoonotic Parasites ◽

The Impact ◽

Phosphate Buffered Saline

AbstractAnisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

Download Full-text

1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1463 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S655-S655

Author(s):

Daniel Navas ◽

Angela Charles ◽

Amy Carr ◽

Jose Alexander

Keyword(s):

Multidrug Resistant ◽

Resistance Mechanisms ◽

Vitro Activity ◽

Clinical Isolates ◽

Resistance Testing ◽

Clinical Samples ◽

In Vitro Activity ◽

Carbapenemase Gene ◽

Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC <4µg/dL). CZA (CLSI MIC <8µg/dL) and I/R (FDA MIC <2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

Download Full-text

Vaccination-induced variation in the 140 kD merozoite surface antigen of Plasmodium knowlesi malaria.

Journal of Experimental Medicine ◽

10.1084/jem.165.2.359 ◽

1987 ◽

Vol 165 (2) ◽

pp. 359-367 ◽

Cited By ~ 31

Author(s):

F W Klotz ◽

D E Hudson ◽

H G Coon ◽

L H Miller

Keyword(s):

Monoclonal Antibodies ◽

Malaria Infection ◽

Rhesus Monkeys ◽

Plasmodium Knowlesi ◽

Rabbit Antiserum ◽

Antigenic Determinants ◽

Partial Protection ◽

Erythrocyte Invasion ◽

Merozoite Surface Antigen

Immunity to 143/140 kD schizont antigens of a monkey malaria, Plasmodium knowlesi, provides partial protection to lethal malaria infection in rhesus monkeys challenged with uncloned parasites. To determine the capacity of a cloned parasite to generate variants of the 143/140 kD antigens, immunized monkeys were challenged with a clone of P. knowlesi. Parasites recovered 8 d after inoculation with a cloned parasite retained the 143/140 kD antigens. Parasites recovered 30 d after challenge had undergone changes in the 143/140 kD antigens. Antibodies that block erythrocyte invasion in vitro of the inoculum parasites did not inhibit invasion of erythrocytes by two isolates recovered from the immunized monkeys. An isolate from one monkey recovered on day 30 contained clones expressing new 76/72 kD antigens reactive with rabbit antiserum against the 143/140 kD proteins, and other clones expressing no antigens crossreactive with antisera against the 143/140 kD proteins. An isolate from another monkey obtained 59 d after challenge expressed new antigens of 160/155, 115/113, and 87/85 kD. Using monoclonal antibodies, we found that epitopes were lost from the variant proteins, but we were unable to determine whether new epitopes had appeared. We conclude that clones of P. knowlesi can rapidly vary antigenic determinants on the 143/140 kD proteins in animals immunized with these antigens.

Download Full-text