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Identification and characterization of Leishmania amastigote and axenic form antigens

Abdelhaleem AA, Elamin EM, Bakheit SM, Mukhtar MM

Trop Biomed, 2019 Dec 01;36(4):866-873.
PMID: 33597459

This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.

Matched MeSH terms: Antigens, Protozoan/analysis*
Fulltext Plasmodium falciparum histidine rich protein 2 (pfhrp2): an additional genetic marker suitable for anti-malarial drug efficacy trials

Atroosh WM, Lau YL, Snounou G, Azzani M, Al-Mekhlafi HM

Malar J, 2022 Jan 04;21(1):2.
PMID: 34983529 DOI: 10.1186/s12936-021-04014-4

BACKGROUND: Genotyping of the three Plasmodium falciparum polymorphic genes, msp1, msp2 and glurp, has been adopted as a standard strategy to distinguish recrudescence from new infection in drug efficacy clinical trials. However, the suitability of a particular gene is compromised in areas where its allelic variants distribution is significantly skewed, a phenomenon that might occur in isolated parasite populations or in areas of very low transmission. Moreover, observation of amplification bias has diminished the value of glurp as a marker.
METHODS: The suitability of the polymorphic P. falciparum histidine-rich protein 2 (pfhrp2) gene was assessed to serve as an alternative marker using a PCR-sequencing or a PCR-RFLP protocol for genotyping of samples in drug efficacy clinical trials. The value of pfhrp2 was validated by side-by-side analyses of 5 admission-recrudescence sample pairs from Yemeni malaria patients.
RESULTS: The outcome of the single pfhrp2 gene discrimination analysis has been found consistent with msp1, msp2 and glurp pool genotyping analysis for the differentiation of recrudescence from new infection.
CONCLUSION: The findings suggest that under the appropriate circumstances, pfhrp2 can serve as an additional molecular marker for monitoring anti-malarials efficacy. However, its use is restricted to endemic areas where only a minority of P. falciparum parasites lack the pfhrp2 gene.

Matched MeSH terms: Antigens, Protozoan/analysis*
A rapid sporozoite ELISA using 3,3',5,5'-tetramethylbenzidine as the substrate chromogen

Lee M, Harrison BA, Lewis GE

Am J Trop Med Hyg, 1990 Apr;42(4):314-9.
PMID: 2184690 DOI: 10.4269/ajtmh.1990.42.314

A modified version of the standard 2-site sporozoite enzyme-linked immunosorbent assay (ELISA) using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate chromogen solution was adapted for rapid detection and identification of Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins. The TMB-ELISA was evaluated using sporozoites from experimentally infected mosquitoes and laboratory colonized uninfected mosquitoes. Our data indicate comparable sensitivity levels between the TMB-ELISA and the standard ELISA, i.e., 50 P. falciparum or P. vivax sporozoites/50 microliters of test solution. Reactions inherent to the method were specific and background reactivity was minimal. The TMB-ELISA is rapid (1 hr), simple, uses a minimal amount of monoclonal antibodies, and is suitable for use in a wide range of laboratories.

Matched MeSH terms: Antigens, Protozoan/analysis*
Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors

Khaw LT, Ball HJ, Mitchell AJ, Grau GE, Stocker R, Golenser J, et al.

Exp Parasitol, 2014 Oct;145:34-41.
PMID: 25045850 DOI: 10.1016/j.exppara.2014.07.002

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Matched MeSH terms: Antigens, Protozoan/analysis
Fulltext Emergence of Undetectable Malaria Parasites: A Threat under the Radar amid the COVID-19 Pandemic?

Beshir KB, Grignard L, Hajissa K, Mohammed A, Nurhussein AM, Ishengoma DS, et al.

Am J Trop Med Hyg, 2020 08;103(2):558-560.
PMID: 32553046 DOI: 10.4269/ajtmh.20-0467

Rapid diagnostic tests (RDTs) play a critical role in malaria diagnosis and control. The emergence of Plasmodium falciparum parasites that can evade detection by RDTs threatens control and elimination efforts. These parasites lack or have altered genes encoding histidine-rich proteins (HRPs) 2 and 3, the antigens recognized by HRP2-based RDTs. Surveillance of such parasites is dependent on identifying false-negative RDT results among suspected malaria cases, a task made more challenging during the current pandemic because of the overlap of symptoms between malaria and COVID-19, particularly in areas of low malaria transmission. Here, we share our perspective on the emergence of P. falciparum parasites lacking HRP2 and HRP3, and the surveillance needed to identify them amid the COVID-19 pandemic.

Matched MeSH terms: Antigens, Protozoan/analysis
Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis

Lokanathan Y, Mohd-Adnan A, Kua BC, Nathan S

J Fish Dis, 2016 Sep;39(9):1069-83.
PMID: 27086498 DOI: 10.1111/jfd.12474

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

Matched MeSH terms: Antigens, Protozoan/analysis
Fulltext Combining parasite lactate dehydrogenase-based and histidine-rich protein 2-based rapid tests to improve specificity for diagnosis of malaria Due to Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

Grigg MJ, William T, Barber BE, Parameswaran U, Bird E, Piera K, et al.

J Clin Microbiol, 2014 Jun;52(6):2053-60.
PMID: 24696029 DOI: 10.1128/JCM.00181-14

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with "species-specific" parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic.

Matched MeSH terms: Antigens, Protozoan/analysis*