ABSTRACT
Leishmaniasis is an important travel-related parasitic infection in the United States. Treatment regimens vary by Leishmania species and require an accurate diagnosis. The sensitivity and specificity of diagnostic methods depend on the type and condition of specimen analyzed. To identify the best algorithm for detection of parasites in fresh and fixed tissue samples, we evaluated parasite cultures, two PCR methods, and Leishmania immunohistochemistry (IHC) in samples received by the CDC from 2012 through 2019. The sensitivity and specificity of IHC assays were evaluated in fresh specimens tested. Diagnostic accuracy for formalin-fixed tissue was evaluated by using PCR-based methods and IHC. Of 100 suspected cases with fresh tissue available, Leishmania spp. infection was identified by PCR in 56% (56/100) of specimens; from these, 80% (45/56) were positive by parasite culture and 59% (33/56) by IHC. Of 420 possible cases where only fixed specimens were available, 58% (244/420) were positive by IHC and/or PCR. Of these, 96% (235/420) were positive by IHC and 84% (204/420) by PCR-based methods. Overall parasite detection using all methodologies was similar for fresh and formalin-fixed tissue specimens (56% versus 58%, respectively). Although PCR-based methods were superior for diagnosis of leishmaniasis and species identification in fresh samples, IHC in combination with PCR increased the accuracy for Leishmania spp. detection in fixed samples. In conclusion, PCR is the most effective method for detecting Leishmania infection in fresh tissue samples, whereas for formalin-fixed samples, IHC and PCR-based methods should be used in combination.
Subject(s)
Algorithms , Leishmania , Leishmaniasis , Polymerase Chain Reaction , Sensitivity and Specificity , Humans , Leishmania/isolation & purification , Leishmania/genetics , Leishmaniasis/diagnosis , Leishmaniasis/parasitology , Polymerase Chain Reaction/methods , ImmunohistochemistryABSTRACT
The rapid emergence of drug-resistant malaria parasites during the course of an infection remains a major challenge for providing accurate treatment guidelines. This is particularly important in cases of malaria treatment failure. Using a previously well-characterized case of malaria treatment failure, we show the utility of using next-generation sequencing for early detection of the rise and selection of a previously reported atovaquone-proguanil (malarone) drug resistance-associated mutation.
Subject(s)
Cytochromes b/genetics , Drug Resistance/genetics , Mutation , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Adult , Antimalarials/therapeutic use , Atovaquone/therapeutic use , Drug Combinations , Gene Expression , High-Throughput Nucleotide Sequencing , Humans , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Male , Nigeria , Plasmodium falciparum/drug effects , Plasmodium falciparum/isolation & purification , Proguanil/therapeutic use , Travel , Treatment Failure , United StatesABSTRACT
Immunization of mice with Plasmodium berghei or Plasmodium yoelii synthetic linear peptide chimeras (LPCs) based on the circumsporozoite protein protects against experimental challenge with viable sporozoites. The immunogenicity of LPCs is significantly enhanced by spontaneous polymerization. To better understand the antigenic properties of polymeric antimalarial peptides, we studied the immune responses elicited in mice immunized with a polymer or a monomer of a linear peptide construct specific for P. yoelii and compared the responses of antigen-presenting cells following incubation with both peptide species. Efficient uptake of the polymeric peptide in vitro resulted in higher expression of the coactivation markers CD80, CD40, and CD70 on dendritic cells and higher proinflammatory cytokine production than with the monomeric peptide. Macropinocytosis seems to be the main route used by polymeric peptides internalized by antigen-presenting cells. Spontaneous polymerization of synthetic antimalarial-peptide constructs to target professional antigen-presenting cells shows promise for simple delivery of subunit malaria vaccines.
Subject(s)
Malaria Vaccines/genetics , Malaria Vaccines/immunology , Plasmodium berghei/immunology , Plasmodium yoelii/immunology , Protein Multimerization/immunology , Protozoan Proteins/immunology , Animals , Antigen-Presenting Cells/immunology , B7-1 Antigen/biosynthesis , CD27 Ligand/biosynthesis , CD40 Antigens/biosynthesis , Cell Line , Cells, Cultured , Cytokines/metabolism , Female , Mice , Mice, Inbred BALB C , Pinocytosis , Plasmodium berghei/genetics , Plasmodium yoelii/genetics , Protozoan Proteins/genetics , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
Synthetic linear peptide chimeras (LPCs(cys+)) show promise as delivery platforms for malaria subunit vaccines. Maximal immune response to LPCs(cys+) in rodent malaria models depends upon formation of cross-linkages to generate homopolymers, presenting challenges for vaccine production. To replicate the immunogenicity of LPCs(cys+) using a recombinant approach, we designed a recombinant LPC (rLPC) based on Plasmodium yoelii circumsporozoite protein-specific sequences of 208 amino acids consisting of four LPC subunits in series. BALB/c or CAF1/J mice were immunized with synthetic or recombinant LPCs. Antibody concentrations, cytokine production and protection against challenge were compared. Recombinant peptide replicated the robust, high avidity antibody responses obtained with the synthetic linear peptide chimera. After in vitro stimulation spleen cells from mice immunized with rLPC or synthetic LPC(cys+) produced gamma interferon and IL-4 suggesting the efficient priming of T cells. Immunization of mice with either recombinant or synthetic LPC(cys+) provided comparable protection against experimental challenge with P. yoelii sporozoites. Recombinant LPCs reproduced the immunogenicity of synthetic LPC(cys+) without requiring polymerization, improving prospects for use as malaria vaccines.
