Molecular diagnosis of human toxoplasmosis: the state of the art.

Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan protozoan that causes toxoplasmosis. Approximately one-third of the world's population is currently T. gondii-seropositive. Although most infections are symptomless, a few can produce retinal lesions and, in immunocompromised persons or when congenitally contracted, can progress to life-threatening central nervous system disseminated infections. Therefore, quick, and precise diagnosis is a must. Molecular techniques nowadays play a crucial role in toxoplasmosis diagnosis, particularly in immunocompromised patients or congenital toxoplasmosis. This review aimed to detail recent advancements in molecular diagnostics of T. gondii infections. The terms "Toxoplasmosis," "Molecular diagnostics," "PCR," "qPCR," "B1," and "rep529" were used to search the English-language literature. In developed nations, conventional PCR (PCR) and nested PCR have been supplanted by quantitative PCR (qPCR), although they are still widely employed in poor nations. The diagnosis of toxoplasmosis has been revolutionized by the emergence of molecular diagnostics. Unfortunately, there is still substantial interlaboratory variability. There is an immediate need for standardization to increase the comparability of results between laboratories and clinical trials. Graphical abstract: A graphical abstract highlighting the summary of Toxoplasma molecular diagnostics, created using Biorender.com.

Amoebicidal effect of Allium cepa against Allovahlkampfia spelaea: A keratitis model.

Allovahlkampfia spelaea (A. spelaea) is a free-living amoeba, proved to cause Acanthamoeba-like keratitis with quite difficult treatment. This study aimed to evaluate the amoebicidal effect of Allium cepa (A. cepa) on A. spelaea trophozoites and cysts both in vitro and in vivo using Chinchilla rabbits as an experimental model of this type of keratitis. Chemical constituents of the aqueous extract of A. cepa were identified using Liquid Chromatography-mass Spectrometry (LC-MS). In vitro, A. cepa showed a significant inhibitory effect on trophozoites and cysts compared to the reference drug, chlorhexidine (CHX) as well as the non-treated control (P < 0.05) with statistically different effectiveness in terms of treatment durations and concentrations. No cytotoxic effect of A. cepa on corneal cell line was found even at high concentrations (32 mg/ml) using agar diffusion method. The in vivo results confirmed the efficacy of A. cepa where the extract enhanced keratitis healing with complete resolution of corneal ulcers in 80% of the infected animals by day 14 (post infection)pi compared to 70% recovery with CHX after 20 treatment days. The therapeutic effect was also approved at histological, immune-histochemical, and parasitological levels. Our findings support the potential use of A. cepa as an effective agent against A. spelaea keratitis.

Allovahlkampfia spelaea Causing Keratitis in Humans.

BACKGROUND: Free-living amoebae are present worldwide. They can survive in different environment causing human diseases in some instances. Acanthamoeba sp. is known for causing sight-threatening keratitis in humans. Free-living amoeba keratitis is more common in developing countries. Amoebae of family Vahlkampfiidae are rarely reported to cause such affections. A new genus, Allovahlkampfia spelaea was recently identified from caves with no data about pathogenicity in humans. We tried to identify the causative free-living amoeba in a case of keratitis in an Egyptian patient using morphological and molecular techniques. METHODS: Pathogenic amoebae were culture using monoxenic culture system. Identification through morphological features and 18S ribosomal RNA subunit DNA amplification and sequencing was done. Pathogenicity to laboratory rabbits and ability to produce keratitis were assessed experimentally. RESULTS: Allovahlkampfia spelaea was identified as a cause of human keratitis. Whole sequence of 18S ribosomal subunit DNA was sequenced and assembled. The Egyptian strain was closely related to SK1 strain isolated in Slovenia. The ability to induce keratitis was confirmed using animal model. CONCLUSIONS: This the first time to report Allovahlkampfia spelaea as a human pathogen. Combining both molecular and morphological identification is critical to correctly diagnose amoebae causing keratitis in humans. Use of different pairs of primers and sequencing amplified DNA is needed to prevent misdiagnosis.

Copper Homeostasis for the Developmental Progression of Intraerythrocytic Malarial Parasite.

Malaria is one of the world's most devastating diseases, particularly in the tropics. In humans, Plasmodium falciparum lives mainly within red blood cells, and malaria pathogenesis depends on the red blood cells being infected with the parasite. Nonesterified fatty acids (NEFAs), including cis-9-octadecenoic acid, and phospholipids have been critical for complete parasite growth in serum-free culture, although the efficacy of NEFAs in sustaining the growth of P. falciparum has varied markedly. Hexadecanoic acid and trans-9-octadecenoic acid have arrested development of the parasite, in association with down-regulation of genes encoding copper-binding proteins. Selective removal of Cu+ ions has blockaded completely the ring-trophozoite-schizont progression of the parasite. The importance of copper homeostasis for the developmental progression of P. falciparum has been confirmed by inhibition of copper-binding proteins that regulate copper physiology and function by associating with copper ions. These data have provided strong evidence for a link between healthy copper homeostasis and successive developmental progression of P. falciparum. Perturbation of copper homeostasis may be, thus, instrumental in drug and vaccine development for the malaria medication. We review the importance of copper homeostasis in the asexual growth of P. falciparum in relation to NEFAs, copperbinding proteins, apoptosis, mitochondria, and gene expression.

Perturbation of copper homeostasis is instrumental in early developmental arrest of intraerythrocytic Plasmodium falciparum.

BACKGROUND: Malaria continues to be a devastating disease. The elucidation of factors inducing asexual growth versus arrest of Plasmodium falciparum can provide information about the development of the parasite, and may help in the search for novel malaria medication. Based on information from genome-wide transcriptome profiling of different developmental stages of P. falciparum, we investigated the critical importance of copper homeostasis in the developmental succession of P. falciparum with regard to three aspects of copper function. These were:1) inhibition of copper-binding proteins, 2) copper-ion chelation, and 3) down-regulated expression of genes encoding copper-binding proteins associated with a specific growth-promoting factor. RESULTS: Inhibition of copper-binding proteins with tetrathiomolybdate (TTM) caused cessation of growth of the parasite. TTM arrested the parasite irreversibly during the trophozoite to schizont stage progression. Target molecules for TTM may be present in P. falciparum. The involvement of copper ions in developmental arrest was also investigated by copper-ion chelating methods, which indicated a critical function of reduced copper ions (Cu1+) in the parasite during the early developmental stage. Copper ions, not only in the parasite but also in host cells, were targets of the chelators. Chelation of Cu1+caused blockage of trophozoite progression from the ring stage. Profound growth arrest was detected in parasites cultured in a chemically defined medium containing hexadecanoic acid alone as a growth-promoting factor. This developmental arrest was associated with down-regulated expression of genes encoding copper-binding proteins. Cis-9-octadecenoic acid completely prevented the down-regulation of gene expression and developmental arrest that were observed with the use of hexadecanoic acid. CONCLUSIONS: The critical importance of copper homeostasis in early developmental stages of P. falciparum was confirmed. Perturbation of copper homeostasis induced profound and early developmental arrest of P. falciparum. These findings should help to elucidate the mechanisms behind the development of P. falciparum, and may be applied in the development of effective antimalarial strategies.

Molecular factors that are associated with early developmental arrest of intraerythrocytic Plasmodium falciparum.

Malaria continues to be a devastating disease. We investigated the factors that control intraerythrocytic development of the parasite Plasmodium falciparum by using a chemically defined medium (CDM) containing non-esterified fatty acid(s) (NEFA) and phospholipids with specific fatty acid moieties, to identify substances crucial for parasite development. Different NEFAs in the CDM played distinct roles by altering the development of the parasite at various stages, with effects ranging from complete growth to growth arrest at the ring stage. We used genome-wide transcriptome profiling to identify genes that were differentially expressed among the different developmental stages of the parasite, cultured in the presence of various NEFAs. We predicted 26 transcripts that were associated with the suppression of schizogony, of which 5 transcripts, including merozoite surface protein 2, a putative DEAD/DEAH box RNA helicase, serine repeat antigen 3, a putative copper channel, and palmitoyl acyltransferase, were particularly associated with blockage of trophozoite progression from the ring stage. Furthermore, the involvement of copper ions in developmental arrest was detected by copper-ion-chelating methods, implying a critical function of copper homeostasis in the early growth stage of the parasite. These results should help to elucidate the mechanisms behind the development of P. falciparum.

Plasmodium falciparum: differing effects of non-esterified fatty acids and phospholipids on intraerythrocytic growth in serum-free medium.

Different combinations of non-esterified fatty acids (NEFA) had variable effects on intraerythrocytic growth of Plasmodium falciparum. All stages of the parasite cultured in medium supplemented with cis-9-octadecenoic acid (C18:1-cis-9), hexadecanoic acid (C16:0), phospholipids (Pld) and bovine albumin free of NEFA were similar to those grown in complete growth medium. Three typical growth patterns indicating suppressed schizogony (SS), suppressed formation of merozoites (SMF), and inhibited invasion of merozoites (IMI) resulted from culture in other combinations of lipids. Unsaturated or saturated NEFA with longer or shorter carbon chains than C18:1-cis-9 or C16:0, higher degree of unsaturation, and trans-forms mainly resulted in SS and SMF effects. However, IMI or partial IMI was observed with tetradecanoic acid or octadecanoic acid enriched with C18:1-cis-9, and cis-9-hexadecenoic acid plus C16:0. Isoforms of C18:1-cis-9 also mainly resulted in partial IMI. SMF also occurred with C18:1-cis-9 plus C16:0 in the absence of Pld. Thus different NEFA exerted distinct roles in erythrocytic growth of the parasite by sustaining development at different stages.