Epidemiology and clinical features of imported malaria: a 14-year retrospective single-centre descriptive study in Prague, Czech Republic.

BACKGROUND: Malaria represents one of the most important imported tropical infectious diseases in European travellers. The objective of the study was to identify changes in the epidemiological features of imported malaria and to analyse the clinical findings and outcomes of imported malaria. METHODS: This single-centre descriptive study retrospectively analysed the medical records of all imported malaria cases in travellers treated at the Department of Infectious Diseases of University Hospital Bulovka in Prague from 2006 to 2019. RESULTS: The study included 203 patients with a median age of 37 years (IQR 30-48) and a male to female ratio of 3.72:1. Plasmodium falciparum was the predominant species (149/203), and its proportion significantly increased from 35/60 cases (58.3%) in 2006-2011 to 69/80 (86.3%) in 2016-2019 (p < 0.001). In contrast, the incidence of Plasmodium vivax malaria decreased from 19/60 cases (31.7%) in 2006-2011 to 5/80 (6.3%) in 2016-2019 (p < 0.001). Malaria was imported from sub-Saharan Africa in 161/203 cases (79.3%). The proportion of travellers from Southeast and South Asia decreased from 16/60 (26.7%) and 6/60 (10.0%) in 2006-2011 to 2/80 (2.5%) and no cases (0.0%) in 2016-2019, respectively (p < 0.001 and p = 0.006). Tourism was the most common reason for travel (82/203), however, the proportion of non-tourists significantly increased over time from 29/60 (48.3%) in 2006-2011 to 55/80 (68.8%) in 2016-2019, p = 0.015. Severe malaria developed in 32/203 (15.8%) patients who were significantly older (p = 0.013) and whose treatment was delayed (p < 0.001). Two lethal outcomes were observed during the study period. CONCLUSIONS: This study demonstrated a significant increase in P. falciparum malaria, which frequently resulted in severe disease, especially in older patients and those with delayed treatment initiation. The rising proportion of imported malaria in non-tourists, including business travellers and those visiting friends and relatives, is another characteristic finding analogous to the trends observed in Western European and North American centres. The described changes in the aetiology and epidemiology of imported malaria may serve to optimize pre-travel consultation practices and improve post-travel diagnostics and medical care.

Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle.

BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

Testing the impact of Whole Genome Amplification on genome comparison using the polyploid flagellated Giardia duodenalis as a model.

The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.

Clone-based haplotyping of Giardia intestinalis assemblage B human isolates.

The level of genetic variability of Giardia intestinalis clinical isolates is an intensively studied and discussed issue within the scientific community. Our collection of G. intestinalis human isolates includes six in vitro-cultured isolates from assemblage B, with extensive genetic variability. Such variability prevents the precise genotype characterisation by the multi-locus genotyping (MLG) method commonly used for assemblage A. It was speculated that the intra-assemblage variations represent a reciprocal genetic exchange or true mixed infection. Thus, we analysed gene sequences of the molecular clones of the assemblage B isolates, each representing a single DNA molecule (haplotype) to determine whether the polymorphisms are present within individual haplotypes. Our results, which are based on the analysis of three standard genetic markers (bg, gdh, tpi), point to haplotype diversity and show numerous single nucleotide polymorphisms (SNPs) mostly in codon wobble positions. We do not support the recombinatory origin of the detected haplotypes. The point mutations tolerated by mismatch repair are the possible cause for the detected sequence divergence. The precise sub-genotyping of assemblage B will require finding more conservative genes, as the existing ones are hypervariable in most isolates and prevent their molecular and epidemiological characterisation.

[Diagnosis, treatment and prophylaxis of malaria in the Czech Republic].

Malaria represents the most important parasitic infection imported from the tropics causing death in 1-2 % of travelers with this diagnosis. Around 30 cases of malaria are diagnosed in the Czech Republic every year. Fever is the most common clinical presentation. The most severe forms of malaria are caused by Plasmodium falciparum. The diagnosis of malaria is based on examination of stained thick and thin blood smears. This method enables determination of Plasmodium species and parasite count. The treatment of ma-laria has to be initiated immediately after the laboratory confirmation. In the Czech Republic, uncomplicated falciparum malaria is treated by oral administration of artemether/lumefantrine or atovaquone/proguanil. Complicated falciparum malaria is treated by parenteral administration of quinine in combination with clindamycin. For the chemoprophylaxis of malaria in travelers to the highly endemic regions, atovaquone/proguanil, doxycycline or mefloquine are recommended.

Structural organization of very small chromosomes: study on a single-celled evolutionary distant eukaryote Giardia intestinalis.

During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 µm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.

Visceral leishmaniasis with cutaneous symptoms in a patient treated with infliximab followed by fatal consequences.

Leishmaniasis is an infectious disease caused by parasitic flagellates of the genus Leishmania. The authors present a case of 44-year-old man with Crohn's disease treated successfully with infliximab. This case report shows rare visceral leishmaniasis with cutaneous symptoms in an immunocompromised patient. Skin manifestations may occur before or after the visceral infection and they are often diverse.

Comprehensive analysis of flavohemoprotein copy number variation in Giardia intestinalis: exploring links to metronidazole resistance.

BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.

Artemether-lumefantrine, mefloquine and atovaquone-proguanil in the treatment of uncomplicated Plasmodium falciparum malaria in travellers: A retrospective comparative study of efficacy and treatment failures.

BACKGROUND: The aim of this study was to evaluate the rates of parasitaemia clearance and the prevalence of treatment failure in patients with uncomplicated Plasmodium falciparum malaria treated with artemether-lumefantrine (AL), mefloquine (MQ), and atovaquone-proguanil (AP). METHOD: The retrospective descriptive study included adult patients with uncomplicated P. falciparum malaria treated at the University Hospital Bulovka in Prague from 2006 to 2019. Parasitaemia clearance was estimated using a linear regression model. RESULTS: The study included 72 patients with a median age of 33 years (IQR 27-45) and a male to female ratio of 3.2:1. Thirty-six patients (50.0%) were treated with AL, 27 (37.5%) with MQ and 9 (12.5%) with AP. The proportion of VFR and migrants was 22.2% with no significant differences among the three groups. The median time to the parasitaemia clearance was two days (IQR 2-3) in patients treated with AL versus four days in the MQ (IQR 3-4) and AP (IQR 3-4) groups, p < 0.001. The clearance rate constant was 3.3/hour (IQR 2.5-4.0) for AL, 1.6/hour (IQR 1.3-1.9) for MQ, and 1.9/hour (IQR 1.3-2.4) for AP, p < 0.001. Malaria recrudescence occurred in 5/36 (13.9%) patients treated with AL and in no patients treated with MQ or AP. CONCLUSIONS: The findings demonstrate the superior efficacy of AL compared to other oral antimalarials in early malaria treatment. However, we observed a higher rate of late treatment failure in patients treated with AL than previously reported. This issue warrants further investigation of possible dose adjustments, extended regimens, or alternative artemisinin-based combinations.

Molecular evidence of Pneumocystis jirovecii reinfection in a haemato-oncology patient.

For many years Pneumocystis pneumonia was thought to be caused by the reactivation of a latent infection, but several studies have demonstrated that Pneumocystis jirovecii infection can be acquired de novo. On the basis of our results obtained from a patient with recurrent pneumocystosis, we support the hypothesis that recurrent episodes are caused by reinfection.

[Case report - pediatric visceral leishmaniasis]. / Kazuistika -visceralni leishmanioza ditete.

The diagnosis of visceral leishmaniasis (VL) is not an easy task. The clinical presentation of VL is similar to lymphomas which have significantly higher incidence in our country. This fact is one of the complications in the diagnosis of VL. Moreover, diagnostic methods have low sensitivity and bone marrow aspiration is necessary. We present a case report of a patient who fell ill in the first year of life and due to atypical lifestyle of his parents was seriously examined after 11 months from the first symptoms of the disease. Many problems with the diagnostic algorithm leading to the definitive diagnosis of VL were encountered. Thereafter, the patient underwent treatment with amphotericin B lipid complex. The course was complicated by febrile neutropenia and anemia, with the necessity for blood transfusion.

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada).

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power.

BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.

Propylene glycol and contact-lens solutions containing this diol induce pseudocyst formation in acanthamoebae.

Propylene glycol used as an ophthalmic demulcent in certain contact-lens care systems has been included recently among factors responsible for increasing Acanthamoeba keratitis. In this study, we provide evidence that propylene glycol as well as examined contact-lens solutions containing it induce rapid differentiation of acanthamoebae into pseudocysts. The partial resistance of the pseudocysts and their reversibility to viable trophozoites even after 24-h exposure to the contact-lens solutions indicate a potential risk of infection to contact-lens users.

DNA content in Acanthamoeba during two stress defense reactions: Encystation, pseudocyst formation and cell cycle.

During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.

Inactivation of Acanthamoeba Cysts in Suspension and on Contaminated Contact Lenses Using Non-Thermal Plasma.

Water suspensions of cysts of a pathogenic clinical isolate of Acanthamoeba sp. were prepared, and the cysts were inactivated either in suspension or placed on the surface of contact lenses by the non-thermal plasma produced by the DC corona transient spark discharge. The efficacy of this treatment was determined by cultivation and the presence of vegetative trophozoites indicating non-inactivated cysts. The negative discharge appeared to be more effective than the positive one. The complete inactivation occurred in water suspension after 40 min and on contaminated lenses after 50 min of plasma exposure. The properties of lenses seem to not be affected by plasma exposure; that is, their optical power, diameter, curvature, water content and infrared and Raman spectra remain unchanged.

Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.

BACKGROUND: Giardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig. RESULTS: We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes. CONCLUSIONS: Our results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.

Giardia intestinalis: aphidicolin influence on the trophozoite cell cycle.

This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal.

Companion animals as a potential source of Giardia intestinalis infection in humans in the Czech Republic - A pilot study.

Giardia intestinalis is a common enteric single-celled parasite infecting both humans and animals. Its eight morphologically identical but genetically distinct groups called assemblages differ from each other in host range. While assemblages A and B infect a wide range of hosts, including humans, the other assemblages (C to H) limit their host preferences to particular animal groups only. In companion animals as Giardia hosts, genotyping data have previously shown various results depending on pet species, location, environmental or breeding conditions, and the study design. To strengthen available epidemiological data from developed countries and to evaluate the role of pets in Giardia zoonotic transmission, we investigated Giardia-positive stool samples of three pet species (54 dogs, 18 cats, and 18 chinchillas) by a sequence-based analysis of three Giardia genes (ß-giardin, glutamate dehydrogenase and triose phosphate isomerase). In dog samples, we confirmed assemblage C (21/54), assemblage D (32/54), and one case of a mixed infection C + D (1/54). In cats, we found assemblage F (16/18) and assemblage A, specifically sub-assemblage AI (2/18). All Giardia samples from chinchillas were characterised as assemblage B, specifically sub-assemblage BIV (18/18). These results indicate that in the Czech Republic, pet dogs may not represent a source of Giardia infection for humans because of the presence of only canid-specific genotypes C and D. In contrast, other pets, namely, chinchillas and, to a lesser extent, cats, may pose a potential risk of Giardia transmission to owners or breeders because they can host zoonotic Giardia genotypes.

A rapid workflow for the characterization of small numbers of unicellular eukaryotes by using correlative light and electron microscopy.

The limited availability of biological samples hinders phylogenetic efforts to define structural differences among various biological groups. A novel workflow enabling the analysis of protists in low cell numbers by electron microscopy (EM) is described with cysts of Giardia intestinalis, a single-celled eukaryotic parasite. Correlative light and electron microscopy (CLEM) allows for the selection of individual cells and is economical in terms of time and cost. We describe a cyst purification protocol in combination with an adhesive coating for fixation and ultrathin embedding that results in excellent preservation of cell morphology. The application of advanced structural and analytical EM methods, such as high-resolution field emission scanning electron microscopy (FESEM), focused ion beam tomography (FIB/SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis, is demonstrated. The workflow represents a new approach for studying the cellular and organelle architecture of rare and "difficult to culture" microorganisms.