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Copyright of General Practitioner / Praktický Lékař is the property of Czech Medical Association of JE Purkyne and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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., Competing Interests: Declaration of competing interest All the authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: This study aims to describe the epidemiological characteristics of imported cases of dengue (DEN), chikungunya (CHIK), and Zika virus (ZIKV) infections in Czech travellers., Materials and Methods: This single-centre descriptive study has retrospectively analysed data of patients with laboratory confirmed DEN, CHIK, and ZIKV infections diagnosed at the Department of Infectious, Parasitic and Tropical Diseases of the University Hospital Bulovka in Prague, Czech Republic from 2004 to 2019., Results: The study included a total of 313 patients with DEN, 30 with CHIK, and 19 with ZIKV infections. Most patients travelled as tourists:263 (84.0%), 28 (93.3%), and 17 (89.5%), respectively (p = 0.337). The median duration of stay was 20 (IQR 14-27), 21 (IQR 14-29), and 15 days (IQR 14-43), respectively (p = 0.935). Peaks of imported DEN and ZIKV infections were noted in 2016, and in 2019 in the case of CHIK infection. Most cases of DEN and CHIKV infections were acquired in Southeast Asia:212 (67.7%) and 15 (50%), respectively, while ZIKV infection was most commonly imported from the Caribbean (11; 57,9%)., Conclusions: Arbovirus infections represent an increasingly significant cause of illness in Czech travellers. Comprehensive knowledge of the specific epidemiological profile of these diseases is an essential prerequisite for good travel medicine practice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Trojánek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2022. The Author(s).)

Objective:  The aim of this study was to evaluate attitudes towards the available preventive measures, risk behaviour and health problems in Czech travellers to tropical and subtropical regions., Materials and Methods:  The prospective study included patients of all ages with a history of recent travel to risky countries who presented to the post-travel clinic of the Hospital Na Bulovce in Prague within two months after return, from February 2009 to June 2018. The data were collec-ted through a questionnaire survey and from electronic medical records., Results:  Nine hundred and thirty-four patients (473 M and 461 F, age median of 32 years, IQR 27-39) participated in the study. The most frequent destinations were South-East Asia (332; 35.5%), South Asia (176, 18.8%), and Sub-Saharan Africa (172; 18.4%). The most common reasons for tra-vel were tourism (772; 82.7%) and business (111; 11.9%). An underlying chronic disease was reported by 317 patients (33.9%). Pre-travel health consultation was sought by 415 travellers (44.4%); however, only 312 (33.4%) of the respondents were properly vaccinated. Preventive malaria chemoprophylaxis was indicated in a total of 151 travellers (16.2%) but was only received by 44/151 patients (29.1%). Risky eating or drinking behaviour was admitted by 832 (89.1%) respondents and low standard accommodation by 525 (56.2 %) travellers. The most commonly reported clinical syndromes were acute and chronic diarrhoea (266; 28.5%), febrile illness (240; 25.7%) and skin lesions (166; 17.8%). A total of 199 patients presented with tropical infection (21.3%) and 63 with a vaccine-preventable infection or malaria (6.7%).  Conclusions: The study shows that the adherence of Czech travellers to preventive measures is relatively low, and along with risky behaviours, has a significant impact on travel-related morbidity. However, vaccine-preventable infections only accounted for a small fraction of travel-related illness, which is in accordance with other epidemiological studies and points out the need for a more comprehensive interdisciplinary approach to pre-travel health consultations.

Aim: The aim of our work was to summarize the most important knowledge about listeriosis in humans and to analyse available epidemiological data on this disease in the Czech Republic in 2008-2018., Methods: We conducted a literature search and descriptive epidemiological analysis of all human Listeria cases reported to the national surveillance system of infectious diseases (EpiDat until 2017, ISIN for 2018) in the Czech Republic in 2008-2018. For data management and analysis, MS Excel 2010 was used. The incidence maps were created using the ECDC Map Maker tool (EMMa)., Results: In total, 380 cases of human listeriosis (mean annual incidence of 0.33/100 000 inhabitants) were reported in the Czech Republic in 2008-2018. The highest age specific incidence was detected in 0-year-olds (1.85/100 000) and then in persons over 60 years (mean incidence of 0.95/100 000). Altogether 222 cases were detected in men (mean incidence of 0.39/100 000) and 158 in women (mean incidence of 0.27/100 000). Geographically, the highest mean annual incidence was reported in the Moravian-Silesian Region (0.6/100 000) and Pilsen Region (0.57/100 000). As many as 96.3% of the reported cases required hospital admission. Of 81 deaths related to listeriosis, 50 were directly caused by listeriosis. The highest monthly incidence was observed in June through October and then in January.  The incidence of listeriosis in the CZ shows an oscillating trend over the last 11 years. Neither epidemics nor imported cases were reported to the national surveillance system during the study period., Conclusions: Listeriosis cases have been on the rise in the European Union (EU) over the last years, while in the CZ, an oscillating and slightly increasing trend has been observed. Generally, cases are reported mostly in the population aged over 64 years (and especially after the age of 84). As there is no vaccine available yet against this disease, the only option is the prevention, i.e., health education of consumers in general and of the risk groups in particular, and compliance with food safety and hygiene standards in food production and handling.

This case report describes the first case of imported scrub typhus in a Czech traveler. The infection was diagnosed in a 38-year-old male traveler returning from a one-week business/tourist trip to Laos who presented with fever, chills, joint and muscle pain, localized inguinal lymphadenopathy, rash and a typical eschar. Although laboratory findings included elevation of CRP, hepatic aminotransferases and lactate dehydrogenase, complete blood count revealed only borderline leukocytosis with mild thrombocytopenia. The diagnosis of scrub typhus was made by serological detection of specific antibodies. The patient was treated with a 200 mg daily dose of oral doxycycline for 20 days. His clinical course was uncomplicated. The case underpins the need for a broader differential diagnosis in patients with travel-related health problems, especially those presenting with fever and rash, including less common or neglected tropical diseases.

Influenza is viral acute respiratory illness. During winter season flu occurs in local epidemics of various magnitude. Influenza causes substantial loss of workdays, human suffering, and significant mortality rate in population, despite the existing treat ment and effective vaccination.

By the end of 2019 the first cases of severe pneumonia of unknown origin were reported in Wuhan, China. The causative agent was identified as a novel b-coronavirus SARS-CoV-2 and the disease was named COVID-19. Since the beginning of 2020, the infection has spread worldwide, which led the WHO to declare COVID-19 a public health emergency of international concern and to characterize the current situation as a pandemic. The transmission occurs mainly via respiratory droplets and the incubation period ranges from 2 to 14 days. Most cases are mild, but some patients develop severe pneumonia with acute respiratory distress, septic shock and multi-organ failure. The most common symptoms include fever, dry cough, myalgia and shortness of breath. Characteristic laboratory findings are normal white blood cell count or mild leukopenia, marked lymphopenia, in severe cases elevated CRP, procalcitonin, LDH, and D-dimer are commonly found. Typical imaging findings include multifocal peripherally distributed ground-glass opacities or consolidations, interlobular septal thickening, crazy paving appearance and cystic changes. The overall case fatality rate is estimated to range from 1 to 3 %, however, it is dependent on age and underlying medical comorbidities. Current potential treatment options include hydroxychloroquine, remdesivir, lopinavir/ritonavir and convalescent plasma.

Amoebic colitis represents a common parasitic infection in developing countries. In western world, it is encountered only sporadically. The clinical presentation is usually non-specific, non-invasive laboratory tests are often false negative and endoscopic and histopathological appearance may mimic other illnesses, especially Crohns disease. The disease therefore harbours a huge risk of misdiagnosing and a proper diagnosis is usually challenging. We present a case of an amoebic colitis with Crohn-like features and negative parasitological testing in a 53-years-old woman, in which the final diagnosis was established on the basis of its histopathological examination.

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.

Human alveolar echinococcosis (AE) is caused by larval stages of the tapeworm Echinococcus multilocularis. In the Czech Republic, screening tests to detect the specific infectious agent have been performed since 1998. The first AE cases were diagnosed in 2007, and until 2014, a total of 21 diseases were recorded. In accordance with radiological, histological, and/or PCR data, serological examinations of 699 individuals helped to reveal 15 additional AE cases in the period of 2015-2016. From the cumulative data for 1998-2016, it appears that of 2,695 patients examined, 36 (18 men and 18 women) were diagnosed with AE. Their age at diagnosis ranged from 20 to 82 years and was lower for women (mean 43.7, median 39.5) than for men (50.9 and 57.5, respectively), but the difference was not statistically significant. In the period of 2007-2016, the mean annual incidence rate was 0.034 cases/100 000 population. Our study indicates an ongoing increase in AE cases. The disease can be autochthonous in nature, as evidenced not only by some case history data but also by the detection of the larval stages in wild boar (Sus scrofa). AE risk to humans in the Czech Republic is discussed in the context of the known data on the presence of various parasite developmental stages in animals.

Background: Dengue fever is a frequent cause of morbidity in travellers. The objective was to describe the epidemiological and clinical characteristics of dengue fever in Czech travellers., Method: This descriptive study includes patients with acute dengue fever diagnosed at Hospital Na Bulovce during 2004-2013. Data were collected and analysed retrospectively., Results: A total of 132 patients (83 males and 49 females) of median age 33 years (IQR 29-40) were included. Diagnosis was established by NS1 antigen detection in 87/107 cases (81.3%) and/or RT-PCR in 50/72 (69.4%) and by serology in 25 cases (18.9%). Dengue was acquired in South-East Asia in 69 cases (52.3%), followed by South Asia (48 cases; 36.3%), Latin America (14; 10.6%) and Sub-Saharan Africa (1; 0.8%). The most frequent symptoms included fever, rash and headache. Initial leukocyte and lymphocyte counts were lower in patients who presented in the early phase (0-4 days), however, platelet count was lower and AST, ALT and LDH activity higher in patients with a longer symptoms duration (≥5 days). The clinical course was mostly uncomplicated., Conclusions: Dengue fever is becoming a frequent cause of fever in Czech travellers. Clinicians should be familiar with the typical clinical findings and novel diagnostic methods., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this study was to describe epidemiological and clinical characteristics of imported enteric fever in Czech travellers and to determine the antimicrobial susceptibility of isolated strains. Retrospective descriptive study included adult patients treated with enteric fever at Hospital Na Bulovce during January 2004-December 2012. A case of typhoid or paratyphoid fever was defined as isolation of Salmonella Typhi or Paratyphi from blood or stool. During the study period, there have been diagnosed 19 cases of enteric fever (12 males and 7 females) with age median of 30 years; 14 cases were caused by Salmonella Typhi and 5 cases by S. Paratyphi A. The infection has been acquired in South Asia (16 patients; 84.2 %), in Africa (Egypt, Angola) in two cases (10.5 %), and in Mexico (1; 5.3 %). Symptoms included fever (all patients), diarrhoea (16 cases; 84.2 %), headache (9; 47.4 %), and abdominal pain (7; 36.8 %). Seventeen patients (89.5 %) were treated with fluoroquinolones; however, the treatment failure was observed in seven of them (41.2 %). Decreased ciprofloxacin susceptibility was detected in eight strains (66.7 %), and one strain (8.3 %) was multidrug resistant. Sequence analysis of quinolone resistance-determining regions (QRDR) of the gyrA gene revealed the presence of amino acid substitutions in all tested isolates with decreased ciprofloxacin susceptibility. Typhoid and paratyphoid fevers represent epidemiologically important diseases that may lead to potentially life-threatening complications. Major issue in the management of enteric fever represents the non-susceptibility of Salmonella strains to fluoroquinolones and other antimicrobials.

Objectives: Problems with importing non-registered medicines for treating rare life-threatening infectious diseases led to establishment of the Emergency Anti-Infective Drug Reserve (EAIDR) for the Czech Republic., Methods: Thirteen anti-infective drugs are included in the project: antisera against rabies virus, varicella-zoster virus, and botulinum toxin; antituberculosis drugs (intravenous rifampicin and isoniazid; capreomycin, cycloserine, and clofazimine); antiparasitics (intravenous quinine, primaquine, meglumine antimoniate, and praziquantel); and pentamidine. These drugs are imported according to the Czech drug legislation (specific drug availability programs). Realization: The project, approved by the Czech Ministry of Health in September 2013, was started in January 2014. The anti-infective drugs sufficient for 2-4 patients are permanently available in the Toxicological Information Center (TIC) in Prague. The medicines can be applied in any hospital throughout the Czech Republic within several hours., Conclusions: All but three drugs are available at present; the remaining ones will be imported after new batches of these drugs are released.

Aim: The objective of this study is to present epidemiological characteristics and clinical symptoms of dengue fever cases in Czech workers who acquired the infection while working on the island of Fushivelavaru, Maldives. Furthermore, the study compares the sensitivity of novel direct detection assays, i.e. Real-Time Polymerase Chain Reaction (RT-PCR) and detection of Dengue NS1 antigen., Material and Methods: The retrospective study evaluated the clinical course of dengue fever cases in Czech workers returning from a job in the Maldives who were diagnosed with dengue virus infection from September 1 to October 31, 2012. The laboratory diagnosis of dengue fever was based on the serological detection of IgM and IgG antibodies, detection of dengue NS1 antigen by enzyme-linked immunosobent assay (ELISA), and detection of dengue virus DNA by RT-PCR., Results: The infection with dengue virus was confirmed in 18 males with a median age of 40 years (IQR 36-47) who returned from a job in the Maldives. Only one patient required admission to the hospital while the others were treated on an outpatient basis. The most frequently observed symptoms were fever (18), headache (9), muscle and joint pain (8 and 7, respectively), and rash (9). Typical laboratory findings were leukocytopenia and thrombocytopenia, a low CRP level, and elevated aminotransferase activity. The clinical course was uncomplicated in all patients. The dengue NS1 antigen detection (positive in all 10 patients with acute dengue fever) showed significantly higher sensitivity than the detection of viral RNA using RT-PCR (positive in 4 patients), p = 0.011., Conclusion: Although the vast majority of dengue fever cases are diagnosed among travellers returning from the tropics, the presented study points out the risks posed by dengue fever to long-term workers in endemic areas. The infection in the serologically naïve hosts is usually uncomplicated; however, infected persons are at significant risk of developing a severe complicated clinical course if challenged by another serotype. Furthermore, sick leaves or premature departures cause a considerable economic burden to employers. In the diagnosis of acute dengue fever, preference should be given to highly sensitive and specific tests for the direct detection of dengue virus (NS1 antigen and RT-PCR assays).

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

The S-adenosylhomocysteine hydrolase from the apicomplexan Cryptosporidium parvum (CpSAHH) has been characterized. CpSAHH is a single-copy, intronless gene of 1479 bp encoding a protein of 493 amino acids with a molecular mass of 55.6 kDa. Reverse transcriptase-polymerase chain reaction analysis confirmed that CpSAHH is expressed both in intracellular stages (in C. parvum-infected HCT-8 cells 24 h after infection) and in sporozoites. CpSAHH was expressed in Escherichia coli TB1 cells as a fusion with maltose-binding protein. The recombinant fusion was cleaved by Factor Xa and the enzymatic activity of both the fusion protein and the purified separated CpSAHH was measured. The enzymatic activity of CpSAHH was inhibited by d-eritadenine, S-DHPA and Ara-A.

The rising number of our tourists, who spend their holiday in tropical countries together with the rising immigration from the countries with low hygienic standard, increases the risk of import of some tropical infections. The most common are diarrhoeal diseases; to the more serious belong febrile infections (malaria, typhoid fever, ricketsioses), that without early treatment can be fatal. The diagnosis is based on the precise history, physical, laboratory and special microbiological examination. Among the laboratory methods of the first choice belong the thin smear, the thick blood film, blood count and differential counting, haemocultivation, and detection of inflammatory markers. Finding of the high eosinophilia suggests helmintosis (schistosomosis, filariosis, cystecerkosis, hydatiodosis). Though the tourists become better informed on the health risks during the stay in tropical countries, it is still possible to encounter persons living for high risk countries without proper vaccination and antimalarian prophylaxis.

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.

The case diagnosis and treatment of hydatic cyst in the right and left lobe of the liver in a 28 year old pregnant woman is described in the article.

Here we report a Narf-like gene from the apicomplexan Cryptosporidium parvum (CpNARF). CpNARF is an intronless, single-copy gene of 1680 bp which encodes a putative protein of 560 amino acids with a calculated molecular mass of 63.1 kDa. This gene contains a single highly conserved N-terminal iron-sulfur cluster ([4Fe-4S]) binding site, as well as most of the H-cluster conserved residues. Reverse transcription polymerase chain reaction analysis indicates that CpNARF is expressed by the intracellular stages of C. parvum. Although the function of this gene is as yet unknown, phylogenetic analyses suggest that CpNARF belongs to the group of NARF-like proteins from aerobic protists and higher eukaryotes, which are thought to have had an ancestor in common with [Fe]-hydrogenases.

Several reports have indicated that the iron-sulfur cluster [Fe-S] assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterized and most highly conserved [Fe-S] assembly proteins are a pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe-S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CpIscS and CpIscU) and phylogenetic studies (CpIscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CpIscS and CpIscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe-S] assembly.

The S-adenosylmethionine synthetase gene of the apicomplexan Cryptosporidium parvum (CpSAMS), an agent of diarrhea in immunocompromised and healthy humans and animals is described. CpSAMS is a single-copy, intronless gene of 1221 bp encoding a polypeptide of 406 amino acids with a molecular mass of 44.8 kDa. The gene is AT-rich (61.8%). CpSAMS was expressed in Escherichia coli TB1 cells as a fusion with maltose binding protein. The activity of the recombinant fusion was assayed, and was found to be inhibited by the methionine analog cycloleucine. In order to determine whether CpSAMS was differentially expressed during the life cycle of C. parvum, HCT-8 cells were infected with C. parvum and assayed over 72 h. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) confirmed the differential expression of CpSAMS.

We are reporting a putative multifunctional Type I polyketide synthase (PKS) gene from the apicomplexan Cryptosporidium parvum (CpPKS1). The 40 kb intronless open reading frame (ORF) predicts a single polypeptide of 13,414 amino acids with a molecular mass of 1516.5 kDa. Sequence analysis identified at least 29 enzymatic domains within this protein. These domains are organized into an N-terminal loading unit, seven polyketide chain elongation modules, and a carboxy terminator unit. The loading domain consists of an acyl-CoA ligase (AL) and an acyl carrier protein (ACP). All seven elongation modules contain between two and five of the six domains required for the elongation of two-carbon (C2) acyl units, i.e. ketoacyl synthase, acyl transferase, dehydrase, enoyl reductase, ketoreductase and/or ACP. The carboxy terminator is homologous to various reductases, suggesting that the final elongated product is not hydrolytically released by thioesterases as observed in most Type I PKS and all fatty acid synthetase (FAS) systems, but by a reducing reaction, which has been demonstrated in some non-ribosomal peptide synthase systems. The protein sequence and domain organization of CpPKS1 protein resembles a previously reported C. parvum fatty acid synthase (CpFAS1), which is encoded by a 25 kb ORF. Maximum likelihood phylogenetic analysis of acyl transferases within PKS/FAS from C. parvum and other organisms clearly differentiates acetate-extending clades from those incorporating propionate. All acyl transferase domains from CpPKS1, and a previously reported CpFAS1, clustered within the acetate-extending group, suggesting the likelihood that only non-methylated C2 units are incorporated by C. parvum polyketide and fatty acid synthases. The expression of CpPKS1 was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Many polyketides are medically significant antibiotics, anticancer agents, toxins, or signaling molecules. Therefore, it is interesting to speculate what role CpPKS1 might play in this apicomplexan and the disease caused by this opportunistic infection of AIDS patients.

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.