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Importance: Acute COVID19 related myocardial, pulmonary and vascular pathology, and how these relate to each other, remains unclear. No studies have used complementary imaging techniques, including molecular imaging, to elucidate this. Objective: We used multimodality imaging and biochemical sampling in vivo to identify the pathobiology of acute COVID19. Design, Setting and Participants: Consecutive patients presenting with acute COVID19 were recruited during hospital admission in a prospective cross sectional study. Imaging involved computed tomography coronary angiography (CTCA - identified coronary disease), cardiac 2deoxy2[fluorine18]fluoroDglucose positron emission tomography/computed tomography (18F FDG PET/CT identified vascular, cardiac and pulmonary inflammatory cell infiltration) and cardiac magnetic resonance (CMR identified myocardial disease), alongside biomarker sampling. Results: Of 33 patients (median age 51 years, 94% male), 24 (73%) had respiratory symptoms, with the remainder having non-specific viral symptoms. Nine patients (35%, n=9/25) had CMR defined myocarditis. 53% (n=5/8) of these patients had myocardial inflammatory cell infiltration. Two patients (5%) had elevated troponin levels. Cardiac troponin concentrations were not significantly higher in patients with myocarditis (8.4ng/L [IQR 4.0, 55.3] vs 3.5ng/L [2.5, 5.5], p=0.07) or myocardial cell infiltration (4.4ng/L [3.4, 8.3] vs 3.5ng/L [2.8, 7.2], p=0.89). No patients had obstructive coronary artery disease or vasculitis. Pulmonary inflammation and consolidation (percentage of total lung volume) was 17% (IQR 5, 31%) and 11% (7, 18%) respectively. Neither were associated with presence of myocarditis. Conclusions and relevance: Myocarditis was present in a third patients with acute COVID-19, and the majority had inflammatory cell infiltration. Pneumonitis was ubiquitous, but this inflammation was not associated with myocarditis. The mechanism of cardiac pathology is non-ischaemic, and not due to a vasculitic process.

Giardia duodenalis captured the attention of Leeuwenhoek in 1681 while he was examining his own diarrheal stool, but, ironically, it did not really gain attention as a human pathogen until the 1960s, when outbreaks were reported. Key technological advances, including in vitro cultivation, genomic and proteomic databases, and advances in microscopic and molecular approaches, have led to an understanding that this is a eukaryotic organism with a reduced genome rather than a truly premitochondriate eukaryote. This has included the discovery of mitosomes (vestiges of mitochondria), a transport system with many of the features of the Golgi apparatus, and even evidence for a sexual or parasexual cycle. Cell biology approaches have led to a better understanding of how Giardia survives with two nuclei and how it goes through its life cycle as a noninvasive organism in the hostile environment of the lumen of the host intestine. Studies of its immunology and pathogenesis have moved past the general understanding of the importance of the antibody response in controlling infection to determining the key role of the Th17 response. This work has led to understanding of the requirement for a balanced host immune response that avoids the extremes of an excessive response with collateral damage or one that is unable to clear the organism. This understanding is especially important in view of the remarkable ranges of early manifestations, which range from asymptomatic to persistent diarrhea and weight loss, and longer-term sequelae that include growth stunting in children who had no obvious symptoms and a high frequency of postinfectious irritable bowel syndrome (IBS).

Objectives: Candida auris emerged as a human pathogen in 2009 and has subsequently been identified around the world as a cause of invasive candidiasis. We did an analysis from a single institution in order to analyze risk factors and outcomes for C. auris candidemia. Methods: Patients with candidemia were identified by the electronic medical record and reviewed for risk factors and outcome. Candida isolates were identified by Vitek2 as Candida haemulonii, but species determinations for 21 of the isolates using published molecular and proteomic methods identified all as C. auris. Findings: From September 2010 to December 2016, C. auris accounted for 38% of 201 patients with candidemia, while C. albicans contributed 25%. C. auris patients had been hospitalized longer (mean 32 days vs. 13 days; p

Background: Remifentanil and dexmedetomidine are common agents used in general anaesthesia, monitored anaesthesia care and critical care. When combined with inhaled or intravenous anaesthetic agents intra-operatively, they provide analgesia, lower general anaesthetic requirements and provide sedation and analgesia in the peri-operative period if indicated. Pharmacodynamically, they cause hypotension and bradycardia which are reversible if well managed. Past studies of these drugs have shown a significant proportion of patients with hypotension when compared with similar agents or in isolation. This study compares these two drugs on the effect of hypotension when used as adjuncts to general anaesthesia at low dose standard rate of infusions. Objective: To compare the proportion of hypotension episodes in a group of adult patients receiving dexmedetomidine infusion at 0.4mcg/kg/hr versus a group receiving remifentanil infusion at 0.2mcg/kg/min, severity of hypotension and physician interventions in each group. Methods: One hundred and four patients scheduled for elective surgery under general anaesthesia were randomized into two groups: Control group; received remifentanil infusion at 0.2mcg/kg/min Intervention group; received dexmedetomidine at 0.4mcg/kg/hr. General anaesthesia was standardized in both groups. The patients were blinded to the study. Baseline blood pressures of all patients were determined prior to induction. The patient’s demographic characteristics were recorded. The number of patients who developed hypotension, the frequency of hypotension and the physician interventions were recorded and analysed. Results: The age and gender characteristics were different between the two groups (p values

BackgroundMany studies have shown that Staphylococcus aureus is a leading cause of both community onset and hospital onset bloodstream infections. However, relatively little is known about the occurrence and outcome of S. aureus bacteremia in sub-Saharan Africa. A previous report indicated that S. aureus accounts for 16% of community onset and 6% of hospital onset bloodstream infections at Aga Khan University Hospital Nairobi (AKUHN). Data about the etiology of S. aureus bacteremia in sub-Saharan Africa will help optimize recognition and treatment. This study was performed in order to understand the etiologies and risk factors for S. aureus bacteremia in a sub-Saharan location.Materials and methodsA review of the electronic record of laboratory results from September 2010 through December 2018 identified 201 patients seen at AKUHN with S. aureus bacteremia. The source and/or focus of infection was identified and in-hospital mortality was determined. Cases with bacteremia after three days of hospitalization were considered hospital acquired. Community onset cases were divided into community acquired and health care associated.ResultsMost cases (71%; 143/201) were community onset, but only 41% (83/201) of these cases were community acquired. The most commonly identified foci of infection for community acquired bacteremia were musculoskeletal (25%; 21/83) and skin and soft tissue (24%; 20/83). The majority of health care associated (70%; 40/57) and hospital acquired cases (74%; 43/58) were associated with invasive vascular devices, with peripheral IVs being the most common for hospital acquired and dialysis catheters being the most common for health care associated infections. In-hospital mortality rates were 23% (19/83) for community acquired, 19% (11/57) for health care associated and 33% (19/58) for hospital acquired infections.ConclusionInvasive vascular devices were associated with a substantial portion of cases of S. aureus bacteremia and provide an important target for infection control efforts.

Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A–H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.

Background Vertebral involvement is a severe complication of infection caused by Coccidioides species. Methods We conducted a retrospective review of patients diagnosed with vertebral coccidioidomycosis at an academic medical center between 1996 and 2009. Results We identified 39 cases of vertebral coccidioidomycosis. Thirty-four patients (79%) were male, and 23 patients (61%) were black. Black patients were overrepresented in comparison with all other patients by a 50-fold odds ratio (95% confidence interval, 26-95). Only 8 patients (20%) were immunocompromised, including 7 who had received systemic steroids. The number of infected vertebrae ranged from 1 to 24; 8 patients (21%) had epidural involvement. All patients received a triazole as part of medical therapy, and 20 patients also received amphotericin B, typically early in the course. Twenty-six patients (67%) required surgery, 18 of whom also required hardware placement. The most common indication for surgery was pain, but 7 patients had neurologic compromise. No patients developed recurrent or refractory infection at the site of surgical debridement, but 6 patients experienced disease relapse after stopping antifungal therapy. Conclusion Vertebral infection caused by Coccidioides species requires a multispecialty approach that always includes medical therapy and frequently requires surgical intervention for debridement or stabilization. A favorable outcome can usually be achieved, but discontinuation of medical therapy is associated with a high risk of relapse, which can occur years later.

Alpha8-giardin (α8-giardin) is a member of the multi-gene α-giardin family in the intestinal parasitic protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium dependent binding to membranes that contain acidic phospholipids. In the present study, the antigenicity, hydrophilicity, flexibility, surface probability, and secondary structure of α8-giardin amino acids were predicted by bioinformatics applications. A specific anti-peptide antiserum, anti-P3, was used to determine the intracellular location of α8-giardin with confocal immunofluorescence microscopy and immunoelectron microscopy. The results indicated that α8-giardin was located on the plasma membrane and flagella, but not on the ventral disk. Reduction of α8-giardin transcript levels by ribozyme-mediated cleavage decreased trophozoite motility and growth rate, indicating the functional importance of α8-giardin to Giardia trophozoite biology.

Background Candida auris emerged as a human pathogen in 2009 and has subsequently been identified around the world as a cause of invasive candidiasis. Published clinical information on this organism consists primarily of case reports and small case series; thus, data from a single institution will allow us to examine risk factors for acquiring C. auris candidemia in comparison to other Candida species. Methods Aga Khan University Hospital Nairobi is a 280-bed referral center with 50 critical care beds. Candida species account for 34% of hospital acquired bloodstream infections (Maina et al., 2016). Blood cultures were monitored continuously using the Bactec and the VitekII was used for identification and susceptibility. The VitekII identified C. auris as Candida haemulonii, but species determinations were done for 21 of the isolates and all were identified as C. auris using published methods (Pfaller et al., 2012). Results From September 2010 to December 2016, 201 patients had 228 episodes of candidemia. Further analyses were performed only for first episodes. C. auris accounted for 38% of candidemia cases and 25% for C. albicans. A case–control analysis was done to compare patients with C. auris vs. Candida albicans fungemia. C. auris patients were more likely to be from critical care beds (78% vs. 52%; P = 0.003) and had been hospitalized longer (mean 33 days vs. 13 days; P < 0.001) prior to the positive blood culture. There was a trend toward more pre-existing renal failure (39% vs. 24%; P = 0.09) in C. auris patients and during the two weeks prior to candidemia, they were more likely to have central lines (84% vs. 54%; P ≤ 0.001). C. auris patients received a mean of 3.35 antibiotic classes vs. 2.6 for C. albicans (P = 0.02). 75% of C. auris patients received carbapenems vs. 54% for C. albicans (P = 0.02). Eighteen percent of C. auris patients had ≥14 days of candidemia, despite frequent lack of followup blood cultures. Prolonged candidemia was not associated with development of in vitro resistance. The crude mortality was 29%, compared with 36% for C. albicans and 39% for other Candida spp. (NS). Conclusion These findings suggest an opportunistic pathogen that may be less virulent, but difficult to eradicate and that control efforts should focus on antimicrobial usage. Disclosures M. Castanheira, Allergan: Research Contractor, Research support. M. A. Pfaller, Allergan: Research Contractor, Research support.

Pneumocystis spp. infect the lungs of multiple mammalian species and cause disease in immunosuppressed individuals. The Pneumocystis isolates that have been studied to date fall into two major clades, those from primates and those from rodents. Within each of these clades, different species have been described on the basis of host specificity and differences in sequence and morphology. Here, we demonstrate that dexamethasone immunosuppression consistently results in histologically apparent lung infection in gerbils (28/35 animals). Sequence analysis of the 18S, 5.8S and internal transcribed spacer regions of the rDNA and a portion of the mitochondrial large subunit rDNA demonstrated that this gerbil Pneumocystis is grouped with other rodent Pneumocystis spp., but is distinct from them. Our results suggest that gerbil Pneumocystis differs sufficiently from Pneumocystis species found in other rodents to be considered a separate species.

Purpose Extrapulmonary dissemination of Coccidioides species is associated with significant morbidity and mortality. The clinical manifestations vary widely according to the host, the severity of illness, and location of dissemination. The morbidity and mortality can be reduced by early recognition and treatment, which in turn depends on understanding the spectrum and presentation of disease. Methods We performed a retrospective analysis of 150 cases with extrapulmonary nonmeningeal disease seen from 1996 to 2007 at a referral medical center in an endemic region. Results Hematogenous dissemination was associated with high mortality and occurred primarily in immunocompromised patients, but only 30% of patients with more limited forms of dissemination were immunocompromised. In keeping with prior studies, there was a preponderance of males (nearly 2:1) and people of African or Asian (especially Pacific Islanders) descent. In contrast, Hispanics and diabetics were not at increased risk. Serology was frequently negative in immunocompromised patients, but the diagnosis could be established by isolation of the organism in culture, or in histologic or cytologic specimens. Conclusions Although coccidioidomycosis is a great imitator, the diagnosis can usually be made readily if a high level of suspicion is maintained and appropriate diagnostic testing is performed. In most patients, that will include serologic testing in addition to cultures and histology or cytology of appropriate samples.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Giardia lamblia is an early branching eukaryote, and although distinctly eukaryotic in its cell and molecular biology, transcription and translation in G. lamblia demonstrate important differences from these processes in higher eukaryotes. The cyclic octapeptide amanitin is a relatively selective inhibitor of eukaryotic RNA polymerase II (RNAP II) and is commonly used to study RNAP II transcription. Therefore, we measured the sensitivity of G. lamblia RNAP II transcription to α-amanitin and found that unlike most other eukaryotes, RNAP II transcription in Giardia is resistant to 1 mg/ml amanitin. In contrast, 50 μg/ml amanitin inhibits 85% of RNAP III transcription activity using leucyl-tRNA as a template. To better understand transcription in G. lamblia, we identified 10 of the 12 known eukaryotic rpb subunits, including all 10 subunits that are required for viability in Saccharomyces cerevisiae. The amanitin motif (amanitin binding site) of Rpb1 from G. lamblia has amino acid substitutions at six highly conserved sites that have been associated with amanitin resistance in other organisms. These observations of amanitin resistance of Giardia RNA polymerase II support previous proposals of the mechanism of amanitin resistance in other organisms and provide a molecular framework for the development of novel drugs with selective activity against G. lamblia.

Giardia lambliais medically important as a cause of diarrhea and malabsorption throughout the world and is thought to be one of the earliest-branching eukaryotes on a phylogenetic tree. Nevertheless, the mechanisms of inheritance are largely unknown. The trophozoites ofGiardiaand other diplomonads are interesting in their possession of two nuclei that are identical or similar in several respects. They replicate at nearly the same time, have similar quantities of DNA, and are both transcriptionally active. We used fluorescence in situ hybridization to demonstrate that genes from each of the five chromosomes are found in both nuclei, confirming that each nucleus has at least one complete copy of the genome. This raises a second question. The alleles of a gene in different nuclei are expected to accumulate different mutations, but surprisingly, the degree of heterozygosity in a clone is very low. One possible mechanism for eliminating sequence differences between nuclei is that each daughter cell receives two copies of the same nucleus at cell division. We used trophozoites with a plasmid transfected into a single nucleus to demonstrate that the two nuclei are partitioned equationally at cytokinesis. The mechanism(s) by which homozygosity is maintained will require further investigation.

As part of the ISO 15189:2007 accreditation process, the Aga Khan University Hospital Nairobi laboratory became the first internationally accredited hospital laboratory in sub-Saharan Africa outside South Africa in 2011 through the South Africa National Accreditation System.Seven preanalytic, 10 analytic, eight postanalytic, and five administrative performance parameters were monitored from 2009 to 2012 to measure the impact of the accreditation process.Most measures in all four categories showed substantial improvement. The seven preanalytic measures all showed major improvement-between a quarter and a half sigma. Real but less dramatic improvement appeared in analytic and postanalytic measures, but greater than one sigma decrease in analytic "procedure violations" and a three-quarter sigma decrease in excessive turnaround time were noted in these categories. Administrative improvements included dramatic decreases in misdirected and missing reports and complaints.This study demonstrates the correlation of the accreditation process with improvement in quality measures in a low-resource region.

Study Objectives. To determine the effect of a high-fat meal, orange juice, and antacids on absorption of a single oral dose of cycloserine and to estimate its population pharmacokinetic parameters. Design. Randomized, four-period, crossover study. Setting. Clinical research center. Patients. Twelve healthy volunteers. Interventions. Subjects received single doses of cycloserine 500 mg after a 12-hour fast (reference), with a high-fat meal, with orange juice, and with antacids. They also received clofazimine 200 mg, ethionamide 500 mg, and p-aminosalicylic acid granules 6000 mg. Measurements and Main Results. Plasma samples were collected for 48 hours and assayed by validated high-performance capillary electrophoresis assay. Concentration-time data were analyzed with noncompartmental, one-compartment, and population methods. The maximum concentration (Cmax) of cycloserine was decreased (p=0.02) by the high-fat meal. No other statistically significant differences were observed for Cmax and area under the curve from time zero to infinity across the four treatments. The high-fat meal significantly (p

SUMMARYGiardia lambliais a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from otherGiardiaspecies by light or electron microscopy. The two major genotypes ofG. lambliathat infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal thatGiardiaspp. are among the most primitive eukaryotes.G. lambliaprobably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5′ and 3′ untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from onevspgene to another have not been associated with sequence alterations or gene rearrangements. TheGiardiagenome project promises to greatly increase our understanding of this interesting and enigmatic organism.

This study was conducted in order to (i) determine the effect of food, orange juice, or antacids on the absorption of a single oral 500-mg dose of ethionamide (ETA) in healthy volunteers, including an assessment of bioequivalence, and (ii) determine ETA population pharmacokinetic (PK) parameters. The pharmacokinetics of ETA in serum was determined for 12 healthy males and females in a randomized, four-period crossover study. Volunteers received single 500-mg doses of ETA either on an empty stomach (reference) or with food, orange juice, or antacids. Serum samples were collected for 48 h and assayed by high-performance liquid chromatography. Data were analyzed by noncompartmental and population methods. Mean test/reference ratios and 90% confidence intervals were determined. No statistically significant differences were seen in the maximum concentration of ETA ( C max ), time to maximum concentration ( T max ), or area under the concentration-time curve from 0 h to infinity (AUC 0–∞ ) between the four treatments ( P > 0.05 by analysis of variance). The least-squares mean ratios (with confidence intervals in parentheses) for C max were 105% (81.2 to 135%) after orange juice, 94% (72.8 to 121%) after food, and 88% (68.4 to 114%) after antacids. The least-squares mean ratios (with confidence intervals is in parentheses) for AUC 0–∞ were 91% (72.7 to 115%) after orange juice, 96% (76.4 to 121%) after food, and 95% (75.5 to 120%) after antacids. The mean T max was slightly prolonged following antacid or food administration (2.3 to 2.6 h) compared to administration on an empty stomach or with juice (1.7 to 1.9 h). The median population PK parameters were as follows: K a = 0.37 to 0.48 h −1 , V/F = 2.0 to 2.8 liters/kg, CL/ F = 56.5 to 72.2 liters/h, and terminal half-life = 1.7 to 2.1 h, where K a is the absorption rate constant, V is the volume of distribution, and CL is clearance. The PK behavior of ETA was not significantly modified by the different conditions studied. Mean ratios for AUC ranged from 0.91 to 0.96 for the orange juice, food, and antacid treatments, indicating a minimal effect on relative bioavailability. ETA can, therefore, be administered with food if tolerance is an issue.

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis . The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis . In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.

Giardia lamblia (also Giardia duodenalis, Giardia intestinalis) isolates have been variably divided into two or three genotypes by different investigators. We have compared the triose phosphate isomerase sequences of the three genotypes (Groups 1, 2, and 3) described by Nash and shown that Groups 1 and 2 are similar, while Group 3 is markedly different from Groups 1 and 2, indicating that Group1/2 and Group 3 correspond to the two major genotypes identified by other investigators. We have also analysed three Chinese isolates and showed that two fit into Group 3, while the third contained a mixture of Groups 1 and 3 isolates. These results confirm the relatedness of G. lamblia isolates from throughout the world, and established the feasibility of using DNA amplification and sequence analysis for detecting mixed isolates.

The surfaces of Giardia lamblia trophozoites contain one of a set of variant-specific surface proteins. The genes encoding these proteins are highly conserved at the 3' terminus, but frequently demonstrate little similarity in the remainder of the coding region. This report describes a family of vsp genes highly similar to a repeat-containing vsp gene (vspC5) at the 5' coding and flanking regions, but which diverge abruptly from vspC5 in the first repeat and do not themselves contain full copies of the repeat. This observation suggests the possibility that recombination among different vsp genes may have played a role in development of the vsp gene repertoire.

The Giardia lamblia genome consists of 12 Mb divided among 5 chromosomes ranging in size from approximately 1 to 4 Mb. The assembled contigs of the genotype A1 isolate, WB, were previously mapped along the 5 chromosomes on the basis of hybridization of plasmid clones representing the contigs to chromosomes separated by PFGE. In the current report, we have generated an MluI optical map of the WB genome to improve the accuracy of the physical map. This has allowed us to correct several assembly errors and to better define the extent of the subtelomeric regions that are not included in the genome assembly.

Our understanding of the transcription and recombination of Giardia has evolved substantially with recent experimental, genomic and population genetic data. Earlier assumptions that Giardia is an asexual organism have been called into serious question by the fi nding of the meiotic genes in the genome, the documentation that genetic material can be transferred from one nucleus to another during the process of encystation, and the evidence from population genetic data supporting the occurrence of meiotic recombination within Genotype A2. The different genotypes vary signifi cantly in the way they handle introduction of plasmid DNA and their levels of allelic heterozygosity, indicating differences in their processes of DNA recombination. Transcription in Giardia is typical of that of other eukaryotes with its use of a 5′ cap, polyadenylated tail, and its (rare) use of introns. However, it follows the minimalist approach that Giardia follows in many areas of metabolism, with an apparently abbreviated set of transcription factors and promoters that vary signifi cantly from those of other eukaryotes and may also be simplifi ed in their structure and function. The answers to how Giardia handles transcription and recombination with its minimalist approach may also shed light on the more complex systems used by other organisms.

Background. Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. Methods. Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. Results. Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. Conclusions. In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.

Giardia species are flagellated parasites of vertebrates and belong to the diplomonads, most of which have two nuclei. These organisms were classified among the earliest branching eukaryotes on the basis of small subunit rDNA sequences and their lack of many canonical organelles. However, some of these organelles have subsequently been identified in rudimentary form, such as Golgi that become apparent during encystation. One of the "missing" organelles has been the nucleolus, the site of rRNA synthesis, since it was not identified in earlier ultrastructural studies. In the current study, we visualized in-vitro-grown Giardia lamblia trophozoites by transmission electron microscopy and laser scanning confocal microscopy. We found that each of the two nuclei contains a single small and deeply stained granular nucleolus, thus demonstrating that Giardia does indeed have nucleoli.

Giardia lamblia is an early branching eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In most organisms, glycolysis is catalyzed by pyruvate kinase (PK), allowing the generation of two ATP molecules from one molecule of pyruvate. Giardia has both PK and pyrophosphate-dependent pyruvate phosphate dikinase (PPDK), which catalyzes the generation of five ATP molecules from pyruvate by pyrophosphate-dependent glycolysis and offers a potential selective advantage. In order to evaluate the importance of pyrophosphate-dependent glycolysis, we used ribozyme-mediated cleavage of the PPDK transcript to decrease PPDK transcript levels to 20% of normal. The accompanying decrease in PPDK enzyme activity decreased ATP levels to 3% of normal and increased glycogen deposition, confirming the importance pyrophosphate-mediated glycolysis that was previously suggested by cell lysate studies. PPDK is not found in vertebrates, so specific inhibitors may be useful for treatment of infections caused by anaerobic protists that depend on pyrophosphate-dependent glycolysis.

SummaryGiardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes [1]. However, the discovery of at least rudimentary forms of certain features, such as Golgi and mitochondria, has refuted the proposal that its emergence from the eukaryotic lineage predated the development of certain eukaryotic features. The recent recognition of many of the genes known to be required for meiosis in the genome has also cast doubt on the idea that Giardia is primitively asexual, but so far there has been no direct evidence of sexual reproduction in Giardia, and population data have suggested clonal reproduction. We did a multilocus sequence evaluation of the genotype A2 reference strain, JH, and five genotype A2 isolates from a highly endemic area in Peru. Loci from different chromosomes yielded significantly different phylogenetic trees, indicating that they do not share the same evolutionary history; within individual loci, tests for recombination yielded significant statistical support for meiotic recombination. These observations provide genetic data supportive of sexual reproduction in Giardia.

Giardia lamblia, a protozoan parasite inhabiting the small intestine, is a common infection worldwide that frequently results in chronic diarrhea, malabsorption and upper gastrointestinal symptoms. Giardia undergoes surface antigenic variation in humans and animal model infections, a phenomenon that may account for both chronicity of infections and the relatively broad mammalian host specificity with genotypically identical organisms found in humans, cats, beavers, and other mammals. The variant-specific surface proteins (VSPs) are an unusual family of related cysteine-rich proteins, from 50 kD to over 200 kD in size, that coat the surface of the trophozoite. Only one VSP of the estimated 150 or more vsp genes is expressed on an individual at any specific time. However, the repertoires of vsp genes may differ depending on the genetic group. VSPs switch spontaneously every 6–12 generations although some Giardia also switch during encystation/excystation. All VSPs have a high cysteine content of about 12 % cysteines that are mostly present as CXXC motifs as well as a highly conserved C-terminus, a surface Zn finger motif, a GGCY motif and other common features. The biological function(s) of VSPs are uncertain but they undergo both immune and biological selection. The molecular mechanism(s) involved in antigenic variation are unknown but because there is the absence of gene movement or gene mutations and only one of 4 practically identical alleles (Giardia are tetraploid) is expressed; epigenetic processes are likely involved. Further studies of the mechanism of antigenic variation and the biological role of the VSPs promise to contribute to our understanding of G. lamblia and its pathogenesis.

Rhinocerebral mucormycosis with intracranial involvement has a high mortality. The standard therapy consists of aggressive surgical débridement accompanied by high doses of amphotericin B deoxycholate. Even with this therapy, the mortality rate has been 48% in the series reported since 1980. We treated a 60-year-old diabetic woman with rhinocerebral mucormycosis involving the cavernous sinus whose infection responded to medical therapy with amphotericin B lipid complex. To our knowledge, this is the only well-documented medical cure of a patient with rhinocerebral mucormycosis and intracranial involvement.

List of contributors Preface 1: Epidemiology 1. Epidemiology and zoonotic potential of Giardia infections R.C. Andrew Thompson 2. Entamoeba histolytica and Entamoeba dispar, the non-identical twins C. Graham Clark 3. Epidemiology and strain variation of Cryptosporidium Rachel M. Chalmers and David P. Casemore 4. Cyclospora cayetanensis: An emergent and still perplexing coccidian parasite Charles R. Sterling and Ynes R. Ortega 2: Host parasite interactions 5. Antigenic variation of the VSP genes of Giardia lamblia Rodney D. Adam and Theodore E. Nash 6. Pathogenesis and immunity to Entamoeba histolytica Jessica L. Tarleton and William A. Petri Jr. 7. Innate and T cell-mediated immune responses in cryptosporidiosis Carol R. Wyatt and Vincent McDonald 3: Treatment and Control 8. Rationale approaches to treating Cryptosporidium, Cyclospora, Giardia and Entamoeba Jan R. Mead and Pablo Okhuysen 9. Inactivation and removal of enteric protozoa in water Frank W. Schaefer, III, Marilyn M. Marshall and Jennifer L. Clancy 10. Monitoring of Giardia and Cryptosporidium in water in the UK and US Jennifer L. Clancy and Paul R. Hunter 4: Genomics 11.Entanioeba histolytica genome James J. McCoy and Barbara J. Mann 12. Ciyptosporidium parvum genomics: Impact on research and control Guan Zhu and Mitchell S. Abrahamsen Index

OBJECTIVE: To determine the pharmacokinetics and relative bioavailability of para-aminosalicylic acid (PAS) granules. DESIGN: Phase I pharmacokinetics study. SETTING: University of Arizona School of Pharmacy. PARTICIPANTS: Sixteen healthy male and female volunteers aged 36 ± 8 years. INTERVENTIONS: Subjects received single doses of PAS granules (6 g) combined with cycloserine 500 mg, clofazimine 200 mg, ethionamide 500 mg, and pyridoxine 100 mg. Drugs were given on an empty stomach after an overnight fast (reference) with high-fat food, with orange juice, and with antacids. MEASUREMENTS AND RESULTS: Four subjects did not complete all four treatments due to adverse events or personal reasons. Plasma and urine samples were collected for 48 hours and measured by a validated HPLC assay. Pharmacokinetic data analysis was performed with WinNonlin using noncompartmental methods and a one-compartmental model. Bioequivalence testing was performed using the mean ratios of the maximum concentrations (Cmax) and AUC0-∞ of PAS, with 90% confidence intervals. Compared with the fasted condition, food increased Cmax 1.5–fold and AUC0-∞ 1.7-fold, and it doubled the time to maximum concentration (tmax). The least-squares mean ratios (treatment/reference) for Cmax were 0.90 (58% to 139% CI), 1.16 (75% to 179% CI), and 0.82 (52% to 127% CI) with orange juice, food, or antacid treatment, respectively. Corresponding ratios for AUC0-∞ were 1.05 (71% to 155% CI), 1.52 (103% to 224% CI), and 0.84 (57% to 125% CI), respectively. CONCLUSIONS: Food significantly enhanced the absorption of PAS, while orange juice and antacids had minor effects.

Giardia lamblia trophozoites are unable to carry out de novo lipid synthesis. It is therefore likely that lipids are acquired from the small intestine of the host, in which the trophozoites are exposed to free and conjugated fatty acids, various sterols, phospholipids, bile acids, and bile-lipid mixed micelles. Here we show that G. lamblia is capable of taking up exogenous phosphatidylcholine (PC), phosphatidylinositol (PI), sphingomyelin (SM), cholesterol, ceramide (Cer), and fatty acids. Results from epifluorescence and high-resolution confocal microscopy suggest that fluorescent analogs of SM and PC were accumulated in the plasma membranes, whereas palmitic acid and Cer were localized intracellularly. Interestingly, many of these analogs were also concentrated in perinuclear regions. Similar labeling patterns were observed when the fluorescent analogs were delivered to the parasite via liposomes. To test whether G. lamblia was capable of esterifying exogenous fatty acids into membrane or cellular phospholipids, trophozoites were pulse-labeled with 3H-labeled palmitic or myristic acids and the phospholipids analyzed by thin-layer chromatography. Results document that G. lamblia was able to incorporate exogenous fatty acids into various phospholipids, i.e., PI, PC, PE, and PG. Interestingly, a major portion of radiolabeled fatty acids was incorporated into PG, a phospholipid characteristic of prokaryotic membranes.

Animals are commonly considered to be potential sources for Giardia lamblia infections in humans, but the extent of zoonotic transmission of G. lamblia remains controversial because of inadequate understanding of its epidemiology. A better understanding of the epidemiology of G. lamblia may be facilitated by a more effective means for classifying G. lamblia isolates. To develop a sequence-based classification system, the gene encoding the metabolic enzyme triose phosphate isomerase (tim) was sequenced from a number of G. lamblia isolates of various host origins. Restriction enzymes were identified that can distinguish among isolates without the need for sequencing, simplifying the application of this approach to the epidemiologic investigation of giardiasis. Isolates from a previously reported epidemic of giardiasis were accurately classified by this technique, further verifying its utility for epidemiologic investigation.

Giardia lamblia trophozoites express on their surfaces one of a set of cysteine-rich antigenically variant proteins, called variant-specific surface proteins, which comprise the majority of proteins detected by surface labeling. While these VSP proteins may be immunodominant proteins important in the host immune response to G. lamblia, the ability to switch expression from one VSP to another may provide a means for the trophozoites to avoid the host immune response. The first VSP characterized, VSPA6 (from the A6 clone of the WB isolate, originally termed CRP170), contains 18-23 copies of a 65 amino acid repeat. We have now used the repeat as a probe to isolate from a WBA6 genomic library two genes related to vspA6 (called vspA6-S1, vspA6-S2). Sequence analysis of the vspA6-S1 gene revealed nearly two complete copies of the 195 bp repeat and substantial nucleotide and translated amino acid similarity in the coding regions 5' and 3' to the repeats. The vspA6-S2 gene, while still related, showed greater divergence from vspA6 than vspA6-S1 in the nonrepeat coding region and contained nearly four copies of a 201 bp repeat that was 75% identical to the 195 bp vspA6 repeat. These results suggest that gene duplication followed by divergence has played a key role in the generation of the vsp gene repertoire.