Illness In Immunocompromised Individuals Research Articles

In silico and in vivo evaluation of the anti-cryptosporidial activity of eugenol.

Cryptosporidiosis is an opportunistic parasitic disease widely distributed worldwide. Although Cryptosporidium sp. causes asymptomatic infection in healthy people, it may lead to severe illness in immunocompromised individuals. Limited effective therapeutic alternatives are available against cryptosporidiosis in this category of patients. So, there is an urgent need for therapeutic alternatives for cryptosporidiosis. Recently, the potential uses of Eugenol (EUG) have been considered a promising novel treatment for bacterial and parasitic infections. Consequently, it is suggested to investigate the effect of EUG as an option for the treatment of cryptosporidiosis. The in silico bioinformatics analysis was used to predict and determine the binding affinities and intermolecular interactions of EUG and Nitazoxanide (NTZ) toward several Cryptosporidium parvum (C. parvum) lowa II target proteins. For animal study, five groups of immunosuppressed Swiss albino mice (10 mice each) were used. Group I was left uninfected (control), and four groups were infected with 1,000 oocysts of Cryptosporidium sp. The first infected group was left untreated. The remaining three infected groups received NTZ, EUG, and EUG + NTZ, respectively, on the 6th day post-infection (dpi). All mice were sacrificed 30 dpi. The efficacy of the used formulas was assessed by counting the number of C. parvum oocysts excreted in stool of infected mice, histopathological examination of the ileum and liver tissues and determination of the expression of iNOS in the ileum of mice in different animal groups. treatment with EUG resulted in a significant reduction in the number of oocysts secreted in stool when compared to infected untreated mice. In addition, oocyst excretion was significantly reduced in mice received a combination therapy of EUG and NTZ when compared with those received NTZ alone. EUG succeeded in reverting the histopathological alterations induced by Cryptosporidium infection either alone or in combination with NTZ. Moreover, mice received EUG showed marked reduction of the expression of iNOS in ileal tissues. Based on the results, the present study signified a basis for utilizing EUG as an affordable, safe, and alternative therapy combined with NTZ in the management of cryptosporidiosis.

Spanish HCMV Seroprevalence in the 21st Century.

Human cytomegalovirus (HCMV) is linked to age-related diseases like cardiovascular disease, neurodegenerative conditions, and cancer. It can also cause congenital defects and severe illness in immunocompromised individuals. Accurate HCMV seroprevalence assessment is essential for public health planning and identifying at-risk individuals. This is the first HCMV seroprevalence study conducted in the general Spanish adult population in 30 years. We studied HCMV seroprevalence and HCMV IgG antibody titres in healthy adult donors (HDs) and HCMV-related disease patients from 2010 to 2013 and 2020 to 2023, categorized by sex and age. We compared our data with 1993 and 1999 studies in Spain. The current HCMV seroprevalence among HDs in Spain is 73.48%. In women of childbearing age, HCMV seroprevalence has increased 1.4-fold in the last decade. HCMV-seropositive individuals comprise 89.83% of CVD patients, 69% of SMI patients, and 70.37% of COVID-19 patients. No differences in HCMV seroprevalence or HCMV IgG antibody titres were observed between patients and HDs. A significant reduction in Spanish HCMV seroprevalence among HDs was observed in 1993. However, women of childbearing age have shown an upturn in the last decade that may denote a health risk in newborns and a change in HCMV seroprevalence trends.

COVID-19 Therapeutics for Nonhospitalized Patients—Updates and Future Directions

In this Viewpoint, the authors summarize the therapeutic landscape for COVID-19, discuss who is most likely to benefit from treatment, provide an update on managing illness in immunocompromised individuals, and highlight how to improve COVID-19 treatment.

Immunogenic multi-epitope-based vaccine development to combat cyclosporiasis of immunocompromised patients applying computational biology method.

Cyclospora cayetanensis infections, also known as cyclosporiasis, persist to be the prevalent emerging protozoan parasite and an opportunist that causes digestive illness in immunocompromised individuals. In contrast, this causal agent can affect people of all ages, with children and foreigners being the most susceptible populations. For most immunocompetent patients, the disease is self-limiting; in extreme circumstances, this illness can manifest as severe or persistent diarrhea as well as colonize on secondary digestive organs leading to death. According to recent reports, worldwide 3.55% of people are infected by this pathogen, with Asia and Africa being more prevalent. For the treatment, trimethoprim-sulfamethoxazole is the only licensed drug and does not appear to work as well in some patient populations. Therefore, the much more effective strategy to avoid this illness is immunization through the vaccine. This present study uses immunoinformatics for identifying a computational multi-epitope-based peptide vaccine candidate for Cyclospora cayetanensis. Following the review of the literature, a highly efficient, secure, and vaccine complex based on multi-epitopes was designed by utilizing the identified proteins. These selected proteins were then used to predict non-toxic and antigenic HTL-epitopes, B-cell-epitopes, and CTL-epitopes. Ultimately, both a few linkers and an adjuvant were combined to create a vaccine candidate with superior immunological epitopes. Then, to establish the vaccine-TLR complex binding constancy, the TLR receptor and vaccine candidates were placed into the FireDock, PatchDock, and ClusPro servers for molecular docking and iMODS server for molecular-dynamic simulation. Finally, this selected vaccine construct was cloned into Escherichia coli strain-K12; thus, the constructed vaccines against Cyclospora cayetanensiscould improve the host immune response and can be produced experimentally.

450: FUSARIUM OXYSPORUM-INDUCED ACUTE RESPIRATORY DISTRESS SYNDROME IN AN IMMUNOCOMPETENT HOST

Introduction: Fusarium species is an environmental mold that historically causes systemic illness in immunocompromised individuals, predominantly those with acute leukemia or allogenic stem cell transplant recipients. Here we present an immunocompetent patient with acute respiratory distress syndrome (ARDS) secondary to Fusarium oxysporum. Description: A 68 y.o. male with no significant past medical history was admitted for acute hypoxic respiratory failure due to multifocal pneumonia. After initial improvement on antibiotics, his respiratory status decompensated to the point of mechanical ventilation with low tidal volume for ARDS. Cell count with differential from bronchoalveolar lavage revealed 99 nucleated cells with 44% neutrophils, 27% lymphocytes, 2% eosinophils, and 5000 red blood cells. Additional studies were negative for viral, fungal, bacterial and malignant causes. Connective tissue causes were ruled out. High-dose steroids were started for possible cryptogenic organizing pneumonia, but the patient failed to recover. Further inquiry revealed that he was caring for two red squirrels, one of which he attempted mouth to mouth resuscitation. Infectious disease was consulted and began anti-fungal therapy given his exposure history. Extensive infectious workup including testing for tularemia, brucella, bartonella, hantavirus, and lymphocytic choriomeningitis virus was negative. Home well water testing was negative. Karius Test™ was performed on serum and positive for 11 pathogens, HSV-1 and Fursarium oxysporum having the highest concentrations. Consulting teams determined that the patient’s presentation was most consistent with a fungal pneumonia, so treatment was narrowed to isavuconazole. He was eventually discharged on voriconazole and a steroid taper. Discussion: There are few reports of Fusarium species related ARDS. Extensive literature review showed only one previous case of ARDS secondary to Fusarium oxysporum. Because of the rarity of this infection, definitive diagnosis is often delayed with send-out testing. This leads to treatment delay of an already highly resistant fungus. Even if an early diagnosis is made, anti-fungal therapies are limited to amphotericin and voriconazole. For this reason, early identification is integral to improving patient outcomes.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles

Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

Scleredema — Rare skin manifestation in diabetic

Scleredema is a connective tissue disorder of unknown etiology. A 44 years diabetic, obese female patient, admitted with edema feet, erythematous, indurated hyper pigmentation with ascending progression to trunk which rapidly transformed to painless dark pigmented blister and hard non-pitting edema of anterior abdominal wall. In a week time. She was managed with broad spectrum antibiotics, was aggressively diuresed along with albumin infusion. Her skin biopsy was done which revealed scleredema. She had poor LV function due to volume overload or cardiomyopathy. After initial improvement on 14 day, she suddenly developed fulminant septicemia. Within hours, she displayed multi organ dysfunction and the heart and kidney which were already delicately balanced, decompensated. There was intractable arrhythmias and severe LV dysfunction, renal failure and ischemic hepatitis following severe hypotension. Scleredema in diabetic subjects, can be an ominous sign for producing fulminant multiorgan dysfunction and can present as cardiomyopathy, myocarditis, liver dysfunction etc., and these patients are more prone to infections or may be vice versa. Scleredema can be considered as a marker of fulminant illness in immunocompromised individuals. There is a need for treatment protocols or research into this a rather enigmatic but rare illness.

Trends of seropositivity and clinical spectra of Toxoplasmosis- A two-year observational study from a tertiary care centre in North India

Background: Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is highly prevalent in man and other warm-blooded animals. Most of the affected adults do not manifest any serious illness, but it can cause mental retardation and blindness in congenitally infected children and serious illness in immunocompromised individuals. We aimed to study the disease burden and clinical manifestations of Toxoplasma seropositive patients in the hilly region of North India. Methods: This is a hospital based retrospective cross-sectional study conducted between January-2018 and December-2019.Sera of suspected cases of toxoplasmosis were examined for presence of anti-Toxoplasma antibodies (IgG and IgM) by enzyme-linked immunosorbent assay. Antibody index of >1.1 was interpreted as positive. Clinical data were retrieved from medical records maintained in the department. Result: Out of 174 included cases, 70 (40.22%) cases showed positivity to both IgG and IgM antibodies. Most of the suspected cases were sent from the Pediatrics department followed by Departments of Obstetrics and Gynecology, Ophthalmology and General Medicine. Majority of the patients (45.7%) presented with mild non-specific symptoms like fever, malaise and generalized weakness and lymphadenopathy in 25.7%. Ocular and neurological manifestations were observed in 21.4% and 20% respectively. Other rare presentations were hepatic, cutaneous, pulmonary and endocrine diseases. More than one clinical feature could be found in one patient. Co-infection with HIV and Tuberculosis was found in 8.6% and 5.7% respectively. 20% cases were associated with congenital anomalies; while 18.6% cases were detected in routine anti-natal screening. Conclusion: Toxoplasmosis is highly prevalent in this hilly region showing wide variety of clinical manifestations. Therefore, prevention and control policies mainly targeting the anti-natal cases are the need of the hour.

Diagnostic Features of Blastocystis Life Cycle Forms in the Small Intestine in an HIV-Infected Patient.

Blastocystis spp. are parasites of the intestinal tract found in many hosts including humans. This pathogen is commonly found in immunocompetent in asymptomatic individuals and in patients with gastrointestinal and extra-intestinal symptoms. Recently, it has been implicated as an important cause of diarrheal illness in immunocompromised individuals, including HIV-infected patients. At least six life cycle stages have been described in faeces and cultures, namely vacuolar, granular, multi-vacuolar, avacuolar, ameboid and cyst forms. The aim of the present study was to describe the histological findings of Blastocystis infection in an adult HIV-infected patient with gastrointestinal symptoms. Parasitological techniques and PCR were applied to stool samples. Histological analysis was performed on duodenal biopsy specimens. Standard parasitological methods revealed vacuolar, granular, cyst and multi-vacuolar forms of Blastocystis in faecal samples with the presence of Blastocystis DNA being confirmed by PCR. DNA sequencing revealed Blastocystis subtype ST1. Histological findings in duodenal samples showed an inflammatory infiltrate with plasma cells and lymphocytes. We identified cyst, granular, ameboid and multi-vacuolar forms in the lumen. To our knowledge, there are no previous peer review reports describing these four different forms of Blastocystis in histological sections from the lumen and the brush border of the enterocytes.

Enabling Cost-Effective Screening for Antimicrobials against Listeria monocytogenes in Ham

Ready-to-eat meat products, such as deli ham, can support the growth of Listeria monocytogenes (LM), which can cause severe illness in immunocompromised individuals. The objectives of this study were to validate a miniature ham model (MHM) against the ham slice method and to screen antimicrobial combinations to control LM on ham by using response surface methology (RSM) as a time- and cost-effective high-throughput screening tool. The effect of nisin (Ni), potassium lactate and sodium diacetate, lauric arginate (LAG), lytic bacteriophage (P100), and ε-polylysine (EPL) added alone, or in combination, were determined on the MHM over 12 days of storage. Results showed the MHM accurately mimics the ham slice method because no statistical differences were found (P = 0.526) in the change of LM cell counts in MHM and slice counts after 12 days of storage at 4°C for treated and untreated hams. The MHM was then used to screen antimicrobial combinations by using an on-face design and three center points in a central composite design. The RSM was tested by using a cocktail of five LM strains isolated from foodborne disease outbreaks. Three levels of the previously mentioned antimicrobials were used in combination for a total of 28 runs performed in triplicate. The change of LM cell counts were determined after 12 days of storage at 4°C. All tested antimicrobials were effective on reducing LM cell counts on ham when added alone. A significant antagonistic interaction (P = 0.002) was identified by the RSM between LAG and P100, where this antimicrobial combination caused a 2.2-log CFU/g change of LM cell counts after 12 days of storage. Two interactions, between Ni and EPL (P = 0.058), and Ni and P100 (P = 0.068), showed possible synergistic effects against LM on the MHM. Other interactions were clearly nonsignificant, suggesting additive effects. In future work, the developed MHM in combination with RSM can be used as a high-throughput method to analyze novel antimicrobial treatments against LM.

Predominance and association risk of Blastocystis hominis subtype I in colorectal cancer: a case control study

BackgroundBlastocystis, a genetically diverse intestinal parasite with controversial pathogenic potential, has increasingly been incriminated for diarrheal illness in immunocompromised individuals including colorectal cancer (CRC) patients. The aim of the current study was to assess the possible association between Blastocystis infection and CRC condition in Makkah, Saudi Arabia (KSA).MethodsStool samples were collected from 80 non-cancer (NC) and 138 cancer subjects including 74 CRC patients and 64 patients with other cancers outside gastrointestinal tract (COGT). Molecularly confirmed Blastocystis isolates were genetically grouped and subtyped using multiplex polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) and sequence-tagged site primers-based PCR (PCR-STS), respectively.ResultsBlastocystis hominis were confirmed in 29.7, 25 and 15% among CRC, COGT and NC patients, respectively. Obtained Blastocystis isolates were initially categorized into 2 groups (A and C), which were subsequently subtyped into 3 different subtypes; subtype-I (38%), subtype-II (44%) and subtype-V (22%). Interestingly, subtype-I was the most predominantly detected subtype (54.5%) among CRC patients with a significant association risk (COR 7.548; 95% CI: 1.629–34.987; P = 0.004).ConclusionTo the best of our knowledge, the current study is the first to provide genetic insights on the prevalence of Blastocystis hominis among CRC patients in Makkah, KSA. Moreover, the study suggests for a possible association between subtype-I of Blastocystis hominis and CRC, which could indicate a potential influence of Blastocystis on CRC condition. Further studies are required to confirm this association risk and to investigate the possible underlying mechanism of postulated carcinogenic influence of Blastocystis hominis subtype-I.

Investigating the Determinants of Toxoplasma gondii Prevalence in Meat: A Systematic Review and Meta-Regression.

BackgroundToxoplasma gondii is one of the most widespread parasites in humans and can cause severe illness in immunocompromised individuals. However, its role in healthy people is probably under-appreciated. The complex epidemiology of this protozoan recognizes several infection routes but consumption of contaminated food is likely to be the predominant one. Among food, consumption of raw and undercooked meat is a relevant route of transmission, but the role of different meat producing animal species and meats thereof is controversial.ObjectivesThe aim of the present work is to summarize and analyse literature data reporting prevalence estimates of T. gondii in meat animals/meats.Data SourcesWe searched Medline, Web of Science, Science Direct (last update 31/03/2015).Eligibility CriteriaRelevant papers should report data from primary studies dealing with the prevalence of T. gondii in meat from livestock species as obtained through direct detection methods. Meta-analysis and meta-regression were performed.ResultsOf 1915 papers screened, 69 papers were included, dealing mainly with cattle, pigs and sheep. Pooled prevalences, based on random-effect models, were 2.6% (CI95 [0.5–5.8]) for cattle, 12.3% (CI95 [7.6–17.8]) for pigs and 14.7% (CI95 [8.9–21.5]) for sheep. Due to the high heterogeneity observed, univariable and multivariable meta-regression models were fitted showing that the geographic area for cattle (p = 0.032), the farming type for pigs (p = 0.0004) and the sample composition for sheep (p = 0.03) had significant effects on the prevalences of Toxoplasma detected/estimated. Moreover, the role of different animal species was dependent on the geographic location of animals’ origin.LimitationsLimitations were due mainly to a possible publication bias.Conclusions and ImplicationsThe present work confirms the role of meat, including beef, as T. gondii sources, and highlights the need for a control system for this parasite to be implemented along the meat production chain. Moreover, consumer knowledge should be strengthened in order to reduce the impact of disease.

Prevalence, enumeration, and pheno- and genotypic characteristics of Listeria monocytogenes isolated from raw foods in South China.

Listeria monocytogenes is an important foodborne pathogen that can cause serious illness in immunocompromised individuals, pregnant women, the elderly, and newborns. The aim of this study was to: (i) evaluate the prevalence and contamination level [most probable number (MPN)] of L. monocytogenes in 567 retail raw foods (fishery products, n = 154; raw/fresh meat, n = 123; frozen foods, n = 110; edible fungi, n = 108; vegetables, n = 72) collected from South China and (ii) to gain further knowledge on the phenotype and genotype distributions of this important foodborne pathogen. Approximately 22% of the samples were positive for L. monocytogenes. The contamination levels were between 0.3 and 10 MPN/g in 75.0%, between 10 and 100 MPN/g in 11.0% and less than 100 MPN/g in 14.0% of the countable samples. Five serogroups were identified among the 177 foodborne L. monocytogenes isolates, with 1/2a-3a (42.4%) and 1/2b-3b (26.0%) serogroups being the most dominant. Serogroups I.1 and II.2 were only found in the edible mushrooms, while serogroup III was dominant in the fishery products, suggesting that specific serogroups of L. monocytogenes may have distinct ecological niches. Ten (5.6%) L. monocytogenes isolates exhibited multidrug resistance. Genetic relatedness analysis revealed the absence of distinct associations between specific food types, antibiotic resistance, serogroups, and genetic diversity. The present study provided the first baseline data on the prevalence, contamination level, and characteristics of L. monocytogenes isolated from raw foods in South China. Some multidrug resistant strains belonged to the epidemiologically important serogroups (I.1 and II.1), implying a potential public health risk. In addition, these findings also provide basic information for the Chinese food safety associated authorities to draft appropriate standards to control L. monocytogenes contamination and improve microbiological safety of raw foods.

Antibacterial Effects of Afzelin Isolated from Cornus macrophylla on Pseudomonas aeruginosa, A Leading Cause of Illness in Immunocompromised Individuals

The crude ethyl acetate extract of the leaves of Cornus macrophylla showed antibacterial activity against Pseudomonas aeruginosa, a leading cause of illness in immunocompromised individuals. Bioactivity-guided separation led to the isolation of kaempferol 3-O-α-L-rhamnopyranoside (afzelin). The structure was determined based on evaluation of its spectroscopic (UV, MS, and NMR) data. The minimum inhibitory concentration (MIC) of afzelin against Pseudomonas aeruginosa was found to be 31 µg/mL. In addition, the results indicated that a hydroxyl group at C3 of the C-ring of the flavone skeleton and the rhamnose group may act as a negative factor and an enhancing factor, respectively, in the antibacterial activities of afzelin.

Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels.

Characterization of viral determinants of herpes simplex virus type 1 pathogenesis by bioimagery

The human pathogen herpes simplex virus 1 (HSV-1) infects mucous membranes leading to cold sores, following which a latent infection is established in neurons of the trigeminal ganglia (TG). While the infection in healthy individuals is usually benign, viral encephalitis may sometimes occur. Furthermore, the infection can lead to severe illness in immunocompromised individuals, and can result in permanent neurological sequelae in newborns. The UL24 protein of HSV-1 is highly conserved throughout the Herpesviridae family, and has been identified as a viral determinant of pathogenesis. In a murine model of ocular infection, a UL24-deficient virus exhibits a modest reduction in viral titers in the eye and a severe reduction of viral titers in the TG. A virus that does not express UL24 also exhibits defects in the establishment of latency and in the efficiency of viral reactivation from latency, as compared to a wild-type virus. Although UL24 is important for the pathogenesis of HSV-1, its exact role in vivo is unknown. We hypothesized that UL24 is important for viral dissemination to the trigeminal ganglia following ocular infection. To investigate this possibility, we generated a recombinant strain of HSV-1 expressing a second generation red fluorescent protein (RFP), mCherry. The RFP expression cassette, driven by the eukaryotic CMV promoter, was inserted within the intergenic region between the viral genes Us7 and Us8. This virus, vUs7-8mCherry, behaved similarly to the wild type virus (KOS) in cell culture as well as in vivo. We did not detect a loss of the RFP cassette over multiple rounds of viral replication in cell culture or following passage of the virus in mice. Following ocular infection, histological cross sections of eyes and TGs harvested three days post-infection were observed by confocal microscopy. Detection of mCherry enabled us to easily visualize infected cells in both the eye and in TG. This virus will be a powerful tool to study the role of UL24 in viral dissemination. In the long term, results from this project will help us further our understanding of the molecular mechanisms involved in viral pathogenesis, and possibly lead to the development of new therapeutic strategies.

A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

BackgroundMicroarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators.ResultsTaking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform future array designs.ConclusionsIn addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.

ChemInform Abstract: Systemic Antifungal Agents Against AIDS-Related Opportunistic Infections: Current Status and Emerging Drugs in Development

No effective drug was available for the treatment of systemic fungal infections until the discovery of Amphotericin B in 1953. Since then flucytosine, azoles and later the triazoles, have now become available. The current interest in the development of new antifungal agents can partially be explained by the dramatic rise in the number of AIDS cases and the subsequent suppression of the immune system in patients with the disease. For example, over 90% of those diagnosed to be HIV-positive contract a fungal infection during the course of their illness. Other conditions that have spurred the development of new systemic antifungal agents include the increase in the frequency of bone marrow and organ transplants, the use of antineoplastic agents, long-term use of corticosteroids and even the indiscriminate use of antibiotics. The emergence of fungi resistant to currently available agents, especially the azoles, has made the need for new and effective antifungal agents more urgent. This review article focuses on agents targeted against opportunistic fungal infections, i.e., fungal infections which, in contrast to immunocompetent individuals, may cause serious life-threatening illness in immunocompromised individuals. Agents currently on the market or undergoing clinical development, as well as potential new agents that have been discovered, are discussed.

Rejoining of gamma-ray-induced DNA damage in Cryptosporidium parvum measured by the comet assay

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25kGy) and were incubated for various periods (6–96h) after exposure to 10kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10kGy, double-strand breaks peaked at 6h postirradiation, and rejoining was highest at 72h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10kGy.

Development of a gene capture method to rescue a large deletion mutant of human cytomegalovirus

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that causes serious clinical illness in immunocompromised individuals. A major breakthrough in the progression of HCMV genetics studies is the development of bacterial artificial chromosome (BAC) clones of the viral genome. Recently, a luciferase reporter gene was inserted in the BAC clone (BAC luc) which facilitates monitoring of the virus growth both in vitro and in vivo. The virus made from the BAC luc grew with the similar kinetics as the wild-type strain indicating that the luciferase gene insertion does not interfere with the virus growth. Although the construction of the BAC clone has eased genetic studies of herpesviruses tremendously, there are still difficulties in cloning large DNA fragments of the virus to rescue mutations with large deletions. This paper describes a novel method termed “gene capture”, which allows easier cloning of large pieces of DNA. As an application of this method, a 15-kb fragment that was deleted from the HCMV genome was rescued back into the viral genome. A mutant HCMV clone with the 15-kb region deletion was generated first using the λ prophage recombination system in E. coli. Utilizing the new rescue method, the deleted fragment was then rescued in two steps: the 15-kb region was captured into a vector by homologous recombination; and the captured DNA fragment from the vector was inserted back into its native site in the mutant viral BAC by second homologous recombination. This method will be useful particularly for cloning large DNA fragments from any genome without introducing undesired mutations by traditional PCR-based approaches.