Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR) and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates TGF-beta expression whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions and EMT induction is unknown. We hypothesized that the EGFR and TGF-beta signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-beta expression as early as 6 h post-infection of epithelial cells and stimulated both the EGFR and TGF-beta signaling pathways. Inhibition of either the EGFR or TGF-betaR1 signaling substantially reduced inclusions development; however, the combined inhibition of both EGFR and TGF-betaR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-beta expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-betaduring infection. Finally, TGF-betaR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3) that stabilizes EGFR signaling, suggesting a reciprocal regulation between TGF-beta and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-beta expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. The finding may provide new targets for chlamydial disease biomarkers and prevention.

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1alpha leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.

PURPOSE: Trachoma, caused by repeated ocular infection with Chlamydia trachomatis, is the leading infectious cause of blindness worldwide and is targeted for elimination as a public health problem. We sought to determine whether a one-time azithromycin mass treatment would reduce trachomatous inflammation-follicular (TF) levels below the elimination threshold of 5% in communities with disease prevalence between 5 and 9.9%. METHODS: The study was conducted in 96 sub-village units (balozis) in the Kongwa district of Tanzania which were predicted from prior prevalence surveys to have TF between 5 and 9.9%. Balozis were randomly assigned to the intervention and control arms. The intervention arm received a single mass drug administration of azithromycin. At baseline and 12-month follow-up, ocular exams for trachoma, ocular swabs for detection of chlamydial DNA, and finger prick blood for analysis of anti-chlamydial antibody were taken. RESULTS: Comparison of baseline and 12-month follow-up showed no significant difference in the overall TF1-9 prevalence by balozi between control and treatment arms. In the treatment arm there was a significant reduction of ocular infection 12 months after treatment (p = 0.004) but no change in the control arm. No change in Pgp3-specific antibody responses were observed after treatment in the control or treatment arms. Anti-CT694 responses increased in both study arms (p = 0.009 for control arm and p = 0.04 for treatment arm). CONCLUSION: These data suggest that a single round of MDA may not be sufficient to decrease TF levels below 5% when TF1-9 is between 5 and 9.9% at baseline.

Emergence of azole resistant Aspergillus fumigatus has become a clinical problem in many parts of the world. Several amino acid mutations in the azole target protein, Cyp51Ap, contribute to this resistance, with the most concerning being the environmentally-derived TR34/L98H and TR46/Y121F/T289A mutations. Here, we performed passive surveillance to assess a sample of the A. fumigatus population in the US for the presence of these mutations. We found 1.4% of those isolates to exhibit elevated MIC via broth microdilution and five of those isolates harbored the TR34/L98H mutation.

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However the molecular pathogenesis of these complications remain poorly understood. The induction of pathogenic epithelial-Mesenchyme Transition (EMT) through miRNA dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydial-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III and IV, TGF-beta, TGF-betaR1, the connective tissue growth factor (CTGF), E-cadherin, SRY-Box 7 (SOX7), and nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts that produced ECM proteins, including collagen type I, III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydial-related complications.

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1alpha (IRE1alpha), and activating transcription factor-6alpha (ATF6alpha)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1alpha mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1alpha promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1alpha resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

Protozoan pathogens, including Entamoeba histolytica, Giardia, Cryptosporidium, Cyclospora, Cystoisospora, and microsporidia such as Enterocytozoon bieneusi, are well-known agents of diarrhea and a major public health problem in developing countries. Infection with Cyclospora cayetanensis and E. bieneusi can occur in immunocompromised and immunocompetent persons. Severe diarrhea and weight loss along with anorexia, nausea, and low-grade fever occur in immunocompromised persons, particularly those with HIV/AIDS and transplant recipients who are taking immunosuppressive drugs (1,2). However, transient diarrhea occurs in immunocompetent persons, notably in travelers returning from countries with poor hygienic standards (1–3). | | We report on a kidney transplant recipient who had uncontrollable diarrhea and weight loss in whom C. cayetanensis and E. bieneusi were detected in biopsy specimens; the diarrhea resolved after treatment with drugs that act specifically on these 2 parasites. The patient was a 55-year-old man from the Dominican Republic living in New York, NY, USA; he had a history of long-term diabetes, coronary disease, and alcoholism. He had undergone a cadaveric renal transplant 14 months earlier and had an uneventful posttransplant course. After returning from visiting family in the Dominican Republic, he sought treatment for acute, profuse watery diarrhea in early November, 2009. He had >10 watery bowel movements daily that were associated with a 20-lb weight loss. His symptoms persisted for 2 months, and he required 2 hospitalizations for the diarrhea.

BACKGROUND: Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. METHODS: PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. RESULTS: Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. CONCLUSION: These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Trichomonas vaginalis infections are usually asymptomatic or can result in non-specific clinical symptoms, which makes laboratory-based detection of this protozoan parasite essential for diagnosis and treatment. We report the development of a battery of highly sensitive and specific PCR assays for detection of T. vaginalis in urine, a non-invasive specimen, and development of a protocol for differentiating among Trichomonas species that commonly infect humans.

Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (Ct) as a major cause. While TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during Ct genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type Ct serovar L2 led to infertility, but the non-inflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3-deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications.

The Phylum Microsporidia comprises >1,200 species, only 15 of which are known to infect humans, including the genera Trachipleistophora, Pleistophora, and Brachiola. We report an infection by Tubulinosema sp. in an immunosuppressed patient.

Despite a high prevalence of sexually transmitted Chlamydia trachomatis infections in Brazil and other countries in South America, very little is known about the distribution of C. trachomatis genovars. In this study, we genotyped C. trachomatis strains from urine or endocervical specimens collected from 163 C. trachomatis-positive female and male youths, and female adults, residing in two different regions of Brazil, the city of Goiania located in the central part of Brazil, and the city of Vitoria in the south-east region. C. trachomatis strains were genotyped by amplifying and sequencing the ompA gene encoding the chlamydial major outer-membrane protein, which is genovar specific. We found nine different C. trachomatis genovars: E (39.3 %), F (16.6 %), D (15.9 %), I (8.6 %), J (7.4 %), G (4.9 %), K (3.1 %), H (2.4 %) and B (1.8 %). The distribution of the C. trachomatis genovars in the two regions of Brazil was similar, and there was no statistically significant association of serovars with age, gender, number of sexual partners or clinical symptoms. The overall distribution of C. trachomatis genovars in Brazil appears similar to that found in other regions of the world, where E, D and F are the most common. This supports the notion that, during the last few decades, the overall distribution of C. trachomatis genovars throughout the world has been relatively stable.

We report 13 cases of Naegleria fowleri primary amebic meningoencephalitis in persons in Karachi, Pakistan, who had no history of aquatic activities. Infection likely occurred through ablution with tap water. An increase in primary amebic meningoencephalitis cases may be attributed to rising temperatures, reduced levels of chlorine in potable water, or deteriorating water distribution systems.

Vaccines are needed to prevent the oculogenital diseases of Chlamydia trachomatis. Infected hosts develop immunity, although temporary, and experimental vaccines have yielded significant protective immunity in animal models, fueling the impetus for a vaccine. Because infections cause sequelae, the functional relationship between infection- and vaccine-induced immunity is unclear. We hypothesized that infection- and vaccine-induced immunity are functionally distinct, particularly in the ability to prevent sequelae. Chlamydia-immune mice, with immunity generated by either a previous infection or vaccination, exhibited a significant degree of protective immunity, marked by a lower-intensity, abbreviated course of infection. However, vaccinated mice were protected from infertility, whereas preinfected mice were not. Thus, infection-induced immunity does not prevent the pathologic process leading to infertility. Furthermore, T cell subsets, especially CD8 T cells, play a major role in Chlamydia-induced infertility. The results have important implications for the immunopathogenesis of chlamydial disease and new vaccine strategies.