Zoonotic Chlamydiaceae species associated with trachoma, Nepal.

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed.

Population genomics of Chlamydia trachomatis: insights on drift, selection, recombination, and population structure.

The large number of sexually transmitted diseases and ocular trachoma cases that are caused globally each year by Chlamydia trachomatis has made this organism a World Health Organization priority for vaccine development. However, there is no gene transfer system for Chlamydia to help identify potential vaccine targets. To accelerate discoveries toward this goal, here we analyzed the broadest diversity of C. trachomatis genomes to date, including 25 geographically dispersed clinical and seven reference strains representing 14 of the 19 known serotypes. Strikingly, all 32 genomes were found to have evidence of DNA acquisition by homologous recombination in their history. Four distinct clades were identified, which correspond to all C. trachomatis disease phenotypes: lymphogranuloma venereum (LGV; Clade 1); noninvasive urogenital infections (Clade 2); ocular trachoma (Clade 3); and protocolitis (Clade 4; also includes some noninvasive urogenital infections). Although the ancestral relationship between clades varied, most strains acted as donor and recipient of recombination with no evidence for barriers to genetic exchange. The niche-specific LGV and trachoma clades have undergone less recombination, although the opportunity for mixing with strains from other clades that infect the rectal and ocular mucosa, respectively, is evident. Furthermore, there are numerous occasions for gene conversion events through sequential infections at the same anatomic sites. The size of recombinant segments is relatively small (~357 bp) compared with in vitro experiments of various C. trachomatis strains but is consistent with in vitro estimates for other bacterial species including Escherichia coli and Helicobacter pylori. Selection has also played a crucial role during the diversification of the organism. Clade 2 had the lowest nonsynonymous to synonymous ratio (dN/dS) but the highest effect of recombination, which is consistent with the widespread occurrence of synonymous substitutions in recombined genomic segments. The trachoma Clade 3 had the highest dN/dS estimates, which may be caused by an increased effect of genetic drift from niche specialization and a reduced effective population size. The degree of drift, selection, and recombination in C. trachomatis suggests that the challenge will remain to identify genomic regions that are stable and cross protective for the development of an efficacious vaccine.

A novel method of data analysis for utilization of red blood cell transfusion.

BACKGROUND: A necessary component of an effective blood management program is the accurate and comprehensive collection and analysis of blood utilization data. This study describes innovative methods for analyzing and presenting data for red blood cell (RBC) utilization that compare hemoglobin (Hb) transfusion triggers and targets to those representing the restrictive transfusion strategy advocated by previous large outcome studies. STUDY DESIGN AND METHODS: From one institution, blood utilization data for 134,456 patients, 23,559 of whom were transfused with RBCs, were analyzed. Hb triggers and targets for transfused patients were plotted and graphically compared to the trigger and target ranges from previously published randomized clinical trials. RESULTS: Nine hospital services with the highest transfusion rates were selected for analysis. The service with the highest Hb trigger and target was further analyzed by comparing transfusion thresholds for individual providers. Differences among services and among individual providers for mean Hb transfusion triggers and targets were significant (up to 1.5 g/dL, p < 0.0001). The variation between the 10th and 90th percentiles for both trigger and target was also significant (up to 3 g/dL, p < 0.0001). If a restrictive transfusion strategy were implemented, the need for transfusion would be reduced or eliminated in 10% to 50% of patients, depending on the service and the individual provider. CONCLUSION: By using these methods for analyzing and presenting RBC utilization data, opportunities can be identified for blood conservation, and educational efforts can be directed toward the appropriate individual hospital services and providers.

Optimizing preoperative blood ordering with data acquired from an anesthesia information management system.

BACKGROUND: The maximum surgical blood order schedule (MSBOS) is used to determine preoperative blood orders for specific surgical procedures. Because the list was developed in the late 1970s, many new surgical procedures have been introduced and others improved upon, making the original MSBOS obsolete. The authors describe methods to create an updated, institution-specific MSBOS to guide preoperative blood ordering. METHODS: Blood utilization data for 53,526 patients undergoing 1,632 different surgical procedures were gathered from an anesthesia information management system. A novel algorithm based on previously defined criteria was used to create an MSBOS for each surgical specialty. The economic implications were calculated based on the number of blood orders placed, but not indicated, according to the MSBOS. RESULTS: Among 27,825 surgical cases that did not require preoperative blood orders as determined by the MSBOS, 9,099 (32.7%) had a type and screen, and 2,643 (9.5%) had a crossmatch ordered. Of 4,644 cases determined to require only a type and screen, 1,509 (32.5%) had a type and crossmatch ordered. By using the MSBOS to eliminate unnecessary blood orders, the authors calculated a potential reduction in hospital charges and actual costs of $211,448 and $43,135 per year, respectively, or $8.89 and $1.81 per surgical patient, respectively. CONCLUSIONS: An institution-specific MSBOS can be created, using blood utilization data extracted from an anesthesia information management system along with our proposed algorithm. Using these methods to optimize the process of preoperative blood ordering can potentially improve operating room efficiency, increase patient safety, and decrease costs.

TRAIL-R1 is a negative regulator of pro-inflammatory responses and modulates long-term sequelae resulting from Chlamydia trachomatis infections in humans.

The immune system eliminates Chlamydia trachomatis infection through inflammation. However, uncontrolled inflammation can enhance pathology. In mice, TNF-related apoptosis-inducing ligand receptor (TRAIL-R), known for its effects on apoptosis, also regulates inflammation. In humans, the four homologues of TRAIL-R had never been investigated for effects on inflammation. Here, we examined whether TRAIL-R regulates inflammation during chlamydial infection. We examined TRAIL-R1 single nucleotide polymorphisms (SNPs) in an Ecuadorian cohort with and without C. trachomatis infections. There was a highly significant association for the TRAIL+626 homozygous mutant GG for infection vs no infection in this population. To confirm the results observed in the human population, primary lung fibroblasts and bone marrow-derived macrophages (BMDMs) were isolated from wildtype (WT) and TRAIL-R-deficient mice, and TRAIL-R1 levels in human cervical epithelial cells were depleted by RNA interference. Infection of BMDMs and primary lung fibroblasts with C. trachomatis strain L2, or the murine pathogen C. muridarum, led to higher levels of MIP2 mRNA expression or IL-1ß secretion from TRAIL-R-deficient cells than WT cells. Similarly, depletion of TRAIL-R1 expression in human epithelial cells resulted in a higher level of IL-8 mRNA expression and protein secretion during C. trachomatis infection. We conclude that human TRAIL-R1 SNPs and murine TRAIL-R modulate the innate immune response against chlamydial infection. This is the first evidence that human TRAIL-R1 is a negative regulator of inflammation and plays a role in modulating Chlamydia pathogenesis.

Sudden Death in a Patient with Carney's Complex.

Carney's complex is a rare autosomal dominantly inherited multiple endocrine neoplasia syndrome that involves spotty skin pigmentations, recurrent cardiac myxomas, endocrine hyperactivity, pituitary adenomas, peripheral nerve tumors, testicular tumors, and ovarian lesions. We present a case of sudden cardiac death in a 40 year old female with a history of Carney's complex with recurrent cardiac myxomas presenting for exploratory laparotomy and enblock adnexal resection of a slowly enlarging right sided ovarian mass. This case highlights the risk for sudden death in these patients as well as the preoperative assessment that should be undertaken by the anesthesiologist as it relates to Carney's complex.

Eukaryotic protein recruitment into the Chlamydia inclusion: implications for survival and growth.

Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen that multiplies within a parasitophorous vacuole called an inclusion. We report that the location of several host-cell proteins present in the cytosol, the nucleus, and membranes was altered during Ct development. The acyl-CoA synthetase enzyme ACSL3 and the soluble acyl-CoA binding protein ACBD6 were mobilized from organelle membranes and the nucleus, respectively, into the lumen of the inclusion. The nuclear protein ZNF23, a pro-apoptosis factor, was also translocated into the inclusion lumen. ZNF23, among other proteins, might be targeted by Ct to inhibit host cell apoptosis, thereby enabling bacterial survival. In contrast, the acyl-CoA:lysophosphatidylcholine acyltransferase LPCAT1, an endoplasmic reticulum membrane protein, was recruited to the inclusion membrane. The coordinated action of ACBD6, ACSL3 and LPCAT1 likely supports remodeling and scavenging of host lipids into bacterial-specific moieties essential to Ct growth. To our knowledge, these are the first identified host proteins known to be intercepted and translocated into the inclusion.

A multiplexed microfluidic PCR assay for sensitive and specific point-of-care detection of Chlamydia trachomatis.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes. METHODOLOGY AND PRINCIPAL FINDINGS: Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively. CONCLUSIONS: This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.