Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

Background

Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots.

Methods and Findings

Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups.

Conclusion

Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.     

Identification of sVSG117 as an Immunodiagnostic Antigen and Evaluation of a Dual-Antigen Lateral Flow Test for the Diagnosis of Human African Trypanosomiasis

Background

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.

Methodology/Principle Findings

Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.

Conclusion/Significance

In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.     

Sensitivity and Specificity of a Prototype Rapid Diagnostic Test for the Detection of Trypanosoma brucei gambiense Infection: A Multi-centric Prospective Study

Background

A major challenge in the control of human African trypanosomiasis (HAT) is lack of reliable diagnostic tests that are rapid and easy to use in remote areas where the disease occurs. In Trypanosoma brucei gambiense HAT, the Card Agglutination Test for Trypanosomiasis (CATT) has been the reference screening test since 1978, usually on whole blood, but also in a 1/8 dilution (CATT 1/8) to enhance specificity. However, the CATT is not available in a single format, requires a cold chain for storage, and uses equipment that requires electricity. A solution to these challenges has been provided by rapid diagnostic tests (RDT), which have recently become available. A prototype immunochromatographic test, the SD BIOLINE HAT, based on two native trypanosomal antigens (VSG LiTat 1.3 and VSG LiTat 1.5) has been developed. We carried out a non-inferiority study comparing this prototype to the CATT 1/8 in field settings.

Methodology/Principal Findings

The prototype SD BIOLINE HAT, the CATT Whole Blood and CATT 1/8 were systematically applied on fresh blood samples obtained from 14,818 subjects, who were prospectively enrolled through active and passive screening in clinical studies in three endemic countries of central Africa: Angola, the Democratic Republic of the Congo and the Central African Republic. One hundred and forty nine HAT cases were confirmed by parasitology. The sensitivity and specificity of the prototype SD BIOLINE HAT was 89.26% (95% confidence interval (CI) = 83.27–93.28) and 94.58% (95% CI = 94.20–94.94) respectively. The sensitivity and specificity of the CATT on whole blood were 93.96% (95% CI = 88.92–96.79) and 95.91% (95% CI = 95.58–96.22), and of the CATT 1/8 were 89.26% (95% CI = 83.27–93.28) and 98.88% (95% CI = 98.70–99.04) respectively.

Conclusion/Significance

After further optimization, the prototype SD BIOLINE HAT could become an alternative to current screening methods in primary healthcare settings in remote, resource-limited regions where HAT typically occurs.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Background

The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT).

Methodology and Results

Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%.

Conclusions/Significance

The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.     

In Silico Identification of a Candidate Synthetic Peptide (Tsgf118–43) to Monitor Human Exposure to Tsetse Flies in West Africa

Background

The analysis of humoral responses directed against the saliva of blood-sucking arthropods was shown to provide epidemiological biomarkers of human exposure to vector-borne diseases. However, the use of whole saliva as antigen presents several limitations such as problems of mass production, reproducibility and specificity. The aim of this study was to design a specific biomarker of exposure to tsetse flies based on the in silico analysis of three Glossina salivary proteins (Ada, Ag5 and Tsgf1) previously shown to be specifically recognized by plasma from exposed individuals.

Methodology/Principal Findings

Synthetic peptides were designed by combining several linear epitope prediction methods and Blast analysis. The most specific peptides were then tested by indirect ELISA on a bank of 160 plasma samples from tsetse infested areas and tsetse free areas. Anti-Tsgf1_18–43 specific IgG levels were low in all three control populations (from rural Africa, urban Africa and Europe) and were significantly higher (p<0.0001) in the two populations exposed to tsetse flies (Guinean HAT foci, and South West Burkina Faso). A positive correlation was also found between Anti-Tsgf1_18–43 IgG levels and the risk of being infected by Trypanosoma brucei gambiense in the sleeping sickness foci of Guinea.

Conclusion/Significance

The Tsgf1_18–43 peptide is a suitable and promising candidate to develop a standardize immunoassay allowing large scale monitoring of human exposure to tsetse flies in West Africa. This could provide a new surveillance indicator for tsetse control interventions by HAT control programs.     

Quantifying the Association between Bovine and Human Trypanosomiasis in Newly Affected Sleeping Sickness Areas of Uganda

Background

Uganda has active foci of both chronic and acute HAT with the acute zoonotic form of disease classically considered to be restricted to southeast Uganda, while the focus of the chronic form of HAT was confined to the northwest of the country. Acute HAT has however been migrating from its traditional disease focus, spreading rapidly to new districts, a spread linked to movement of infected cattle following restocking. Cattle act as long-term reservoirs of human infective T. b. rhodesiense showing few signs of morbidity, yet posing a significant risk to human health. It is important to understand the relationship between infected cattle and infected individuals so that an appropriate response can be made to the risk posed to the community from animals infected with human pathogens in a village setting.

Methodology/Principal Findings

This paper examines the relationship between human T. b. rhodesiense infection and human infective and non-human T. brucei s.l. circulating in cattle at village level in Kaberamaido and Dokolo Districts, Uganda. The study was undertaken in villages that had reported a case of sleeping sickness in the six months prior to sample collection and those villages that had never reported a case of sleeping sickness.

Conclusions and Significance

The sleeping sickness status of the villages had a significant effect with higher odds of infection in cattle from case than from non-case villages for T. brucei s.l. (OR: 2.94, 95%CI: 1.38–6.24). Cattle age had a significant effect (p<0.001) on the likelihood of T. brucei s.l. infection within cattle: cattle between 18–36 months (OR: 3.51, 95%CI: 1.63–7.51) and cattle over 36 months (OR: 4.20, 95%CI: 2.08–8.67) had significantly higher odds of T. brucei s. l. infection than cattle under 18 months of age. Furthermore, village human sleeping sickness status had a significant effect (p<0.05) on the detection of T. b. rhodesiense in the village cattle herd, with significantly higher likelihood of T. b. rhodesiense in the village cattle of case villages (OR: 25, 95%CI: 1.2–520.71). Overall a higher than average T. brucei s.l. prevalence (>16.3%) in a village herd over was associated with significantly higher likelihood of T. b. rhodesiense being detected in a herd (OR: 25, 95%CI: 1.2–520.71).     

Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of Drug-Resistant Tuberculosis in Harare,Zimbabwe

Introduction

Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings.

Methods

We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method.

Results

One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69–95%) and specificity was 93% (95% CI, 84–98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7–15 days) than MGIT (12 days; IQR 6–16 days) or LJ (28 days; IQR 21–35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68–97%), 96% (95% CI, 79–100%), and 91% (95% CI, 72–99%), respectively; specificity was 89% (95% CI, 52–100%), 89% (95% CI, 52–100%), and 90% (95% CI, 56–100%), respectively.

Conclusion

In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

Risk Factors for Periprosthetic Joint Infection after Total Hip Arthroplasty and Total Knee Arthroplasty in Chinese Patients

Purpose

The purpose of this hospital-based case–control study was to evaluate the risk factors for periprosthetic joint infection (PJI) of total hip arthroplasty (THA) and total knee arthroplasty (TKA) in Chinese patients.

Method

From January 2000 to December 2012, 45 patients undergoing THA and TKA who developed PJI were recruited for case subjects; controls were 252 without PJI, matched by year of index for surgery and type of surgery. Conditional logistic regressions were run to compute odds ratios (ORs) and 95% confidence intervals (CIs).

Results

Demographic factors and comorbid conditions associated with an increased adjusted risk of PJI (in decreasing order of significance) were diabetes (OR = 5.47, 95% CI: 1.77–16.97; p = 0.003), age (65–75 vs. 45–65 years) (OR = 3.36, 95% CI: 1.30–8.69; p = 0.013), BMI (≥28 vs. 18.5–28 kg/m²) (OR = 2.77, 95% CI: 1.20–6.40; p = 0.017), place of residence (rural) (OR = 2.63, 95% CI: 1.13–6.10; p = 0.025) and alcohol abuse (OR = 2.95, 95% CI: 1.06–8.23; p = 0.039).

Conclusion

Patients with diabetes, older age, BMI of ≥28 kg/m² and alcohol abuse or living in rural areas, had increased PJI risk. Additional systematic large-scale studies are needed to verify these results.     

A CATT Negative Result after Treatment for Human African Trypanosomiasis Is No Indication for Cure

Background

Cure after treatment for human African trypanosomiasis (HAT) is assessed by examination of the cerebrospinal fluid every 6 months, for a total period of 2 years. So far, no markers for cure or treatment failure have been identified in blood. Trypanosome-specific antibodies are detectable in blood by the Card Agglutination Test for Trypanosomiasis (CATT). We studied the value of a normalising, negative post-treatment CATT result in treated Trypanosoma brucei (T.b.) gambiense sleeping sickness patients as a marker of cure.

Methodology/Principal Findings

The CATT/T.b. gambiense was performed on serum of a cohort of 360 T.b. gambiense patients, consisting of 242 primary and 118 retreatment cases. The CATT results during 2 years of post-treatment follow-up were studied in function of cure or treatment failure. At inclusion, sensitivity of CATT was 98% (234/238) in primary cases and only 78% (91/117) in retreatment cases. After treatment, the CATT titre decreased both in cured patients and in patients experiencing treatment failure.

Conclusions/Significance

Though CATT is a good test to detect HAT in primary cases, a normalising or negative CATT result after treatment for HAT does not indicate cure, therefore CATT cannot be used to monitor treatment outcome.     

Adiponectin-11377CG Gene Polymorphism and Type 2 Diabetes Mellitus in the Chinese Population: A Meta-Analysis of 6425 Subjects

Background

Although adiponectin −11377CG gene polymorphism is implied to be associated with increased type 2 diabetes mellitus (T2DM) risk, results of individual studies are inconsistent.

Objective and Methods

A meta-analysis consisting of 12 individual studies, including a total of 6425 participants, was carried out in order to investigate the association of adiponectin −11377CG gene polymorphism with T2DM. The pooled odds ratio (OR) and its corresponding confidence interval (CI) at 95% were assessed through the random- or fixed- effect model.

Results

A significant relationship was observed between adiponectin −11377CG gene polymorphism and T2DM under allelic (OR: 1.150, 95% CI: 1.060 to 1.250, P = 0.001), recessive (OR: 1.450, 95% CI: 1.180–1.770, P = 0.0004), dominant (OR: 1.071, 95% CI: 1.013–1.131, P = 0.015), additive (OR: 1.280, 95% CI: 1.090–1.510, P = 0.002), and homozygous genetic models (OR: 1.620, 95% CI: 1.310–1.990, P<0.00001). No significant association was found between them under the heterozygous genetic model (OR: 1.640, 95% CI: 0.850–3.170, P = 0.140).

Conclusions

Adiponectin −11377CG gene polymorphism was significantly associated with T2DM risk susceptibility. G allele carriers are predisposed to T2DM risk.     

Effects of Gender on Severity,Management and Outcome in Acute Biliary Pancreatitis

Background

We conducted a population-based cross-sectional study to examine gender differences in severity, management, and outcome among patients with acute biliary pancreatitis (ABP) because available data are insufficient and conflicting.

Methods

We analyzed 13,110 patients (50.6% male) with first-attack ABP from Taiwan’s National Health Insurance Research Database between 2000 and 2009. The primary outcome was hospital mortality. Secondary outcomes included the development of severe ABP and the provision of treatment measures. Gender difference was assessed using multivariable analyses with generalized estimating equations models.

Results

The odds of gastrointestinal bleeding (adjusted odds ratio [aOR] 1.44, 95% confidence interval [CI] 1.18–1.76) and local complication (aOR 1.38, 95% CI 1.05–1.82) were 44% and 38% higher in men than in women, respectively. Compared with women, men had 24% higher odds of receiving total parenteral nutrition (aOR 1.24, 95% CI 1.00–1.52), but had 18% and 41% lower odds of receiving cholecystectomy (aOR 0.82, 95% CI 0.72–0.93) and hemodialysis (aOR 0.59, 95% CI 0.42–0.83), respectively. Hospital mortality was higher in men than in women (1.8% vs. 1.1%, p = 0.001). After adjustment for potential confounders, men had 81% higher odds of in-hospital death than women (aOR 1.81, 95% CI 1.15–2.86). Among patients with severe ABP, hospital mortality was 11.0% and 7.5% in men and women (p<0.001), respectively. The adjusted odds of death remained higher in men than in women with severe ABP (aOR 1.72, 95% CI 1.10–2.68).

Conclusions

Gender is an important determinant of outcome in patients with ABP and may affect their treatment measures.     

MLH1 Promoter Methylation Frequency in Colorectal Cancer Patients and Related Clinicopathological and Molecular Features

Purpose

To describe the frequency of MLH1 promoter methylation in colorectal cancer (CRC); to explore the associations between MLH1 promoter methylation and clinicopathological and molecular factors using a systematic review and meta-analysis.

Methods

A literature search of the PubMed and Embase databases was conducted to identify relevant articles published up to September 7, 2012 that described the frequency of MLH1 promoter methylation or its associations with clinicopathological and molecular factors in CRC. The pooled frequency, odds ratio (OR) and 95% confidence intervals (95% CI) were calculated.

Results

The pooled frequency of MLH1 promoter methylation in unselected CRC was 20.3% (95% CI: 16.8–24.1%). They were 18.7% (95% CI: 14.7–23.6%) and 16.4% (95% CI: 11.9–22.0%) in sporadic and Lynch syndrome (LS) CRC, respectively. Significant associations were observed between MLH1 promoter methylation and gender (pooled OR = 1.641, 95% CI: 1.215–2.215; P = 0.001), tumor location (pooled OR = 3.804, 95% CI: 2.715–5.329; P<0.001), tumor differentiation (pooled OR = 2.131, 95% CI: 1.464–3.102; P<0.001), MSI (OR: 27.096, 95% CI: 13.717–53.526; P<0.001). Significant associations were also observed between MLH1 promoter methylation and MLH1 protein expression, BRAF mutation (OR = 14.919 (95% CI: 6.427–34.631; P<0.001) and 9.419 (95% CI: 2.613–33.953; P = 0.001), respectively).

Conclusion

The frequency of MLH1 promoter methylation in unselected CRC was 20.3%. They were 18.7% in sporadic CRC and 16.4% in LS CRC, respectively. MLH1 promoter methylation may be significantly associated with gender, tumor location, tumor differentiation, MSI, MLH1 protein expression, and BRAF mutation.     

Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis

Objective

To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract.

Study Design

Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria.

Results

A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0–76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0–341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6–205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis.

Conclusion

Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

Efficacy and Safety of Adding Clopidogrel to Aspirin on Stroke Prevention among High Vascular Risk Patients: A Meta-Analysis of Randomized Controlled Trials

Objectives

Whether clopidogrel should be added to aspirin for stroke prevention remained controversial for the risk of hemorrhagic complications. This meta-analysis was aimed to assess the efficacy and safety of adding clopidogrel to aspirin on stroke prevention in high vascular risk patients, and to provide evidence for a suitable duration of dual antiplatelet therapy.

Methods

We searched PubMed, EMBase, OVID and Cochrane Central Register of Controlled Trials (up to June, 2013) for randomized controlled trials evaluating the efficacy and safety of clopidogrel plus aspirin versus aspirin alone in high vascular risk patients. Comparisons of stroke and hemorrhagic complications between treatment groups were expressed by the pooled Relative Risks (RRs) with 95% Confidence Intervals (CIs).

Results

Fifteen trials with a total of 97692 intention-to-treat participants were included with duration of follow-up ranging from 7 days to 3.6 years. Dual antiplatelet therapy reduced all stroke by 21% (RR: 0.79, 95% CI: 0.73–0.85) with no evidence of heterogeneity across the trials (P = 0.27, I ² = 17%).The effects were consistent between short-term subgroup (≤1 month, RR: 0.76, 95% CI: 0.67–0.85) and long-term subgroup (≥3 months, RR: 0.81, 95% CI: 0.73–0.89). The risk of major bleeding was not significantly increased by dual antiplatelet therapy in short-term subgroup (RR: 1.11, 95% CI: 0.91–1.36), while significantly increased in long-term subgroup (RR: 1.52, 95% CI: 1.36–1.69). Long-term dual antiplatelet therapy substantially increased the risk of intracranial bleeding (RR: 1.76, 95% CI: 1.22–2.54).

Conclusions

This meta-analysis demonstrates that short-term combination of clopidogrel and aspirin is effective and safe for stroke prevention in high vascular risk patients. Long-term combination therapy substantially increases the risk of major bleeding and intracranial bleeding.     

Remote magnetic navigation vs. manual navigation for ablation of ventricular tachycardia: a meta-analysis

Background

The purpose of this study was to prospectively evaluate the efficacy and safety of remote magnetic navigation (RMN) in comparison with manual catheter navigation (MCN) in performing ventricular tachycardia ablation.

Methods

An electronic search was performed using PubMed (1948–2013) and EMBASE (1974–2013) studies comparing RMN with MCN which were published prior to 31 December 2013. Outcomes of interest were as follows: acute success, recurrence rate, complications, total procedure and fluoroscopic times. Standard mean difference (SMD) and its 95 % confidence interval (CI) were used for continuous outcomes; odds ratios (OR) were reported for dichotomous variables.

Results

Four non-randomised studies, including a total of 328 patients, were identified. RMN was deployed in 191 patients. Acute success and long-term freedom from arrhythmias were not significantly different between the RMN and control groups (OR 1.845, 95 % CI 0.731–4.659, p = 0.195 and OR 0.676, 95 % CI 0.383–1.194, p = 0.177, respectively). RMN was associated with less peri-procedural complications (OR 0.279, 95 % CI 0.092–0.843, p = 0.024). Shorter procedural and fluoroscopy times were achieved (95 % CI -0.487 to -0.035, p = 0.024 and 95 % CI -1.467 to -0.984, p<0.001, respectively).

Conclusion

The acute and long-term success rates for VT ablation are equal between RMN and MCN, whereas the RMN-guided procedure can be performed with a lower complication rate and less procedural and fluoroscopic times. More prospective randomised trials will be needed to better evaluate the superior role of RMN for catheter ablation of ventricular tachycardia.     

The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

Background

The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients.

Methodology/Principal Findings

We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test.

Conclusions/Significance

Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.