Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein

Background

The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes.

Methods and Findings

We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions.

Conclusions

Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.     

Temporal trends in the impact factor of European versus USA biomedical journals

Background

The impact factors of biomedical journals tend to rise over time. We sought to assess the trend in the impact factor, during the past decade, of journals published on behalf of United States (US) and European scientific societies, in four select biomedical subject categories (Biology, Cell Biology, Critical Care Medicine, and Infectious Diseases).

Methods

We identified all journals included in the above-mentioned subject categories of Thomson Reuters Journal Citation Reports® for the years 1999, 2002, 2005, and 2008. We selected those that were published on behalf of US or European scientific societies, as documented in journal websites.

Results

We included 167 journals (35 in the subject category of Biology, 79 in Cell Biology, 27 in Critical Care Medicine, and 26 in Infectious Diseases). Between 1999 and 2008, the percentage increase in the impact factor of the European journals was higher than for the US journals (73.7±110.0% compared with 39.7±70.0%, p = 0.049). Regarding specific subject categories, the percentage change in the factor of the European journals tended to be higher than the respective US journals for Cell Biology (61.7% versus 16.3%), Critical Care Medicine (212.4% versus 65.4%), Infectious Diseases (88.3% versus 48.7%), whereas the opposite was observed for journals in Biology (41.0% versus 62.5%).

Conclusion

Journals published on behalf of European scientific societies, in select biomedical fields, may tend to close the “gap” in impact factor compared with those of US societies.

What''s Already Known About This Topic?

The impact factors of biomedical journals tend to rise through years. The leading positions in productivity in biomedical research are held by developed countries, including those from North America and Western Europe.

What Does This Article Add?

The journals from European biomedical scientific societies tended, over the past decade, to increase their impact factor more than the respective US journals.     

Comparative brain stem lesions on MRI of acute disseminated encephalomyelitis, neuromyelitis optica, and multiple sclerosis

Background

Brain stem lesions are common in patients with acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO), and multiple sclerosis (MS).

Objectives

To investigate comparative brain stem lesions on magnetic resonance imaging (MRI) among adult patients with ADEM, NMO, and MS.

Methods

Sixty-five adult patients with ADEM (n = 17), NMO (n = 23), and MS (n = 25) who had brain stem lesions on MRI were enrolled. Morphological features of brain stem lesions among these diseases were assessed.

Results

Patients with ADEM had a higher frequency of midbrain lesions than did patients with NMO (94.1% vs. 17.4%, P<0.001) and MS (94.1% vs. 40.0%, P<0.001); patients with NMO had a lower frequency of pons lesions than did patients with MS (34.8% vs. 84.0%, P<0.001) and ADEM (34.8% vs. 70.6%, P = 0.025); and patients with NMO had a higher frequency of medulla oblongata lesions than did patients with ADEM (91.3% vs. 35.3%, P<0.001) and MS (91.3% vs. 36.0%, P<0.001). On the axial section of the brain stem, the majority (82.4%) of patients with ADEM showed lesions on the ventral part; the brain stem lesions in patients with NMO were typically located in the dorsal part (91.3%); and lesions in patients with MS were found in both the ventral (44.0%) and dorsal (56.0%) parts. The lesions in patients with ADEM (100%) and NMO (91.3%) had poorly defined margins, while lesions of patients with MS (76.0%) had well defined margins. Brain stem lesions in patients with ADEM were usually bilateral and symmetrical (82.4%), while lesions in patients with NMO (87.0%) and MS (92.0%) were asymmetrical or unilateral.

Conclusions

Brain stem lesions showed various morphological features among adult patients with ADEM, NMO, and MS. The different lesion locations may be helpful in distinguishing these diseases.     

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.     

A fly in the ointment: evaluation of traditional use of plants to repel and kill blowfly larvae in fermented fish

Introduction

In rural areas in Laos, fly larvae infestations are common in fermenting fish. Blowflies (Chrysomya megacephala, Diptera: Calliphoridae) are attracted to oviposit (and/or larviposit) onto fermenting fish which results in infestations with fly larvae. Knowledge of traditional use of plants to repel larvae during the production of fermented fish is common and widespread in Lao PDR.

Research Questions

How effective are the most salient species in repelling, and killing fly larvae in fermenting fish?

Material and Methods

The three plant species most frequently reported to repel fly larvae during an ethnobotanical survey throughout Lao PDR were tested for repellence and larvicidal activity of fly larvae infesting fermented fish. The lethality and repellence of Tadehagi triquetrum (L.) H. Ohashi (Fabaceae), Uraria crinita (L.) Desv. ex DC. (Fabaceae) and Bambusa multiplex (Lour.) Raeusch. ex Schult. & Schult. f. (Poaceae) were tested in an experimental design using fermenting fish in Vientiane, Lao PDR.

Results

The repellent effect of fresh material of T. triquetrum and U. crinita, and the larvicidal effect of fresh B. multiplex, is significantly more effective than that of dried material of the same species, and the total effect (repellence and larvicidal effect combined) for each of the three species was significantly more effective for fresh than for dry material. Fresh material of T. triquetrum, U. crinita, or B. multiplex added on top of the fermenting fish repelled 50%, 54%, 37%, and killed 22%, 28%, and 40% of fly larvae. The total effect was not significantly different per species at 72%, 82%, and 77%, respectively.

Discussion and Conclusions

The three most salient species are effective in repelling and killing fly larvae in the production of fermented fish, and may be essential to augment food safety during traditional fermentation in open jars.     

Comparison of HIV-, HBV-, HCV- and co-infection prevalence between Chinese and Burmese intravenous drug users of the China-Myanmar border region

Background

Co-infection with HIV and HCV and/or HBV is highly prevalent in intravenous drug users (IDUs). Because of the proximity to the “Golden Triangle”, HIV prevalence among the IDUs is very high in the China-Myanmar border region. However, there are few studies about co-infection with HIV and HCV and/or HBV, especially in the region that belongs to Myanmar.

Methods

721 IDUs, including 403 Chinese and 318 Burmese, were investigated for their HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) serological status. Statistical analysis was performed to evaluate the differences of the epidemic situation between the Chinese IDUs and the Burmese IDUs.

Results

Among the Chinese IDUs and the Burmese IDUs, HCV infection was the most prevalent (69.0% vs 48.1%, P<0.001), followed by HBV (51.6% vs 43.1%, P<0.05) and HIV (33.7% vs 27.0%, P>0.05). Besides, there were more HIV-HBV co-infected IDUs (20.1% vs 11.3%, P<0.005), and HIV-HCV co-infected IDUs (31.8% vs 23.9%, P<0.05) in China than in Myanmar, as well as HIV-HBV-HCV triple infection (19.1% vs 10.4%, P<0.005).

Conclusion

Co-infection with HIV and HCV and/or HBV is highly prevalent among the IDUs in the China-Myanmar border region. The HIV epidemic appears to be in a downward trend, compared with previous reports. However, all infections were more prevalent among the Chinese IDUs than among the Burmese.     

Co-deletion of chromosome 1p/19q and IDH1/2 mutation in glioma subsets of brain tumors in Chinese patients

Objective

To characterize co-deletion of chromosome 1p/19q and IDH1/2 mutation in Chinese brain tumor patients and to assess their associations with clinical features.

Methods

In a series of 528 patients with gliomas, pathological and radiological materials were reviewed. Pathological constituents of tumor subsets, incidences of 1p/19q co-deletion and IDH1/2 mutation in gliomas by regions and sides in the brain were analyzed.

Results

Overall, 1p and 19q was detected in 339 patients by FISH method while the sequence of IDH1/2 was determined in 280 patients. Gliomas of frontal, temporal and insular origin had significantly different pathological constituents of tumor subsets (P<0.001). Gliomas of frontal origin had significantly higher incidence of 1p/19q co-deletion (50.4%) and IDH1/2 mutation (73.5%) than those of non-frontal origin (27.0% and 48.5%, respectively) (P<0.001), while gliomas of temporal origin had significantly lower incidence of 1p/19q co-deletion (23.9%) and IDH1/2 mutation (41.7%) than those of non-temporal origin (39.9% and 63.2%, respectively) (P = 0.013 and P = 0.003, respectively). Subgroup analysis confirmed these findings in oligoastrocytic and oligodendroglial tumors, respectively. Although the difference of 1p/19q co-deletion was not statistically significant in temporal oligodendroglial tumors, the trend was marginally significant (P = 0.082). However, gliomas from different sides of the brain did not show significant different pathological constituents, incidences of 1p/19q co-deletion or IDH1/2 mutation.

Conclusion

Preferential distribution of pathological subsets, 1p/19q co-deletion and IDH1/2 mutation were confirmed in some brain regions in Chinese glioma patients, implying their distinctive tumor genesis and predictive value for prognosis.     

Persistence of Mycoplasma genitalium following azithromycin therapy

Background

To determine clinical outcomes and cure rates for M.genitalium genital infection in men and women following azithromycin 1 g.

Methodology

Patients attending Melbourne Sexual Health Centre between March 2005 and November 2007 with urethritis/epididymitis, cervicitis/pelvic inflammatory disease and sexual contacts of M.genitalium were tested for M.genitalium by polymerase chain reaction (PCR). M.genitalium-infection was treated with 1 g of azithromycin and a test-of-cure (toc) was performed one month post-azithromycin. Response to azithromycin, and response to moxifloxacin (400 mg daily for 10 days) in individuals with persistent infection post-azithromycin, was determined.

Principal Findings

Of 1538 males and 313 females tested, 161 males (11%) and 30 females (10%) were infected with M.genitalium. A toc was available on 131 (69%) infected individuals (median = 36 days [range 12-373]). Of 120 individuals prescribed azithromycin only pre-toc, M.genitalium was eradicated in 101 (84%, 95% confidence intervals [CI]: 77–90%) and persisted in 19 (16%, 95% CI: 10–23%). Eleven individuals with persistent infection (9%, 95% CI: 5–15%) had no risk of reinfection from untreated-partners, while eight (7%, 95% CI: 3–12%) may have been at risk of reinfection from doxycycline-treated or untreated-partners. Moxifloxacin was effective in eradicating persistent infection in all cases not responding to azithromycin. Patients with persistent-M.genitalium were more likely to experience persistent symptoms (91%), compared to patients in whom M.genitalium was eradicated (17%), p<0.0001.

Conclusion

Use of azithromycin 1 g in M.genitalium-infected patients was associated with unacceptable rates of persistent infection, which was eradicated with moxifloxacin. These findings highlight the importance of follow-up in M.genitalium-infected patients prescribed azithromycin, and the need to monitor for the development of resistance. Research to determine optimal first and second-line therapeutic agents for M.genitalium is needed.     

Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey

Background

This study compared diagnostic methods for identifying Blastocystis in stool samples, and evaluated the frequency of detection of Blastocystis in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD).

Results and Discussion

From a set of 105 stool specimens submitted for routine parasitological analysis, 30 were identified as positive for Blastocystis by the culture method. From that group of 30 positives, Lugol''s stain, trichrome staining, and an immunofluorescence assay identified 11, 15, and 26 samples as positive respectively. Using culture as a standard, the sensitivity of Lugol''s stain was 36.7%, trichrome staining was 50%, and the IFA stain was 86.7%. The specificity of Lugol''s stain was 91%, trichrome staining was 100%, and the IFA stain was 97.3%. In the group of 27 IBS and IBD patients, using all methods combined, we detected Blastocystis in 67% (18/27) of the patients. Blastocystis was detected in 33% (2/6) of IBD patients and 76% (16/21) of IBS patients. For comparison, trichrome staining alone, the method most frequently used in many countries, would have only identified Blastocystis infection in 29% (6/21) of the IBS patients. No parasitic co-infections were identified in the IBS/IBD patients. Most Blastocystis-positive IBS/IBD patients were over 36 with an average length of illness of 4.9 years.

Conclusions

Most IBS patients in this study were infected with Blastocystis. IFA staining may be a useful alternative to stool culture, especially if stool specimens have been chemically preserved.     

The burden of selected chronic non-communicable diseases and their risk factors in Malawi: nationwide STEPS survey

Background

Chronic non-communicable diseases (NCDs) are becoming significant causes of morbidity and mortality, particularly in sub-Saharan African countries, although local, high-quality data to inform evidence-based policies are lacking.

Objectives

To determine the magnitude of NCDs and their risk factors in Malawi.

Methods

Using the WHO STEPwise approach to chronic disease risk factor surveillance, a population-based, nationwide cross-sectional survey was conducted between July and September 2009 on participants aged 25–64 years. Socio-demographic and behaviour risk factors were collected in Step 1. Physical anthropometric measurements and blood pressure were documented in Step 2. Blood cholesterol and fasting blood glucose were measured in Step 3.

Results and Conclusion

A total of 5,206 adults (67% females) were surveyed. Tobacco smoking, alcohol drinking and raised blood pressure (BP) were more frequent in males than females, 25% vs 3%, 30% vs 4% and 37% vs 29%. Overweight, physical inactivity and raised cholesterol were more common in females than males, 28% vs 16%, 13% vs 6% and 11% vs 6%. Tobacco smoking was more common in rural than urban areas 11% vs 7%, and overweight and physical inactivity more common in urban than rural areas 39% vs 22% and 24% vs 9%, all with p<0.05. Overall (both sexes) prevalence of tobacco smoking, alcohol consumption, overweight and physical inactivity was 14%, 17%, 22%, 10% and prevalence of raised BP, fasting blood sugar and cholesterol was 33%, 6% and 9% respectively. These data could be useful in the formulation and advocacy of NCD policy and action plan in Malawi.     

The blind nasotracheal aspiration method is not a useful tool for pathogen detection of pneumonia in children

Background

Acute lower respiratory infection (ALRI) is a major cause of hospitalization for children in China, while the etiological diagnosis of ALRI remains a challenge. This study was performed to evaluate the utility of the blind Nasotracheal aspiration (NTA) in the pathogen detection in ALRI through an evaluation of the test''s specificity.

Methodology/Principal Findings

A hospital-based study of children ≤3 years was carried out from March 2006 through March 2007 in Suzhou University Affiliated Children''s Hospital, including 379 cases with ALRI from the respiratory wards, and 394 controls receiving elective surgery. Nasopharyngeal swabs (NPS) and NTA specimens were taken on admission. S. pneumoniae was isolated from 10.3% of NTA samples from ALRI children, H. influenzae from 15.3%, and M. catarrhalis from 4.7%. The false positive rate—the strains from NTA in control group children—was 8.4% (95% CI: 5.8%–11.4%) for S. pneumoniae, 27.2% (95% CI: 22.7–31.5%) for H. influenzae, and 22.1% (95% CI: 18.0%–26.2%) for M. catarrhalis. The agreement between NPS and NTA in the control group was over 70%.

Conclusion/Significance

The blind NTA test is not a useful test for etiologic diagnosis of ALRI.     

Association of gender with clinical expression, quality of life, disability, and depression and anxiety in patients with systemic sclerosis

Objectives

To assess the association of gender with clinical expression, health-related quality of life (HRQoL), disability, and self-reported symptoms of depression and anxiety in patients with systemic sclerosis (SSc).

Methods

SSc patients fulfilling the American College of Rheumatology and/or the Leroy and Medsger criteria were assessed for clinical symptoms, disability, HRQoL, self-reported symptoms of depression and anxiety by specific measurement scales.

Results

Overall, 381 SSc patients (62 males) were included. Mean age and disease duration at the time of evaluation were 55.9 (13.3) and 9.5 (7.8) years, respectively. One-hundred-and-forty-nine (40.4%) patients had diffuse cutaneous SSc (dcSSc). On bivariate analysis, differences were observed between males and females for clinical symptoms and self-reported symptoms of depression and anxiety, however without reaching statistical significance. Indeed, a trend was found for higher body mass index (BMI) (25.0 [4.1] vs 23.0 [4.5], p = 0.013), more frequent dcSSc, echocardiography systolic pulmonary artery pressure >35 mmHg and interstitial lung disease in males than females (54.8% vs 37.2%, p = 0.010; 24.2% vs 10.5%, p = 0.003; and 54.8% vs 41.2%, p = 0.048, respectively), whereas calcinosis and self-reported anxiety symptoms tended to be more frequent in females than males (36.0% vs 21.4%, p = 0.036, and 62.3% vs 43.5%, p = 0.006, respectively). On multivariate analysis, BMI, echocardiography PAP>35 mmHg, and anxiety were the variables most closely associated with gender.

Conclusions

In SSc patients, male gender tends to be associated with diffuse disease and female gender with calcinosis and self-reported symptoms of anxiety. Disease-associated disability and HRQoL were similar in both groups.     

Diagnostic value of EBUS-TBNA for lung cancer with non-enlarged lymph nodes: a study in a tuberculosis-endemic country

Background

In tuberculosis (TB)-endemic areas, contrast-enhanced computed tomography (CT) and positron emission tomography (PET) findings of lung cancer patients with non-enlarged lymph nodes are frequently discrepant. Endobronchial ultrasound-guided transbronchial aspiration (EBUS-TBNA) enables real-time nodal sampling, and thereby improves nodal diagnosis accuracy. This study aimed to compare the accuracy of nodal diagnosis by using EBUS-TBNA, and PET.

Methods

We studied 43 lung cancer patients with CT-defined non-enlarged mediastinal and hilar lymph nodes and examined 78 lymph nodes using EBUS-TBNA.

Results

The sensitivity, specificity, positive predictive value, and negative predictive value of EBUS-TBNA were 80.6%, 100%, 100%, and 85.7%, respectively. PET had low specificity (18.9%) and a low positive predictive value (44.4%). The diagnostic accuracy of EBUS-TBNA was higher than that of PET (91% vs. 47.4%; p<0.001). Compared to CT-based nodal assessment, PET yielded a positive diagnostic impact in 36.9% nodes, a negative diagnostic impact in 46.2% nodes, and no diagnostic impact in 16.9% nodes. Patients with lymph nodes showing negative PET diagnostic impact had a high incidence of previous pulmonary TB. Multivariate analysis indicated that detection of hilar nodes on PET was an independent predictor of negative diagnostic impact of PET.

Conclusion

In a TB-endemic area with a condition of CT-defined non-enlarged lymph node, the negative diagnostic impact of PET limits its clinical usefulness for nodal staging; therefore, EBUS-TBNA, which facilitates direct diagnosis, is preferred.     

Effect on adherence to nicotine replacement therapy of informing smokers their dose is determined by their genotype: a randomised controlled trial

Background

The behavioural impact of pharmacogenomics is untested. We tested two hypotheses concerning the behavioural impact of informing smokers their oral dose of NRT is tailored to analysis of DNA.

Methods and Findings

We conducted an RCT with smokers in smoking cessation clinics (N = 633). In combination with NRT patch, participants were informed that their doses of oral NRT were based either on their mu-opioid receptor (OPRM1) genotype, or their nicotine dependence questionnaire score (phenotype). The proportion of prescribed NRT consumed in the first 28 days following quitting was not significantly different between groups: (68.5% of prescribed NRT consumed in genotype vs 63.6%, phenotype group, difference  =  5.0%, 95% CI −0.9,10.8, p  =  0.098). Motivation to make another quit attempt among those (n  =  331) not abstinent at six months was not significantly different between groups (p  =  0.23). Abstinence at 28 days was not different between groups (p = 0.67); at six months was greater in genotype than phenotype group (13.7% vs 7.9%, difference  =  5.8%, 95% CI 1.0,10.7, p  =  0.018).

Conclusions

Informing smokers their oral dose of NRT was tailored to genotype not phenotype had a small, statistically non-significant effect on 28-day adherence to NRT. Among those still smoking at six months, there was no evidence that saying NRT was tailored to genotype adversely affected motivation to make another quit attempt. Higher abstinence rate at six months in the genotype arm requires investigation.

Trial registration

Controlled-Trials.com ISRCTN14352545.     

A genomic survey of proteases in Aspergilli

Background

Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far.

Results

We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli.

Conclusions

Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-523) contains supplementary material, which is available to authorized users.     

Interferon-gamma release assay performance in pulmonary and extrapulmonary tuberculosis

Background

The diagnosis of tuberculosis remains difficult. This study aimed to assess performance of interferon-gamma release assay (IGRA) in diagnosis of active tuberculosis (ATB) with pulmonary and extrapulmonary involvements, and to determine the diagnostic role of IGRA (T-SPOT.TB) and tuberculin skin test (TST) in BCG-vaccinated population.

Methods and Findings

Two hundred twenty-six ATB suspects were recruited and examined with T-SPOT.TB. Among them, fifty-two and seventy-six subjects were simultaneously tested by TST with 5TU or 1TU of purified protein derivative (PPD). The sensitivity of T-SPOT.TB was 94.7% (71/75), comparable in pulmonary and extrapulmonary disease groups (95.6% vs. 93.3%, P>0.05), while the specificity was 84.10% (90/107) but differed in two groups (69.2% vs. 88.9%, P = 0.02). Compared to T-SPOT.TB, TST with 5TU-PPD showed less sensitivity (92.3% vs. 56.4%) and specificity (84.6% vs. 61.5%) (both P<0.01); the sensitivity of TST with 1TU-PPD was 27.8%, and despite its specificity identical to T-SPOT.TB (both 82.8%) positive predictive value (PPV) was only 33.3%. By combining T-SPOT.TB with TST (1TU), the specificity rose to 95%, but the PPV stayed unchanged.

Conclusions

IGRA could function as a powerful immunodiagnostic test to explore pulmonary and extrapulmonary TB, while TST failed to play a reliable or auxiliary role in identifying TB disease and infection in the BCG-vaccinated population.     

Sounds Scary? Lack of Habituation following the Presentation of Novel Sounds

Background

Animals typically show less habituation to biologically meaningful sounds than to novel signals. We might therefore expect that acoustic deterrents should be based on natural sounds.

Methodology

We investigated responses by western grey kangaroos (Macropus fulignosus) towards playback of natural sounds (alarm foot stomps and Australian raven (Corvus coronoides) calls) and artificial sounds (faux snake hiss and bull whip crack). We then increased rate of presentation to examine whether animals would habituate. Finally, we varied frequency of playback to investigate optimal rates of delivery.

Principal Findings

Nine behaviors clustered into five Principal Components. PC factors 1 and 2 (animals alert or looking, or hopping and moving out of area) accounted for 36% of variance. PC factor 3 (eating cessation, taking flight, movement out of area) accounted for 13% of variance. Factors 4 and 5 (relaxing, grooming and walking; 12 and 11% of variation, respectively) discontinued upon playback. The whip crack was most evocative; eating was reduced from 75% of time spent prior to playback to 6% following playback (post alarm stomp: 32%, raven call: 49%, hiss: 75%). Additionally, 24% of individuals took flight and moved out of area (50 m radius) in response to the whip crack (foot stomp: 0%, raven call: 8% and 4%, hiss: 6%). Increasing rate of presentation (12x/min ×2 min) caused 71% of animals to move out of the area.

Conclusions/Significance

The bull whip crack, an artificial sound, was as effective as the alarm stomp at eliciting aversive behaviors. Kangaroos did not fully habituate despite hearing the signal up to 20x/min. Highest rates of playback did not elicit the greatest responses, suggesting that ‘more is not always better’. Ultimately, by utilizing both artificial and biological sounds, predictability may be masked or offset, so that habituation is delayed and more effective deterrents may be produced.     

Serum antibody levels to the Pneumocystis jirovecii major surface glycoprotein in the diagnosis of P. jirovecii pneumonia in HIV+ patients

Background

Pneumocystis jirovecii remains an important cause of fatal pneumonia (Pneumocystis pneumonia or PcP) in HIV+ patients and other immunocompromised hosts. Despite many previous attempts, a clinically useful serologic test for P. jirovecii infection has never been developed.

Methods/Principal Findings

We analyzed serum antibody responses to the P. jirovecii major surface glycoprotein recombinant fragment C1 (MsgC1) in 110 HIV+ patients with active PcP (cases) and 63 HIV+ patients with pneumonia due to other causes (controls) by an enzyme-linked immunosorbent assay (ELISA). The cases had significantly higher IgG and IgM antibody levels to MsgC1 than the controls at hospital admission (week 0) and intervals up to at least 1 month thereafter. The sensitivity, specificity and positive predictive value (PPV) of IgG antibody levels increased from 57.2%, 61.7% and 71.5% at week 0 to 63.4%, 100%, and 100%, respectively, at weeks 3–4. The sensitivity, specificity and PPV of IgM antibody levels rose from 59.7%, 61.3%, and 79.3% at week 0 to 74.6%, 73.7%, and 89.8%, respectively, at weeks 3–4. Multivariate analysis revealed that a diagnosis of PcP was the only independent predictor of high IgG and IgM antibody levels to MsgC1. A high LDH level, a nonspecific marker of lung damage, was an independent predictor of low IgG antibody levels to MsgC1.

Conclusions/Significance

The results suggest that the ELISA shows promise as an aid to the diagnosis of PCP in situations where diagnostic procedures cannot be performed. Further studies in other patient populations are needed to better define the usefulness of this serologic test.     

High plasma level of long pentraxin 3 (PTX3) is associated with fatal disease in bacteremic patients: a prospective cohort study

Introduction

Long pentraxin 3 (PTX3) is an acute-phase protein secreted by various cells, including leukocytes and endothelial cells. Like C-reactive protein (CRP), it belongs to the pentraxin superfamily. Recent studies indicate that high levels of PTX3 may be associated with mortality in sepsis. The prognostic value of plasma PTX3 in bacteremic patients is unknown.

Methods

Plasma PTX3 levels were measured in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococcae and Escherichia coli, using a commercial solid-phase enzyme-linked immunosorbent assay (ELISA). Values were measured on days 1–4 after positive blood culture, on day 13–18 and on recovery.

Results

The maximum PTX3 values on days 1–4 were markedly higher in nonsurvivors compared to survivors (44.8 vs 6.4 ng/ml, p<0.001) and the AUC^ROC in the prediction of case fatality was 0.82 (95% CI 0.73–0.91). PTX3 at a cut-off level of 15 ng/ml showed 72% sensitivity and 81% specificity for fatal disease. High PTX3 (>15 ng/ml) was associated with hypotension (MAP <70 mmHg)(OR 7.9;95% CI 3.3–19.0) and high SOFA score (≥4)(OR 13.2; 95% CI 4.9–35.4). The CRP level (maximum value on days 1 to 4) did not predict case fatality at any cut-off level in the ROC curve (p = 0.132). High PTX3 (>15 ng/ml) remained an independent risk factor for case fatality in a logistic regression model adjusted for potential confounders.

Conclusions

PTX3 proved to be a specific independent prognostic biomarker in bacteremia. PTX3 during the first days after diagnosis showed better prognostic value as compared to CRP, a widely used biomarker in clinical settings. PTX3 measurement offers a novel opportunity for the prognostic stratification of bacteremia patients.     

Performance of local light microscopy and the ParaScreen Pan/Pf rapid diagnostic test to detect malaria in health centers in Northwest Ethiopia

Background

Diagnostic tests are recommended for suspected malaria cases before treatment, but comparative performance of microscopy and rapid diagnostic tests (RDTs) at rural health centers has rarely been studied compared to independent expert microscopy.

Methods

Participants (N = 1997) with presumptive malaria were recruited from ten health centers with a range of transmission intensities in Amhara Regional State, Northwest Ethiopia during October to December 2007. Microscopy and ParaScreen Pan/Pf® RDT were done immediately by health center technicians. Blood slides were re-examined later at a central laboratory by independent expert microscopists.

Results

Of 1,997 febrile patients, 475 (23.8%) were positive by expert microscopists, with 57.7% P.falciparum, 24.6% P.vivax and 17.7% mixed infections. Sensitivity of health center microscopists for any malaria species was >90% in five health centers (four of which had the highest prevalence), >70% in nine centers and 44% in one site with lowest prevalence. Specificity for health center microscopy was very good (>95%) in all centers. For ParaScreen RDT, sensitivity was ≥90% in three centers, ≥70% in six and <60% in four centers. Specificity was ≥90% in all centers except one where it was 85%.

Conclusions

Health center microscopists performed well in nine of the ten health centers; while for ParaScreen RDT they performed well in only six centers. Overall the accuracy of local microscopy exceeded that of RDT for all outcomes. This study supports the introduction of RDTs only if accompanied by appropriate training, frequent supervision and quality control at all levels. Deficiencies in RDT use at some health centers must be rectified before universal replacement of good routine microscopy with RDTs. Maintenance and strengthening of good quality microscopy remains a priority at health center level.