Diagnostic Performance of Various Tests and Criteria Employed in Allergic Bronchopulmonary Aspergillosis: A Latent Class Analysis

Aim

The efficiency of various investigations and diagnostic criteria used in diagnosis of allergic bronchopulmonary aspergillosis (ABPA) remain unknown, primarily because of the lack of a gold standard. Latent class analysis (LCA) can provide estimates of sensitivity and specificity in absence of gold standard. Herein, we report the performance of various investigations and criteria employed in diagnosis of ABPA.

Methods

Consecutive subjects with asthma underwent all the following investigations Aspergillus skin test, IgE levels (total and A.fumigatus specific), Aspergillus precipitins, eosinophil count, chest radiograph, and high-resolution computed tomography (HRCT) of the chest. We used LCA to estimate the performance of various diagnostic tests and criteria in identification of ABPA.

Results

There were 372 asthmatics with a mean age of 35.9 years. The prevalence of Aspergillus sensitization was 53.2%. The sensitivity and specificity of various tests were Aspergillus skin test positivity (94.7%, 79.7%); IgE levels>1000 IU/mL (97.1%, 37.7%); A.fumigatus specific IgE levels>0.35 kUA/L (100%, 69.3%); Aspergillus precipitins (42.7%, 97.1%); eosinophil count>1000 cells/µL (29.5%, 93.1%); chest radiographic opacities (36.1%, 92.5%); bronchiectasis (91.9%, 80.9%); and, high-attenuation mucus (39.7%, 100%). The most accurate criteria was the Patterson criteria using six components followed by the Agarwal criteria. However, there was substantial decline in accuracy of the Patterson criteria if components of the criteria were either increased or decreased from six.

Conclusions

A.fumigatus specific IgE levels and high-attenuation mucus were found to be the most sensitive and specific test respectively in diagnosis of ABPA. The Patterson criteria remain the best diagnostic criteria however they have good veridicality only if six criteria are used.     

A Preliminary Study Examining the Binding Capacity of Akkermansia muciniphila and Desulfovibrio spp., to Colonic Mucin in Health and Ulcerative Colitis

Background

Akkermansia muciniphila and Desulfovibrio spp. are commensal microbes colonising the mucus gel layer of the colon. Both species have the capacity to utilise colonic mucin as a substrate. A. muciniphila degrades colonic mucin, while Desulfovibrio spp. metabolise the sulfate moiety of sulfated mucins. Altered abundances of these microorganisms have been reported in ulcerative colitis (UC). However their capacity to bind to human colonic mucin, and whether this binding capacity is affected by changes in mucin associated with UC, remain to be defined.

Methods

Mucin was isolated from resected colon from control patients undergoing resection for colonic cancer (n = 7) and patients undergoing resection for UC (n = 5). Isolated mucin was purified and printed onto mucin microarrays. Binding of reference strains and three clinical isolates of A. muciniphila and Desulfovibrio spp. to purified mucin was investigated.

Results

Both A. muciniphila and Desulfovibro spp. bound to mucin. The reference strain and all clinical isolates of A. muciniphila showed increased binding capacity for UC mucin (p < .005). The Desulfovibrio reference strain showed increased affinity for UC mucin. The mucin binding profiles of clinical isolates of Desulfovibrio spp. were specific to each isolate. Two isolates showed no difference in binding. One UC isolate bound with increased affinity to UC mucin (p < .005).

Conclusion

These preliminary data suggest that differences exist in the mucin binding capacity of isolates of A. muciniphila and Desulfovibrio spp. This study highlights the mucin microarray platform as a means of studying the ability of bacteria to interact with colonic mucin in health and disease.     

Deciphering the Niches of Colonisation of Vitis vinifera L. by the Esca-Associated Fungus Phaeoacremonium aleophilum Using a gfp Marked Strain and Cutting Systems

Introduction

Esca disease has become a major threat for viticulture. Phaeoacremonium aleophilum is considered a pioneer of the esca complex pathosystem, but its colonisation behaviour inside plants remains poorly investigated.

Material and Methods

In this study, P. aleophilum::gfp7 colonisation was assessed six and twelve weeks post-inoculation in two different types of tissues: in the node and the internode of one year-old rooted cuttings of Cabernet Sauvignon. These processes of colonisation were compared with the colonisation by the wild-type strain using a non-specific lectin probe Alexa Fluor 488-WGA.

Results

Data showed that six weeks post-inoculation of the internode, the fungus had colonised the inoculation point, the bark and xylem fibres. Bark, pith and xylem fibres were strongly colonised by the fungus twelve weeks post-inoculation and it can progress up to 8 mm from the point of inoculation using pith, bark and fibres. P. aleophilum was additionally detected in the lumen of xylem vessels in which tyloses blocked its progression. Different plant responses in specific tissues were additionally visualised. Inoculation of nodes led to restricted colonisation of P. aleophilum and this colonisation was associated with a plant response six weeks post-inoculation. The fungus was however detected in xylem vessels, bark and inside the pith twelve weeks post-inoculation.

Conclusions

These results demonstrate that P. aleophilum colonisation can vary according to the type of tissues and the type of spread using pith, bark and fibres. Woody tissues can respond to the injury and to the presence of this fungus, and xylem fibres play a key role in the early colonisation of the internode by P. aleophilum before the fungus can colonise xylem vessels.     

Emerging Antifungal Drug Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Among Other Species of <Emphasis Type="Italic">Aspergillus</Emphasis>

Purpose of Review

The purpose of this review is to give an overview of recent findings on antifungal resistance in Aspergillus fumigatus (the major causative agent of aspergillosis) and sibling Aspergillus species, which can be hidden agents of aspergillosis.

Recent Findings

Azole resistance by Cyp51A mutation in A. fumigatus is a growing problem worldwide. The resistance can occur in patients or in the environment. The former occurs by drug selection in the host, inducing mutations in Cyp51A. The latter is characterized by a tandem repeat in the promoter region of cyp51A gene and mutation(s) in Cyp51A. Environmental resistant strains are prevailing rapidly and globally. Moreover, efflux pump and biofilm formation are closely related with antifungal resistance of A. fumigatus. Finally, sibling species of Aspergillus are described with regard to antifungal resistance.

Summary

Environmental azole-resistant strains have newly emerged and been dispersed globally, and continuous survey and countermeasures are urgently needed against these strains. Although the contributions of Cyp51A and efflux pumps to antifungal resistance are becoming clear, other resistance mechanisms remain unclear. Further investigations including genome comparisons will help to clarify the novel resistant mechanisms and to develop countermeasures or novel antifungal drugs against resistant strains of A. fumigatus and other Aspergillus species that have low susceptibility to antifungal therapeutics.

     

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.     

Risk and Prognostic Factors for Multidrug-Resistant Acinetobacter Baumannii Complex Bacteremia: A Retrospective Study in a Tertiary Hospital of West China

Background

The increasing prevalence and mortality of multidrug-resistant (MDR) Acinetobacter baumannii complex-associated infections, especially bacteremia, in health care settings poses a great threat to public health. We proceeded to investigate the risk and prognostic factors for MDR A. baumannii complex bacteremia in mainland China.

Methods

This retrospective study was conducted at West China Hospital from January 2009 to December 2013. Using a computer-assisted microbiology laboratory database, patients with MDR A. baumannii complex bacteremia were included as the case group, while those infected with non-MDR A. baumannii complex were selected as the control group. The clinical data were collected and analyzed.

Results

There were 241 non-duplicated A. baumannii complex blood isolates identified in our research, with the overall rate of multidrug resistance reaching 75.52% over the past five years. Using multivariate logistic analysis, being in the intensive care unit (ICU) (adjusted odds ratio [aOR], 5.84; 95% confidence interval [CI], 1.67-20.44), increased Pittsburgh bacteremia score (aOR, 6.55; 95% CI, 1.27-33.70) and use of carbapenem (aOR, 8.90; 95% CI, 1.71-46.30) were independent risk factors for MDR acquisition among patients with A. baumannii complex bacteremia. Older age (aOR, 1.02; 95% CI, 1.00-1.04), being post-transplantation (aOR, 5.21; 95% CI, 1.13-24.04), having a higher Pittsburgh bacteremia score (aOR, 2.19; 95% CI, 1.08-4.47) and having a lower level of albumin (aOR, 0.93; 95% CI, 0.88-0.99) were identified as independent risk factors for 30-day mortality in patients with MDR A. baumannii complex bacteremia.

Conclusion

In conclusion, our research revealed the risk factors associated with acquisition of and mortality from MDR A. baumannii complex bacteremia, which may be used to prioritize infection control practices and prognostic evaluations.     

Release of Lungworm Larvae from Snails in the Environment: Potential for Alternative Transmission Pathways

Background

Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host.

Methodology/Principal Findings

Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior.

Conclusions

Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context.     

Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism

Background

Resistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.

Methods and Findings

We investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.

Conclusions

Azole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.     

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.Invasive aspergillosis is a fungal disease caused by Aspergillus species that primarily affects immunocompromised patients, such as those treated for hematological malignancy. Patients may become infected by inhalation of ambient air that contains fungal spores. The Aspergillus conidia can penetrate into the alveoli and if not effectively removed, may germinate, proliferate, and cause invasive aspergillosis. Mortality and morbidity due to invasive aspergillosis remain a significant problem.Triazoles, such as itraconazole (ITZ), voriconazole, and posaconazole, are used increasingly in the management of patients with this disease. Although the risk of resistance due to the increased use of triazoles is considered low (11), we recently observed ITZ resistance rapidly emerging in clinical Aspergillus fumigatus isolates (19, 22, 24, 25). Azole resistance was observed in up to 6% of patients in our hospital and in up to 14.5% of isolates sent to our laboratory from other hospitals in The Netherlands, which were obtained from patients with aspergillus disease (19). Furthermore, azole resistance has been reported in other European countries (3, 13, 19). The ITZ-resistant isolates also showed significantly reduced susceptibility to the other mold-active medical triazoles voriconazole and posaconazole (19). A substitution of leucine for histidine at codon 98 (L98H), combined with a 34-bp tandem repeat (designated TR) in the promoter region of the cyp51A gene (TR/L98H), which is the target for antifungal azoles, was found in 94% of isolates (14, 19, 24).Azole resistance can develop through the exposure of the fungus to azole compounds, which may occur in azole-treated patients or through the use of azole compounds in the environment. The dominance of a single resistance mechanism is difficult to explain by resistance development in individual azole-treated patients, as one would expect multiple resistance mechanisms to develop. Also, spread by person-to-person transmission of any Aspergillus isolate is highly unlikely. As inhalation of airborne aspergillus spores is the common route of infection for aspergillus diseases, we hypothesized that the dominance of a single resistance mechanism in clinical ITZ-resistant isolates was more consistent with acquisition from a common environmental source (19). If azole-resistant A. fumigatus is present in our environment, patients could inhale resistant spores and subsequently develop azole-resistant disease. Indeed, azole-resistant aspergillosis was reported in azole-naïve patients, indicating that resistance does not exclusively develop during azole therapy (24).Favorable conditions for resistance development are exposure to azole compounds and the presence of reproducing fungus (1). A. fumigatus is abundantly present in our environment as saprophytic, reproducing fungi, most notably in soil and compost. Furthermore, azoles are commonly used for plant protection as well as material preservation. Therefore, it appears that resistance development in A. fumigatus is feasible in the environment, and isolates that develop resistance to fungicides might be cross-resistant to medical triazoles.We investigated if A. fumigatus isolates that are present in our environment are resistant to medical triazoles and if they are cross-resistant to azole fungicides. Furthermore, we characterized the isolates by microsatellite typing in order to determine if they were genetically related to clinical A. fumigatus isolates previously obtained from patients cared for in our University Medical Center.     

Epidemiology and Clinical Features of Pulmonary Nontuberculous Mycobacteriosis in Nagasaki,Japan

Background and Objectives

Recent reports indicate that the incidence of nontuberculous mycobacterial-lung disease (NTM-LD) is increasing. This study aimed to investigate the epidemiology and clinical features of NTM-LD patients in Nagasaki prefecture, Japan to identify the negative prognostic factors for NTM-LD in Japan.

Methods

The medical records of patients newly diagnosed with NTM-LD in eleven hospitals in Nagasaki prefecture between January 2001 and February 2010 were reviewed. Data regarding the annual population of each region and the incidence of all forms of tuberculosis were collected to assess geographic variations in NTM-LD incidence, isolates, and radiological features.

Results

A total 975 patients were diagnosed with NTM-LD. The incidence increased over the study period and reached 11.0 and 10.1 per 100,000 population in 2008 and 2009, respectively. M. intracellulare was the most common pathogen in the southern region, and M. avium most common in other regions. The most common radiographic pattern was the nodular-bronchiectatic pattern. Age >60 years, body mass index <18.5 kg/m², underlying lung disease, and cavitary pattern were the negative prognostic factors at the 1-year follow-up.

Conclusions

The incidence of NTM-LD has been increasing in Nagasaki prefecture. The isolates and radiographic features of patients vary markedly by region.     

Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

Introduction

Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB.

Objective

To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs.

Methods

An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012.

Results

In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031).

Conclusions

A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied.     

Vibrio cholerae Serogroup O139: Isolation from Cholera Patients and Asymptomatic Household Family Members in Bangladesh between 2013 and 2014

Background

Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere.

Methods

Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup.

Findings

Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups.

Conclusion

These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.     

Biodeterioration Risk Threatens the 3100 Year Old Staircase of Hallstatt (Austria): Possible Involvement of Halophilic Microorganisms

Background

The prosperity of Hallstatt (Salzkammergut region, Austria) is based on the richness of salt in the surrounding mountains and salt mining, which is documented as far back as 1500 years B.C. Substantial archaeological evidence of Bronze and Iron Age salt mining has been discovered, with a wooden staircase (1108 B.C.) being one of the most impressive and well preserved finds. However, after its discovery, fungal mycelia have been observed on the surface of the staircase, most probably due to airborne contamination after its find.

Objective

As a basis for the further preservation of this valuable object, the active micro-flora was examined to investigate the presence of potentially biodegradative microorganisms.

Results

Most of the strains isolated from the staircase showed to be halotolerant and halophilic microorganisms, due to the saline environment of the mine. Results derived from culture-dependent assays revealed a high fungal diversity, including both halotolerant and halophilic fungi, the most dominant strains being members of the genus Phialosimplex (synonym: Aspergillus). Additionally, some typical cellulose degraders, namely Stachybotrys sp. and Cladosporium sp. were detected. Numerous bacterial strains were isolated and identified as members of 12 different genera, most of them being moderately halophilic species. The most dominant isolates affiliated with species of the genera Halovibrio and Marinococcus. Halophilic archaea were also isolated and identified as species of the genera Halococcus and Halorubrum. Molecular analyses complemented the cultivation assays, enabling the identification of some uncultivable archaea of the genera Halolamina, Haloplanus and Halobacterium. Results derived from fungi and bacteria supported those obtained by cultivation methods, exhibiting the same dominant members in the communities.

Conclusion

The results clearly showed the presence of some cellulose degraders that may become active if the requirements for growth and the environmental conditions turn suitable; therefore, these microorganisms must be regarded as a threat to the wood.     

Interactions between Environmental Factors and Melatonin Receptor Type 1A Polymorphism in Relation to Oral Cancer Susceptibility and Clinicopathologic Development

Background

The purpose of this study was to explore the combined effect of melatonin receptor type 1A (MTNR1A) gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of oral cancer.

Methodology and Principal Findings

Three polymorphisms of the MTNR1A gene from 618 patients with oral cancer and 560 non-cancer controls were analyzed by real-time polymerase chain reaction (PCR). The CTA haplotype of the studied MTNR1A polymorphisms (rs2119882, rs13140012, rs6553010) was related to a higher risk of oral cancer. Moreover, MTNR1A gene polymorphisms exhibited synergistic effects of environmental factors (betel quid and tobacco use) on the susceptibility of oral cancer. Finally, oral-cancer patients with betel quid-chewing habit who had T/T allele of MTNR1A rs13140012 were at higher risk for developing an advanced clinical stage and lymph node metastasis.

Conclusion

These results support gene-environment interactions of MTNR1A polymorphisms with smoking and betel quid-chewing habits possibly altering oral-cancer susceptibility and metastasis.     

Emerging,Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro

Background

Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS).

Methodology/Principal Findings

We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed ≥45% of the biofilm produced by the non-encapsulated M11 strain.

Conclusions/Significance

This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.     

Serotypes,Antibiotic Susceptibilities,and Multi-Locus Sequence Type Profiles of Streptococcus agalactiae Isolates Circulating in Beijing,China

Background

To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections.

Methods

All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles.

Results

In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB.

Conclusion

S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.     

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection,Identification and Genotyping in Clinical Specimens

Objective

Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.

Methods

To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.

Results

A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.

Conclusions

The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.     

Determination of Azole Resistance and TR<Subscript>34</Subscript>/L98H Mutations in Isolates of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Fumigati</Emphasis> from Turkish Cystic Fibrosis Patients

Background

Aspergillus fumigatus is the species section Fumigati most frequently isolated from the respiratory tract of cystic fibrosis (CF) patients. Recent studies suggest that mutations in the Cyp51 gene, particularly TR₃₄/L98H, are responsible for azole resistance.

Objectives and Methods

The focus of this study was on section Fumigati isolates isolated from the respiratory tract samples of CF patients. More specifically, the goal was to detect A. fumigatus isolates, test their antifungal susceptibility to itraconazole, voriconazole and posaconazole, and finally determine the presence of TR₃₄/L98H and other mutations in the isolates Cyp51A gene.

Results and Conclusions

A set of 31 isolates of Aspergillus section Fumigati were obtained from the sputum samples of 6 CF patients and subsequently identified to species level by microsatellite genotyping. All isolates were determined as A. fumigatus and involved 14 different genotypes. The minimal inhibitory concentrations to the three azoles were determined by the E-test method, and the Cyp51A gene was sequenced. One of the genotypes was found to be resistant to all azoles but no mutations were detected in the Cyp51A gene, especially the TR₃₄/L98H mutation. Therefore, mutations in genes other than Cyp51A or other distinct mechanisms may be responsible for this reported multiazole resistance found in a Turkish CF patient.

     

Functional Characterization of AbeD,an RND-Type Membrane Transporter in Antimicrobial Resistance in Acinetobacter baumannii

Background

Acinetobacter baumannii is becoming an increasing menace in health care settings especially in the intensive care units due to its ability to withstand adverse environmental conditions and exhibit innate resistance to different classes of antibiotics. Here we describe the biological contributions of abeD, a novel membrane transporter in bacterial stress response and antimicrobial resistance in A. baumannii.

Results

The abeD mutant displayed ~ 3.37 fold decreased survival and >5-fold reduced growth in hostile osmotic (0.25 M; NaCl) and oxidative (2.631 μM–6.574 μM; H₂O₂) stress conditions respectively. The abeD inactivated cells displayed increased susceptibility to ceftriaxone, gentamicin, rifampicin and tobramycin (~ 4.0 fold). The mutant displayed increased sensitivity to the hospital-based disinfectant benzalkonium chloride (~3.18-fold). In Caenorhabditis elegans model, the abeD mutant exhibited (P<0.01) lower virulence capability. Binding of SoxR on the regulatory fragments of abeD provide strong evidence for the involvement of SoxR system in regulating the expression of abeD in A. baumannii.

Conclusion

This study demonstrates the contributions of membrane transporter AbeD in bacterial physiology, stress response and antimicrobial resistance in A. baumannii for the first time.