16S PCR Research Articles

Added diagnostic value of broad-range 16S PCR on periprosthetic tissue and clinical specimens from other normally sterile body sites.

Evaluation of 16S PCR in addition to the standard culture to improve the pathogen detection rate in clinical specimens. Microbiological culture and direct 16S PCR was performed on specimens from suspected prosthetic joint infection patients (cohort-1) and on tissues and fluids from other normally sterile body sites (cohort-2). Based on clinical and microbiological data, the detection rate for both methods was assessed, assuming no superiority of either 16S PCR or culture. In cohort-1, 469 specimens were obtained. Culture was positive in 170 (36·2%) specimens, 16S PCR detected 70 (41·2%) of those pathogens. Additionally, 16S PCR detected pathogens in 13 of 299 (4·3%) culture-negative specimens. In cohort-2, pathogens were cultured in 52 of 430 (12·1%) specimens and 16S PCR revealed those pathogens in 32 (61·5%) specimens. 16S PCR detected pathogens in 31 of 378 (8·2%) culture-negative specimens. Overall, the yield with 16S PCR was low. For cohort-1 16S PCR detected pathogens in 4·3% of culture-negative specimens, where this was 8·2% for cohort-2. Although direct 16S PCR cannot replace culture, it may offer a valuable additional diagnostic option for detection of difficult to culture micro-organisms in culture-negative clinical specimens.

Bacterial profile of infant feces associated with lactation infectious breasts.

BackgroundMastitis is a common complication in lactating women. However, the diversity of intestinal bacteria in infant exclusively fed infectious milk remains uncharacterized. Our colleagues recently established a method based on 16S and 23S rRNA-targeted reverse transcription–quantitative PCR (RT-qPCR) for detecting bacteria.Materials and methodsIn the present study, the bacteria present in 14 samples of milk and infant feces were characterized using the RT-qPCR method, and concentrations of fecal organic acids were measured during the period of breast massage using HPLC.ResultsStreptococcus agalactiae and Str. parasanguinis were detected in milk from mastitis patients, whereas Str. salivarius and Str. thermophilus were the predominant bacteria in milk from engorged breasts. In feces of breastfed infants, Str. salivarius, Str. thermophiles, and Str. parasanguinis were isolated. Levels of lactate were high in fecal samples, whereas the pH of infant feces stabilized during breast massage. The bacterial diversity of milk from lactation infectious breasts was similar to that in feces of infant fed milk from lactation infectious breasts. Streptococcus species isolated from the feces of breastfed infants are related to oral cavity health.ConclusionThese results suggest that Streptococcus species, which are part of the healthy oral microflora, may play an important role in preserving the intestinal bacterial flora in infants fed infectious milk.

Development of loop-mediated isothermal amplification (LAMP) assay for detection of Hepatozoon canis infection in dogs

The laboratory diagnosis of canine hepatozoonosis, caused by Hepatozoon canis is tedious, especially in chronic and latent infections. In the present investigation, a loop mediated isothermal amplification (LAMP) assay was developed and standardized targeting the partial 18S rRNA gene (GenBank accession no. KU096058). The LAMP primers specifically amplified H. canis DNA, whereas no amplification was detected in DNA samples from dogs infected with Babesia vogeli, B. gibsoni, Ehrlichia canis and Trypanosoma evansi, and no amplification was observed in DNA samples from H. canis-free dogs. The threshold sensitivity level of the assay was determined to be 15 fg of genomic DNA of H. canis. Furthermore, evaluation of blood samples collected from 250 dogs presented at Small Animal Clinics, Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab (India) was carried out for the presence of H. canis by microscopy, 18S PCR assay and LAMP assay. Of the total samples subjected to these tests, LAMP detected H. canis in 75 samples, while 18S PCR and microscopy detected H. canis in 28 and 9 samples, respectively. The present investigation has developed, for the first time, a highly sensitive, specific and rapid LAMP assay for the detection of H. canis, which has practical applications for the screening of field samples.

2297. The Diagnostic Yield of 16/18S rRNA PCR of Sterile Site Samples in Pediatric Patients

Background16S ribosomal RNA (rRNA) and 18S rRNA gene polymerase chain reaction (16/18S PCR) with sequencing can provide expeditious bacterial or fungal pathogen identification from sterile site samples (cost $474/PCR). Our objective was to assess the utilization and diagnostic yield of 16/18S PCR of sterile site samples in pediatric patients.MethodsPatients’ sterile site fluid or direct tissue specimens were collected and cultured at Lurie Children’s Hospital of Chicago and sent to Northwestern Memorial Hospital for 16/18S PCR as clinically indicated. Clinical data were reviewed including PCRs, cultures, and medical conditions.Results16/18S PCR testing increased over the study period. In total, 177 samples were sent for 16S and/or 18S PCR from 146 patients (January 2016–April 2018). Osteoarticular, CSF, pleural fluid and organ tissue (n = 28; lung=19, chest mass=2, liver=2, spleen=2, etc.) sites were most frequent. The yield of 16/18S PCR by source is listed in Table 1. Twenty-eight of 156 samples for 16S PCR were positive (17.9%); 21 with a single organism ID, one with two organisms, and 6 indeterminate. (Table 2). Of negative 16S PCR samples, one grew Mycobacterium avium complex in culture. 18S PCR was performed on 108 unique samples; 7 were positive (6.5%, Table 3). For 4 positive 18S PCRs, a fungus also grew in culture with 3 concordant results and one discordant. Two negative 18S PCR samples grew molds (Phellinus spp.; Blastomyces dermatitidis). All patients (100%) with positive 18S PCR were immunocompromised compared with 21% (6/28) with positive 16S PCR. Both 16S and 18S PCRs were sent on 87 samples of which 16S PCR was positive in 5, 18S PCR was positive in 3, and none had both 16/18S PCRs positive.Conclusion16/18S PCR can provide important infectious pathogen diagnostics. 16S PCR should be sent only if bacterial culture is negative with higher yield sites being brain, abscess, pleural effusion, bone/joint and CSF. 16S PCR appears useful if an anaerobic pathogen is likely but conditions are not optimal for recovery. 18S PCR is highest yield in patients at risk of fungal disease. 16 and 18S PCRs were often sent together, likely reflexively. Selective or sequential testing may be advisable for most cases, guided by the clinical index of suspicion. Best practices to optimize resource utilization and clinical impact are evolving. Disclosures All authors: No reported disclosures.

Antibiotics for amniotic-fluid colonization by Ureaplasma and/or Mycoplasma spp. to prevent preterm birth: A randomized trial.

ObjectiveTo assess whether antibiotics used for treatment in asymptomatic second-trimester women positive for Mycoplasma or Ureaplasma spp. detected by amniotic-fluid PCR prevents preterm delivery.DesignA randomized, double-blind, placebo-controlled trial.Setting10 maternal fetal medicine centers in France.PopulationWomen with a singleton pregnancy who underwent amniocentesis between 16 and 20 weeks’ gestation (weeks) for Down syndrome screening. A sample of 238 women with PCR-positive findings per treatment group was needed to show a 50% reduction in the preterm delivery rate.MethodsAmniotic fluid was tested. Women with positive findings on real-time PCR of amniotic fluid for Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum and Ureaplasma parvum were randomized to receive josamycin or placebo. Amniotic fluid was also tested for 16S PCR.Main outcome measuresThe primary outcome was delivery before 37 weeks.ResultsIn total, 1043 women underwent amniotic-fluid screening with specific PCR detection between July 2008 and July 2011: PCR detection failed in 27 (2.6%), and 20 (1.9%) underwent termination of pregnancy. Among the 1016 women with PCR results, 980 had available data for the primary outcome (delivery before 37 weeks) and 29 (3.0%) were positive for Ureaplasma and/or Mycoplasma spp. Because of the low rate of women with PCR-positive findings, the trial was stopped prematurely. In total, 19 women were randomized to receive placebo (n = 8) or josamycin (n = 11) and their characteristics were comparable, as was the rate of preterm delivery and secondary outcomes. In comparing all PCR-positive and -negative women regardless of treatment, PCR positivity for Ureaplasma and/or Mycoplasma spp. was not associated with any adverse pregnancy or neonatal outcome. Amniotic-fluid screening by 16S PCR showed no other bacterial colonization associated with preterm birth.ConclusionsBecause of a low amniotic fluid colonization rate, the trial was interrupted. Maternal amniotic-fluid colonization by Mycoplasma and/or Ureaplasma spp. at 16–20 weeks in asymptomatic women is rare and not associated with adverse pregnancy outcomes.Trial registrationClinicalTrials.gov NCT00718705

Genotypic Characterization of Cryptosporidium Species in Humans and Peri-Domestic Animals in Ekiti and Oyo States, Nigeria.

Cryptosporidiosis is one of the leading causes of diarrhea in humans and several other vertebrate species. Because surveys of Cryptosporidium genotypes from animals and humans living in the same region are rare, our understanding of the importance of zoonotic transmission in the epidemiology of cryptosporidiosis remains superficial. PCR was used to amplify a portion of the Cryptosporidium 18S small subunit ribosomal RNA gene from fecal DNA from humans and livestock living in Ekiti and Oyo states, Nigeria. PCR-positive samples were further analyzed using PCR targeting the heat-shock protein HSP-70, the actin, and the sporozoite glycoprotein gene gp60. A questionnaire was used to collect demographic information. Sixteen of 187 samples collected were Cryptosporidium 18S PCR positive. Of these, 5 samples originating from HIV-positive patients, 5 from otherwise healthy children, 2 from chickens, 3 from goats, and 1 from a dog were positive for at least 1 marker. Sequencing of the 18S rRNA amplicons revealed the presence of Cryptosporidium parvum in 2 HIV positive patients and in a child; the actin sequence confirmed the presence of this species. Two samples of HIV-positive patients amplified Cryptosporidium hominis 18S rRNA, one of them confirmed by the HSP-70, actin, and gp60 sequences. Cryptosporidium meleagridis was found in another HIV patient, while C. hominis was detected in 3 children (of which 2 were confirmed by gp60). Cryptosporidium muris was found in 1 child. In birds, we found C. meleagridis and, significantly, C. parvum, whereas we detected C. parvum and C. muris in 1 goat each. The only dog sampled was positive for Cryptosporidium canis. We conclude that, in the environment we surveyed, humans and animals are a potential part of the same transmission cycle. Measures to prevent zoonotic transmission should therefore be considered to reduce the prevalence of cryptosporidiosis.

The Molecular identification of root nodule bacteria from edible crops of Fabaceae family, Kerala

The root nodule bacteria were isolated from three different leguminous plants belonging to the Fabaceae family such as Cajanus cajan, Lablab purpureus and Vigna unguiculata using YEMA medium and identified. The isolates were submitted to 16S rRNA and PCR–RFLP typing. A representative sample was further submitted to sequence analysis of 16S rRNA. Isolates were assigned to two genera and were related to Rhizobium spp. (75%) and Bacillus spp. (25%) respectively. This study opens the doors to researchers to do specific studies on this root nodule bacteria which are symbiotic with these legume plants in this area for their antibacterial activity, medicine and food application, and sustainable agriculture. Rhizobium and other beneficial microbes may be used as biofertilizers as a substitute for chemical fertilizers.

Use of Pristinamycin for Macrolide-Resistant Mycoplasma genitalium Infection.

High levels of macrolide resistance and increasing fluoroquinolone resistance are found in Mycoplasma genitalium in many countries. We evaluated pristinamycin for macrolide-resistant M. genitalium in a sexual health center in Australia. Microbiologic cure was determined by M. genitalium–specific 16S PCR 14–90 days after treatment began. Of 114 persons treated with pristinamycin, infection was cured in 85 (75%). This percentage did not change when pristinamycin was given at daily doses of 2 g or 4 g or at 3 g combined with 200 mg doxycycline. In infections with higher pretreatment bacterial load, treatment was twice as likely to fail for each 1 log10 increase in bacterial load. Gastrointestinal side effects occurred in 7% of patients. Pristinamycin at maximum oral dose, or combined with doxycycline, cured 75% of macrolide-resistant M. genitalium infections. Pristinamycin is well-tolerated and remains an option where fluoroquinolones have failed or cannot be used.

Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba. It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis, however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi, and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba. The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts.

Halobacterium sp. NRC-1: A Review

Halobacteriumsp. NRC-1 is the halophilic bacterium. This halophile has a great potential to survive on high salinity conditions. The genome of this bacterium has sequenced. The studies have done at metagenomics level, which include the Identification and Molecular Characterization. Identification of this extreme halophile was done by 16S ribotyping and PCR amplification using universal primers FD1 and rP1. The sequences than compared with already sequenced genomes of halophiles. The post genomic tools and microarrays were applied for sequencing. Halobacteriumsp. NRC-1 transcriptional profiling showed that it is highly responsive to the environment and give specific responses at gene expression level. There is a need for extensive research at metaproteomic level. To study the expression and function of proteins, whole protein profiling of this specie need to be studied.

Primary pyogenic ventriculitis caused by Neisseria meningitidis: case report and review of the literature.

Background. Pyogenic ventriculitis is a well-known complication of meningitis, brain abscesses and intraventricular drains. Primary pyogenic ventriculitis is a rare entity and few cases have been described so far. We report the first case of primary pyogenic ventriculitis in an adult caused by Neisseria meningitidis and present an overview of all reported adult primary pyogenic ventriculitis cases in the English literature.Methods. A PubMed search was performed using the terms ependymitis, ventricular empyema, pyocephalus and ventriculitis. Filter was set for adults and English. Articles in which pyogenic ventriculitis was a complication of well-known risk factors were excluded. A total of five cases of primary pyogenic ventriculitis were identified.Results. There were seven adult patients. Only one patient showed signs of meningeal irritation. Four patients had positive blood cultures with Escherichia coli (one patient), methicillin-resistant Staphylococcus aureus (one patient), one patient was bacteraemic with Enterococcus faecalis, Escherichia coli and Peptostreptococcus spp., and N. meningitidis (our patient). In four patients cerebrospinal fluid was sent for culture, which yielded methicillin-sensitive Staphylococcus aureus (one patient), Peptostreptococcus spp. (one patient), Streptococcus intermedius (one patient, identified via 16S PCR) and Listeria monocytogenes (one patient). Cerebrospinal fluid cell count was determined in four patients and showed pleocytosis in all four cases. Ventricular drainage was performed in four patients. Five patients survived.Discussion. We report the first case of pyogenic ventriculitis caused by N. meningitidis. Primary pyogenic ventriculitis is a rare entity with various clinical presentations caused by various bacterial species. Treatment consists of adequate antimicrobial therapy, and ventricular drainage may be necessary.

Metabolomic and Metagenomic Analysis of Two Crude Oil Production Pipelines Experiencing Differential Rates of Corrosion.

Corrosion processes in two North Sea oil production pipelines were studied by analyzing pig envelope samples via metagenomic and metabolomic techniques. Both production systems have similar physico-chemical properties and injection waters are treated with nitrate, but one pipeline experiences severe corrosion and the other does not. Early and late pigging material was collected to gain insight into the potential causes for differential corrosion rates. Metabolites were extracted and analyzed via ultra-high performance liquid chromatography/high-resolution mass spectrometry with electrospray ionization (ESI) in both positive and negative ion modes. Metabolites were analyzed by comparison with standards indicative of aerobic and anaerobic hydrocarbon metabolism and by comparison to predicted masses for KEGG metabolites. Microbial community structure was analyzed via 16S rRNA gene qPCR, sequencing of 16S PCR products, and MySeq Illumina shotgun sequencing of community DNA. Metagenomic data were used to reconstruct the full length 16S rRNA genes and genomes of dominant microorganisms. Sequence data were also interrogated via KEGG annotation and for the presence of genes related to terminal electron accepting (TEA) processes as well as aerobic and anaerobic hydrocarbon degradation. Significant and distinct differences were observed when comparing the ‘high corrosion’ (HC) and the ‘low corrosion’ (LC) pipeline systems, especially with respect to the TEA utilization potential. The HC samples were dominated by sulfate-reducing bacteria (SRB) and archaea known for their ability to utilize simple carbon substrates, whereas LC samples were dominated by pseudomonads with the genetic potential for denitrification and aerobic hydrocarbon degradation. The frequency of aerobic hydrocarbon degradation genes was low in the HC system, and anaerobic hydrocarbon degradation genes were not detected in either pipeline. This is in contrast with metabolite analysis, which demonstrated the presence of several succinic acids in HC samples that are diagnostic of anaerobic hydrocarbon metabolism. Identifiable aerobic metabolites were confined to the LC samples, consistent with the metagenomic data. Overall, these data suggest that corrosion management might benefit from a more refined understanding of microbial community resilience in the face of disturbances such as nitrate treatment or pigging, which frequently prove insufficient to alter community structure toward a stable, less-corrosive assemblage.

The Prevalence of Canine Oral Protozoa and Their Association with Periodontal Disease

Periodontal disease is one of the most important health concerns for companion animals. Research into canine forms of periodontitis has focused on the identification and characterization of the bacterial communities present. However, other microorganisms are known to inhabit the oral cavity and could also influence the disease process. A novel, broad spectrum 18S PCR was developed and used, in conjunction with next-generation sequencing analyses to target the identification of protists. Trichomonas sp. and Entamoeba sp. were identified from 92 samples of canine plaque. The overall prevalence of trichomonads was 56.52% (52/92) and entamoebae was 4.34% (4/92). Next-generation sequencing of pooled healthy, gingivitis, early-stage periodontitis, and severe periodontitis samples revealed the proportion of trichomonad sequences to be 3.51% (health), 2.84% (gingivitis), 6.07% (early periodontitis), and 35.04% (severe periodontitis), respectively, and entamoebae to be 0.01% (health), 0.01% (gingivitis), 0.80% (early-stage periodontitis), and 7.91% (severe periodontitis) respectively. Both genera of protists were statistically associated with plaque from dogs with periodontal disease. These findings provide the first conclusive evidence for the presence of oral protozoa in dog plaque and suggest a possible role for protozoa in the periodontal disease process.

PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S)

Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections.

Molecular characterization of Ehrlichia canis infecting dogs, Buenos Aires

Canine monocytic ehrlichiosis (CME) is a worldwide potentially fatal tick-borne rickettsial disease of dogs caused by Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato. CME diagnosis includes indirect (serology) and direct (e.g. blood smears and PCR) methods. PCR is more sensitive and specific than direct microscopic examination and positive PCR results confirm infection, whereas positive serologic test results only confirm exposure.The aim of the present study was to perform a molecular characterization of E. canis from canine samples of the Metropolitan Area of Buenos Aires. We studied 223 blood samples of dogs submitted to our institute for CME diagnoses. The samples were initially screened for Anaplasmataceae family by PCR, resulting in 30 positive dogs (13.4%). Subsequently, positive DNAs were analyzed by nested PCR 16S rRNA specific for E. canis or Anaplasma platys, resulting in 15 (6.7%) and 16 (7.2%) positive dogs, respectively.For molecular characterization, samples positive for E. canis were subjected to amplification of a fragment of the dsb and p28 genes. The nucleotide sequences obtained for the dsb fragment resulted in 100% identity with others E. canis found in dogs from different regions of worldwide. The nucleotide sequences obtained for p28 gene resulted in 100% of identity with each other and closely with E. canis str. Jaboticabal (Brazil). Identity with others sequences of E. canis ranged from 76.9 to 79.7%.The occurrence of canine cases molecularly confirmed in Metropolitan Area of Buenos Aires highlights the need for more studies in order to understand epidemiological factors associated with CME, especially the disease transmission dynamic in South America given the existence of two lineages of R. sanguineus sensu lato with different vectorial capacity for transmission of E. canis.

Accurate quantification of supercoiled DNA by digital PCR.

Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

Molecular Diagnosis of Urinary Tract Infections by Semi-Quantitative Detection of Uropathogens in a Routine Clinical Hospital Setting.

BackgroundThe objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and Pseudomonas aeruginosa. 16S based PCR was performed in parallel to detect Gram-positive and Gram-negative bacteria. Firstly to identify non-targeted agents of infection in the same urine specimen, and secondly to quantify background flora. The method was evaluated in comparison with standard bacterial culture, and a commercial PCR kit for detection of uropathogens.FindingsAnalysis with a known panel of 116 clinical isolates yielded a PCR specificity of 100%. Analysis of urine specimens from 211 patients revealed a high correlation of PCR Cq values with both culture positivity and quantity. Concordance between PCR and culture was 98% when both methods yielded results. PCR was found to be more sensitive than culture. With a cut-off Cq value of 33, the negative predictive value of PCR was 94%. The 16S PCR confirmed most results. One specimen was positive by 16S PCR suggesting another cause of infection not detected by the specific PCR assays.ConclusionWe conclude that it is feasible to detect and identify uropathogens by multiplex real-time PCR assay.

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

Actinomycosis-like Propionibacterium propionicum infection associated with use of intrauterine contraceptive device: A case report

Introduction: Intrauterine contraceptive devices generally carry a low risk of complications. Infection, possibly due to damage caused upon insertion, can occur and actinomycetaceae are the most likely cause of pelvic actinomycosis. Case report: A 41-year-old female presented with a two-month history of raised temperature, weight loss and abdominal tenderness. Contrast tomography imaging indicated pyometria and pelvic actinomycosis-like abscess linked to intrauterine contraceptive device use. Full microbiological reporting, including anaerobic cultures and 16S PCR, identified the bacterium Propionibacterium propionicum. A six-month course of amoxicillin resolved the infection and the patient made a full recovery. Conclusion: Increased IUCD-related actinomycosis awareness and improved diagnostics may increase detection of P. propionicum, a pathogen rarely reported on to date.

Gut Dysbiosis in Patients with Anorexia Nervosa.

Anorexia nervosa (AN) is a psychological illness with devastating physical consequences; however, its pathophysiological mechanism remains unclear. Because numerous reports have indicated the importance of gut microbiota in the regulation of weight gain, it is reasonable to speculate that AN patients might have a microbial imbalance, i.e. dysbiosis, in their gut. In this study, we compared the fecal microbiota of female patients with AN (n = 25), including restrictive (ANR, n = 14) and binge-eating (ANBP, n = 11) subtypes, with those of age-matched healthy female controls (n = 21) using the Yakult Intestinal Flora-SCAN based on 16S or 23S rRNA–targeted RT–quantitative PCR technology. AN patients had significantly lower amounts of total bacteria and obligate anaerobes including those from the Clostridium coccoides group, Clostridium leptum subgroup, and Bacteroides fragilis group than the age-matched healthy women. Lower numbers of Streptococcus were also found in the AN group than in the control group. In the analysis based on AN subtypes, the counts of the Bacteroides fragilis group in the ANR and ANBP groups and the counts of the Clostridium coccoides group in the ANR group were significantly lower than those in the control group. The detection rate of the Lactobacillus plantarum subgroup was significantly lower in the AN group than in the control group. The AN group had significantly lower acetic and propionic acid concentrations in the feces than the control group. Moreover, the subtype analysis showed that the fecal concentrations of acetic acid were lower in the ANR group than in the control group. Principal component analysis confirmed a clear difference in the bacterial components between the AN patients and healthy women. Collectively, these results clearly indicate the existence of dysbiosis in the gut of AN patients.