Broad-range 16S rRNA Gene PCR Research Articles

Development of a scoring system to identify high-yield specimens for bacterial broad-range 16S rRNA gene PCR with sequencing at a tertiary care medical center.

Broad-range bacterial polymerase chain reaction with sequencing (BRBPS) provides valuable diagnostic data, especially in cases of culture-negative infections. However, as BRBPS testing demonstrates generally low positivity, cost per impactful result can be high and commonly involves longer turnaround times compared with other methods, targeting use of this assay to high-yield situations remains a challenging goal. Diagnostic stewardship can help alleviate these challenges and increase clinical utility, yet not all laboratories have a dedicated stewardship team, and little formal guidance exists on identifying high-yield samples outside of specific clinical syndromes. In this study, we performed a retrospective review of 86 BRBPS orders from a tertiary care medical center, with a focus on identifying high-yield cases using an infectious markers scoring system, visualized inflammation or organism (VIO) score, to predict return of actionable diagnostic data. Using chart review, we evaluated how results from high VIO score or low VIO score specimens contributed to clinical management. Testing low VIO score samples identified an organism in only 10% of samples, and of these positive results, 33% were considered to represent contamination. Despite negative routine workup and no identified pathogen via BRBPS, broad antimicrobial treatment was continued in 85% of cases with a low VIO score. In contrast, specimens with high VIO scores were more predictably positive by BRBPS, identified organisms that were universally considered pathogens, and provided opportunities to target or de-escalate antimicrobial therapy. This study describes the VIO scoring system to guide the identification of high-yield samples and steward the appropriate use of BRBPS testing.

Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

Molecular Identification of Cervical Microbes in HIV-Negative and HIV-Positive Women in an African Setting Using a Customized Bacterial Vaginosis Microbial DNA Quantitative PCR (qPCR) Array.

ABSTRACTBacterial vaginosis (BV) is a common polymicrobial vaginal disorder that is associated with sexually transmitted infections (STIs), including HIV. Several studies have utilized broad-range 16S rRNA gene PCR assays with sequence analysis to characterize cervicovaginal bacterial communities of women with healthy and diseased conditions. With the high burden of BV and STIs among African women, there is a need for targeted PCR assays that can rapidly determine the true epidemiological profile of key cervical microbes, including BV-associated bacteria, and a need to explore the utility of such assays for microbiological diagnosis of BV. Here, we used a taxon-directed 16S rRNA gene quantitative PCR (qPCR) assay to examine the prevalences and determinants of specific cervical microbes among African women with and without HIV infection. Cervical samples were collected using a cytobrush from 162 women (aged ≥30 years) attending a community-based clinic in Eastern Cape, South Africa. The samples were screened for specific microbes (i.e., STIs, emerging sexually transmitted pathogens [pathobionts], and BV-associated bacteria) using a customized bacterial vaginosis microbial DNA qPCR array. Statistical analyses were performed using GraphPad Prism v6.01. Chi-square/Fisher’s exact tests were used to evaluate the determinants associated with specific cervical microbes. Only 145 women had any detectable microbes and were included in the analysis. Lactobacillus iners (62.8%) and specific BV-associated bacteria, namely, Gardnerella vaginalis (58.6%), Atopobium vaginae (40.7%), and the pathobiont Ureaplasma parvum (37.9%), were the most prevalent microbes. Hierarchical clustering analysis revealed that 42.8% of the women (62/145) had a diverse array of heterogeneously distributed bacteria typically linked to BV. Women with detectable Lactobacillus species, specifically Lactobacillus crispatus and Lactobacillus jensenii, and to a lesser extent L. iners, had very low prevalence of BV-associated bacteria. Although the cumulative burden of STIs/pathobionts was 62.8%, Chlamydia trachomatis (3.4%), Neisseria gonorrhoeae (4.8%), and Trichomonas vaginalis (4.8%) were detected at low rates. HIV infection was associated with the presence of STIs/pathobionts (P = 0.022) and L. iners (P = 0.003). Prevalent STIs/pathobionts were associated with having multiple partners in the past 12 months (n ≥ 2, P = 0.015), high number of lifetime sexual partners (n ≥ 3, P = 0.007), vaginal sex in the past month (P = 0.010), and decreasing age of women (P = 0.005). C. trachomatis was associated with increasing age among HIV-positive women (P = 0.016). The pathobiont Ureaplasma urealyticum was inversely associated with age of women in the whole cohort (P = 0.018). The overall prevalence of STIs/pathobionts was high and was associated with HIV infection and sexual behavior. Our study helps us to understand the epidemiological trend of STIs and pathobionts and highlights the need to understand the impact of sexual networks on STI and pathobiont transmission and prevention among women in an African setting.IMPORTANCE Bacterial vaginosis (BV), whose etiology remains a matter of controversy, is a common vaginal disorder among reproductive-age women and can increase the risk for sexually transmitted infections (STIs). African women bear a disproportionately high burden of STIs and BV. Using a targeted quantitative PCR (qPCR) assay, a customized bacterial vaginosis microbial DNA qPCR array, we examined the prevalences and determinants of key cervical microbes, including BV-associated bacteria and emerging sexually transmitted pathogens (pathobionts) among women of African descent aged between 30 and 75 years. High-risk behaviors were associated with a higher prevalence of STIs/pathobionts, suggesting the need to better understand the influence of sexual networks on STI and pathobiont transmission and prevention among women. Our molecular assay is important in the surveillance of BV-associated bacteria, pathobionts, and STIs as well as diagnostic microbiology of BV. Furthermore, our research contributes to a better understanding of the epidemiology of STIs and pathobionts in Africa.

Impact of preconception vaginal microbiota on women’s risk of spontaneous preterm birth: protocol for a prospective case-cohort study

IntroductionBacterial vaginosis (BV) and vaginal microbiota disruption during pregnancy are associated with increased risk of spontaneous preterm birth (SPTB), but clinical trials of BV treatment during pregnancy have shown little...

A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene of Kingella kingae and Application to 201 Pediatric Clinical Specimens.

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting the groEL gene and the RTX locus of K. kingae However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack specificity because they could not distinguish between K. kingae and the recently described Kingella negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingaegroEL-based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of the K. kingae mdh gene from 20 distinct sequence types of K. kingae This novel K. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Increased Pathogen Identification in Vascular Graft Infections by the Combined Use of Tissue Cultures and 16S rRNA Gene Polymerase Chain Reaction.

Background: Vascular graft infections (VGI) are difficult to diagnose and treat, and despite redo surgery combined with antimicrobial treatment, outcomes are often poor. VGI diagnosis is based on a combination of clinical, radiological, laboratory and microbiological criteria. However, as many of the VGI patients are already under antimicrobial treatment at the time of redo surgery, microbiological identification is often difficult and bacterial cultures often remain negative rendering targeted treatment impossible. We aimed to assess the benefit of 16S rRNA gene polymerase chain reaction (broad-range PCR) for better microbiological identification in patients with VGI.Methods: We prospectively analyzed the clinical, microbiological, and treatment data of patients enrolled in the observational Vascular Graft Cohort Study (VASGRA), University Hospital Zurich, Switzerland. The routine diagnostic work-up involved microbiological cultures of minced tissue samples, and the use of molecular techniques in parallel. Patient-related and microbiological data were assessed in descriptive analyses, and we calculated sensitivity, specificity, negative and positive predictive value for broad-range 16S rRNA gene PCR versus culture (considered as gold standard).Results: We investigated 60 patients (median age 66 years (Interquartile range [IQR] 59–75)) with confirmed VGI between May 2013 and July 2017. The prevalence of antimicrobial pretreatment at the time of sampling was high [91%; median days of antibiotics 7 days (IQR 1–18)]. We investigated 226 microbiological specimens. Thereof, 176 (78%) were culture-negative and 50 (22%) were culture-positive. There was a concordance of 70% (158/226) between conventional culture and broad-range PCR (sensitivity 58% (95% CI 43–72); specificity 74% (67–80%)). Among the group of 176 culture-negative specimens, 46 specimens were broad-range PCR-positive resulting in identification of overall 69 species. Among the culture and/or broad-range PCR-positive specimens (n = 96), 74 (77%) were monomicrobial and 22 (23%) polymicrobial, whereas the rate of polymicrobial samples was higher in broad-range PCR-positive specimens (93%).Conclusions: Combined cultures and broad-range 16S rRNA gene PCR from periprosthetic tissue and/or explanted vascular grafts increased the diagnostic accuracy in VGI, particularly in patients already under antimicrobial treatment at the time of redo surgery. Ideally, antimicrobial treatment should be withheld until surgical sampling in order to optimize microbiological diagnostics.Clinical trials.gov identifier: NCT01821664

Parallel detection of lactobacillus and bacterial vaginosis-associated bacterial DNA in the chorioamnion and vagina of pregnant women at term

Background: The majority of early preterm births are associated with intrauterine infections, which are thought to occur when microbes traffic into the uterus from the lower genital tract and seed the placenta. Bacterial vaginosis (BV) is associated with heterogeneous bacterial communities in the vagina and is linked to preterm birth. The extent to which trafficking into the uterus of normal and BV-associated vaginal bacteria occurs is unknown. The study objective was to characterize in parallel the distribution and quantities of bacteria in the vagina, uterus, and placental compartments.Methods: Pregnant women at term (≥37 weeks) presenting for delivery were recruited prospectively. Swabs were collected in parallel from the vagina, chorioamnion. Choriodecidual swabs were collected if a cesarean section was performed. Samples were analyzed by culture, broad-range 16S rRNA gene PCR, and bacterial species-specific quantitative PCR (qPCR) for DNA from Lactobacillus and a panel of BV-associated bacteria. Results were correlated with placental histopathology.Results: Of the 23 women enrolled, 15 were delivered by cesarean section (N = 10 without labor; N = 5 in labor) and eight were delivered vaginally. BV was diagnosed in two women not in labor. Placental histopathology identified chorioamnionitis or funisitis in six cases [1/10 (10%) not in labor; 5/13 (38%) in labor]. Among non-laboring women, broad-range 16S qPCR detected bacteria in the chorioamnion and the choriodecidua (4/10; 40%). Among laboring women, Lactobacillus species were frequently detected in the chorioamnion by qPCR (4/13; 31%). In one case, mild chorioamnionitis was associated with qPCR detection of similar microbes in the chorioamnion and vagina (e.g. Leptotrichia/Sneathia, Megasphaera), along a quantitative gradient.Conclusions: Microbial trafficking of lactobacilli and fastidious bacteria into the chorioamniotic membranes and choriodecidua occurs at term in normal pregnancies. In one case, we demonstrated a quantitative gradient between multiple bacterial species in the lower genital tract and placenta. Not all bacterial colonization is associated with placental inflammation and clinical sequelae. Further studies of the role of placental colonization with Lactobacillus in normal pregnancy and fastidious bacteria in chorioamnionitis may improve prevention and treatment approaches for preterm labor.

Evaluation of the association between the concentrations of key vaginal bacteria and the increased risk of HIV acquisition in African women from five cohorts: a nested case-control study

Disruptions of vaginal microbiota might increase women's susceptibility to HIV infection. Advances in molecular microbiology have enabled detailed examination of associations between vaginal bacteria and HIV acquisition. Therefore, this study aimed to evaluate the association between the concentrations of specific vaginal bacteria and increased risk of HIV acquisition in African women. We did a nested case-control study of participants from eastern and southern Africa. Data from five cohorts of African women (female sex workers, pregnant and post-partum women, and women in serodiscordant relationships) were used to form a nested case-control analysis between women who acquired HIV infection versus those who remained seronegative. Deep sequence analysis of broad-range 16S rRNA gene PCR products was applied to a subset of 55 cases and 55 controls. From these data, 20 taxa were selected for bacterium-specific real-time PCR assays, which were examined in the full cohort as a four-category exposure (undetectable, first tertile, second tertile, and third tertile of concentrations). Conditional logistic regression was used to generate odds ratios (ORs) and 95% CIs. Regression models were stratified by cohort, and adjusted ORs (aORs) were generated from a multivariable model controlling for confounding variables. The Shannon Diversity Index was used to measure bacterial diversity. The primary analyses were the associations between bacterial concentrations and risk of HIV acquisition. Between November, 2004, and August, 2014, we identified 87 women who acquired HIV infection (cases) and 262 controls who did not acquire HIV infection. Vaginal bacterial community diversity was higher in women who acquired HIV infection (median 1·3, IQR 0·4-2·3) than in seronegative controls (0·7, 0·1-1·5; p=0·03). Seven of the 20 taxa showed significant concentration-dependent associations with increased odds of HIV acquisition: Parvimonas species type 1 (first tertile aOR 1·67, 95% CI 0·61-4·57; second tertile 3·01, 1·13-7·99; third tertile 4·64, 1·73-12·46; p=0·005) and type 2 (first tertile 3·52, 1·63-7·61; second tertile 0·85, 0·36-2·02; third tertile 2·18, 1·01-4·72; p=0·004), Gemella asaccharolytica (first tertile 2·09, 1·01-4·36; second tertile 2·02, 0·98-4·17; third tertile 3·03, 1·46-6·30; p=0·010), Mycoplasma hominis (first tertile 1·46, 0·69-3·11; second tertile 1·40, 0·66-2·98; third tertile 2·76, 1·36-5·63; p=0·048), Leptotrichia/Sneathia (first tertile 2·04, 1·02-4·10; second tertile 1·45, 0·70-3·00; third tertile 2·59, 1·26-5·34; p=0·046), Eggerthella species type 1 (first tertile 1·79, 0·88-3·64; second tertile 2·62, 1·31-5·22; third tertile 1·53, 0·72-3·28; p=0·041), and vaginal Megasphaera species (first tertile 3·15, 1·45-6·81; second tertile 1·43, 0·65-3·14; third tertile 1·32, 0·57-3·05; p=0·038). Differences in the vaginal microbial diversity and concentrations of key bacteria were associated with greater risk of HIV acquisition in women. Defining vaginal bacterial taxa associated with HIV risk could point to mechanisms that influence HIV susceptibility and provide important targets for future prevention research. National Institute of Child Health and Human Development.

Molecular identification of pathogens from excised heart valves of infective endocarditis cases

Infective endocarditis is a life-threatening disease and identification of the pathogenic organisms is crucial for better prognosis. However, in some conditions, such as previous antibiotic usage or infection with fastidious organisms, obtaining positive cultures results is difficult. In addition to conventional culture methods, a molecular approach has been developed over several decades, such as broad-range 16S rRNA gene PCR and sequencing using excised tissues, to detect the pathogens. The current consensus is that this method is useful for the diagnosis and management of infective endocarditis patients and it has been suggested that these molecular results should be included as a new Deke criterion. Although molecular technique cannot replace culture methods and should be interpreted carefully, broad-range 16S rRNA/sequencing has great value in complementing conventional methods. Tissue samples from infective endocarditis cases should be examined by this method for better management of the patients.

Is Ureaplasma spp. the leading causative agent of acute chorioamnionitis in women with preterm birth?

ObjectivesAim of this study was to detect microorganisms in fetal membranes and placental tissue in preterm chorioamnionitis by combining fluorescence in situ hybridization (FISH) with broad range PCR. The combination of the two molecular techniques enables identification and localization of the microorganisms within the tissue, confirming their clinical relevance. MethodsIn a prospective cohort study, we compared 31 women with preterm premature rupture of membranes or preterm labour and preterm delivery by caesarean section with a control group of 26 women undergoing elective caesarean section at term. Fetal membranes and placental tissue were analysed by FISH and broad range 16S rRNA-gene PCR and sequencing. ResultsFor 20 women in the preterm group, caesarean section was performed because of a clinical diagnosis of chorioamnionitis. Microorganisms were detected in the tissues by both molecular techniques in 11 out of 20 women. Among those, Ureaplasma spp. was most abundant, with five cases that remained culture-negative and would have been missed by routine diagnostic procedures. Other infections were caused by Staphylococcus aureus, Streptococcus mitis or Escherichia coli. FISH and PCR were negative for all women without suspected chorioamnionitis and for the control group. ConclusionsCombination of FISH with broad-range PCR and sequencing permitted unambiguous identification of the causative microorganisms in chorioamnionitis. The high prevalence of Ureaplasma spp. should lead to a re-evaluation of its clinical significance and possible therapeutic consequences.

Relationship of Specific Bacteria in the Cervical and Vaginal Microbiotas With Cervicitis.

Cervicitis is an inflammatory condition of the cervix associated with upper genital tract infection and reproductive complications. Although cervicitis can be caused by several known pathogens, the etiology frequently remains obscure. Here we investigate vaginal bacteria associated with bacterial vaginosis as potential causes of cervicitis. Associations between vaginal bacteria and cervicitis were assessed in a retrospective case-control study of women attending a Seattle sexually transmitted disease clinic. Individual bacterial species were detected using 2 molecular methods: quantitative polymerase chain reaction (qPCR) and broad-range 16S rRNA gene PCR with pyrosequencing. The primary finding from this initial study was evaluated using qPCR in a second cohort of Kenyan women. The presence of Mageeibacillus indolicus, formerly BVAB3, in the cervix was associated with cervicitis, whereas the presence of Lactobacillus jensenii was inversely associated. Quantities of these bacteria did not differ between cervicitis cases and controls, although in a model inclusive of presence and abundance, M. indolicus remained significantly associated with cervicitis after adjustment for other cervicitis-causing pathogens. M. indolicus was not associated with cervicitis in our study of Kenyan women, possibly due to differences in the clinical definition of cervicitis. Colonization of the endocervix with M. indolicus may contribute to the clinical manifestations of cervicitis, but further study is needed to determine whether this finding is repeatable and applicable to diverse groups of women. Colonization of the cervix with L. jensenii could be a marker of health, perhaps reducing inflammation or inhibiting pathogenic infection.

Dynamic vaginal microbiota in pigtail macaques is associated with menstrual cycle and inflammation (MUC9P.753)

Abstract A challenge in prevention of mucosal HIV transmission is incomplete understanding of correlates of vaginal transmission, including mucosal inflammation. Vaginal SIV/SHIV transmission in pigtail macaques (Macaca nemestrina) is an excellent model for HIV transmission, however little is known about how genital microbes may influence inflammation and transmission. Broad-range 16S rRNA gene PCR and pyrosequencing was performed on vaginal swabs to assess vaginal microbiome in healthy pigtail macaques. Bacteria were identified to the species or genus level. Luminex was used to measure cytokine and chemokine levels in vaginal swabs. Pigtail macaques had diverse bacterial communities at all phases of the menstrual cycle, with a range of dominant bacteria. Several species were identified that are commonly found in bacterial vaginosis (BV) in humans, and three animals exhibitied Lactobacillus-dominance. Longitudinal sampling demonstrated that the vaginal microbiome is dynamic and possibly influenced by the menstrual cycle. Animals with Lactobacillus-dominant vaginal microbiota had decreased IL-8 (p=0.009) and IL-1Ra (p=0.044). In conclusion, the macaque vaginal microbiota is diverse, dynamic, and can resemble the human vaginal microbiota, including BV-associated bacteria as well as Lactobacillus spp, which is associated with decreased innate inflammation. These data provide a foundation for understanding how the vaginal microbiome may impact risk of HIV/SIV transmission.

Propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen.

Propionibacterium acnes is known primarily as a skin commensal. However, it can present as an opportunistic pathogen via bacterial seeding to cause invasive infections such as implant-associated infections. These infections have gained more attention due to improved diagnostic procedures, such as sonication of explanted foreign materials and prolonged cultivation time of up to 14 days for periprosthetic biopsy specimens, and improved molecular methods, such as broad-range 16S rRNA gene PCR. Implant-associated infections caused by P. acnes are most often described for shoulder prosthetic joint infections as well as cerebrovascular shunt infections, fibrosis of breast implants, and infections of cardiovascular devices. P. acnes causes disease through a number of virulence factors, such as biofilm formation. P. acnes is highly susceptible to a wide range of antibiotics, including beta-lactams, quinolones, clindamycin, and rifampin, although resistance to clindamycin is increasing. Treatment requires a combination of surgery and a prolonged antibiotic treatment regimen to successfully eliminate the remaining bacteria. Most authors suggest a course of 3 to 6 months of antibiotic treatment, including 2 to 6 weeks of intravenous treatment with a beta-lactam. While recently reported data showed a good efficacy of rifampin against P. acnes biofilms, prospective, randomized, controlled studies are needed to confirm evidence for combination treatment with rifampin, as has been performed for staphylococcal implant-associated infections.

임상미생물 검출을 위한 광대한 범위와 특이도를 가지는 16S rRNA PCR법 개발

16S rRNA gene PCR법은 환자 검체로부터 병원성 미생물을 검출 및 동정에 사용되어진다. 본 연구는 대량의 임상미생물 진단을 위해 bacterial 16S rRNA 부위 유전자 서열을 이용하여 광대한 범위와 높은 특이도를 가지는 primer을 포함한 PCR법을 개발하였다. 10개 표준 균주 16S rRNA 보존 부위의 유전자 서열을 기반으로 primer set를 구축하였다. 98명 환자 검체에서 임상 미생물을 분리하였다. 98개 균주는 phenotypic 방법을 이용하여 확인하고, 개발된 primer set와 universal primer set를 이용한 PCR법으로 확인하였다. 획득한 PCR 산물은 forward primer, reverse primer, 그리고 자동화 DNA 분석기를 이용하여 각 균주의 16S rRNA 유전자 서열을 분석 및 확인하였다. 본 연구에서 개발된 primer set와 universal primer set의 임상미생물 검출에 대한 효율성을 평가하였고, 또한 phenotypic 방법과 분자생물학적 방법을 비교했다. 분리된 98개 균주를 대상으로 개발된 primer set로 16S rRNA PCR을 진행하여 778 bp 크기의 단일밴드로 증폭 되었음을 확인했다. 총 98개중 94개 균주(95.9%)는 phenotypic 결과와 동일함을 확인했다. 새로 개발된 primer set를 이용한 결과는 universal primer set를 이용한 98개 균주(100%)의 결과와 동일함을 확인하였다. 개발된 16S rRNA gene PCR법은 임상미생물 검출 및 동정에서 신속성, 정확성, 그리고 검사 비용 절감의 장점을 가진다. 개발된 primer set는 병원성 미생물 동정에서 효율성을 확인했다. Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

Comparison of molecular and culture method in diagnosis of prosthetic joint infection

Diagnosis of prosthetic joint infection with culture technique can be problematic since the causative agent(s) are not possible to cultivate in all cases. Molecular methods had been evaluated in many studies but their inclusion in routine diagnostics is still controversial. The purpose of our prospective study was to compare the diagnostic accuracy of broad-range (BR)-PCR and culture technique. Intraoperative samples of periprosthetic tissue were retrieved in 67 patients undergoing revision arthroplasty. Samples were analyzed with culture technique, immunohistochemistry and BR 16S rRNA gene PCR. Bacteria in PCR-positive samples were identified using two different methods: direct sequencing of PCR products and specific TaqMan assays. In 63 cases, full concordance was found between BR-PCR and culture technique. Specific TaqMan assays failed to identify bacteria in four culture- and BR-PCR-positive cases and therefore had a lower sensitivity in comparison with BR-PCR. Molecular methods detected bacteria with the same accuracy as culture; however, identification of bacteria was inferior to culture. Further development of species-recognition techniques is required to improve identification of causative microorganisms.

Close Geographic Association of Human Neoehrlichiosis and Tick Populations Carrying “Candidatus Neoehrlichia mikurensis” in Eastern Switzerland

Neoehrlichiosis caused by "Candidatus Neoehrlichia mikurensis" is an emerging zoonotic disease. In total, six patients have been described in Europe, with the first case detected in 2007. In addition, seven patients from China were described in a report published in October 2012. In 2009, we diagnosed the first human case of "Ca. Neoehrlichia mikurensis" infection in the Zurich area (Switzerland). Here, we report two additional human cases from the same region, which were identified by broad-range 16S rRNA gene PCR. Both patients were immunocompromised and presented with similar clinical syndromes, including fever, malaise, and weight loss. A diagnostic multiplex real-time PCR was developed for specific detection of "Ca. Neoehrlichia mikurensis" infections. The assay is based on the signature sequence of a 280-bp fragment of the "Ca. Neoehrlichia mikurensis" 16S rRNA gene and incorporates a "Ca. Neoehrlichia mikurensis" species, a "Ca. Neoehrlichia" genus, and an Anaplasmataceae family probe for simultaneous screening. The analytical sensitivity was determined to be below five copies of the "Ca. Neoehrlichia mikurensis" 16S rRNA gene. Our results show that the assay is suitable for the direct detection of "Ca. Neoehrlichia mikurensis" DNA in clinical samples from, for example, blood and bone marrow. In addition, it allows for monitoring treatment response during antibiotic therapy. Using the same assay, DNA extracts from 1,916 ticks collected in four forests in close proximity to the patients' residences (<3 km) were screened. At all sampling sites, the minimal prevalence of "Ca. Neoehrlichia mikurensis" was between 3.5 to 8% in pools of either nymphs, males, or females, showing a strong geographic association between the three patients and the assumed vector.

Streptococcus tigurinus, a Novel Member of the Streptococcus mitis Group, Causes Invasive Infections

We recently described the novel species Streptococcus tigurinus sp. nov. belonging to the Streptococcus mitis group. The type strain AZ_3a(T) of S. tigurinus was originally isolated from a patient with infective endocarditis. According to its phenotypic and molecular characteristics, S. tigurinus is most closely related to Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus oralis, and Streptococcus infantis. Accurate identification of S. tigurinus is facilitated by 16S rRNA gene analysis. We retrospectively analyzed our 16S rRNA gene molecular database, which contains sequences of all clinical samples obtained in our institute since 2003. We detected 17 16S rRNA gene sequences which were assigned to S. tigurinus, including sequences from the 3 S. tigurinus strains described previously. S. tigurinus originated from normally sterile body sites, such as blood, cerebrospinal fluid, or heart valves, of 14 patients and was initially detected by culture or broad-range 16S rRNA gene PCR, followed by sequencing. The 14 patients had serious invasive infections, i.e., infective endocarditis (n = 6), spondylodiscitis (n = 3), bacteremia (n = 2), meningitis (n = 1), prosthetic joint infection (n = 1), and thoracic empyema (n = 1). To evaluate the presence of Streptococcus tigurinus in the endogenous oral microbial flora, we screened saliva specimens of 31 volunteers. After selective growth, alpha-hemolytic growing colonies were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and subsequent molecular methods. S. tigurinus was not identified among 608 strains analyzed. These data indicate that S. tigurinus is not widely distributed in the oral cavity. In conclusion, S. tigurinus is a novel agent of invasive infections, particularly infective endocarditis.

Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of > 1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of < or = 0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco

A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.