Culture-negative Cases Research Articles

Detection of bacteria by 16S rRNA PCR and sequencing in culture-negative chronic rhinosinusitis.

Current studies on the association between bacteria and chronic rhinosinusitis (CRS) have relied solely on microbiological culture, which can sometimes be negative due to prior antibiotic therapy or lack of transport media. The objective of the study was to understand whether bacteria are associated with culture-negative cases. We investigated 20 cases of CRS undergoing functional endoscopic sinus surgery by both culture and 16S rRNA gene polymerase chain reaction (PCR) followed by sequencing. The disease severity was assessed using Sino-nasal Outcome Test-22 (SNOT-22) scoring preoperatively. Of the 20 cases studied, only five were culture-positive. Bacteria were detected in all 15 culture-negative cases by 16S rRNA gene PCR. Sequencing indicated that Staphylococcus is the dominant group, followed by Enterobacter and Pseudomonas. In two cases, from which Pseudomonas was detected, SNOT-22 scores were above 80, indicating high disease severity. 16S rRNA gene PCR, followed by sequencing, would help the clinician understand the association between bacteria and CRS. Molecular detection and identification of bacteria would help the clinician better predict the outcome and plan further management accordingly. NA. Laryngoscope, 128:2223-2225, 2018.

Diagnostic performance and problem analysis of commercial tuberculosis antibody detection kits in China

BackgroundThe diagnosis of bacterium-negative pulmonary tuberculosis (TB) and extra-pulmonary TB is challenging clinically. The detection of the anti-TB antibody has an important, auxiliary, clinical diagnostic value. Therefore, TB antibody detection kits should be screened and evaluated, and the reagents with the highest sensitivity and specificity should be chosen and used clinically.MethodsThe diagnostic performance of 7 commercially available TB antibody detection kits (kits A, B, C, D, E, F and G) based on the gold immunoassay detection of immunoglobulin (Ig) G or IgM antibodies were simultaneously evaluated and compared in 62 TB cases and 56 non-TB cases in a laboratory. A retrospective analysis including 2549 cases was carried out to assess the clinical diagnosis values of bacteriological examinations and TB antibody tests (kits B and H used in the clinic).ResultsThe sensitivities of TB antibody kits A, B, C, D, E, F and G in the sera from 62 TB patients were 50.0%, 83.9%, 38.7%, 9.7%, 48.4%, 69.4% and 79.0%, respectively; the sensitivities in the sera from 24 smear-negative TB patients were 29.2%, 79.2%, 29.2%, 12.5%, 29.2%, 54.2% and 79.2%, respectively; the specificities in the sera from 56 non-TB patients were 73.2%, 25.0%, 85.7%, 96.4%, 78.6%, 78.6% and 50.0%, respectively. Of the 2549 clinically diagnosed cases, there were 1752 pulmonary TB cases, 505 extra-pulmonary TB cases, 87 old pulmonary TB cases and 205 non-TB cases. The positive results for smear, culture, TB antibody kit B and kit H in pulmonary TB cases were 39.8% (543/1365), 48.6% (372/765), 45.8% (802/1752) and 25.2% (442/1752), respectively; the results in extra-pulmonary TB cases were 3.4% (6/178), 5.8% (4/69), 35.4% (179/505), and 11.3% (57/505), respectively; the results in old pulmonary TB cases were 0% (0/64), 0% (0/30), 32.2% (28/87), and 9.2% (8/87), respectively; and the results in non-TB cases were 0% (0/121), 0% (0/56), 21.5% (44/205), and 2.4% (5/205), respectively. Of 624 smear-positive and/or culture-positive pulmonary TB cases, the sensitivities of antibody test kits B and H were 53.0% and 36.4%, respectively. Of 901 smear-negative and/or culture-negative pulmonary TB cases, the sensitivities of antibody test kits B and H were 42.5% and 19.0%, respectively. The positive rate of antibody detection in the bacterium-positive pulmonary TB cases was significantly higher than that in the bacterium-negative pulmonary TB cases (P < 0.05).ConclusionsThe colloidal gold-labeled TB antibody IgG detection assay is a simple, rapid and economical method that provides a better clinical auxiliary diagnosis value on TB, especially in smear-negative pulmonary TB and extra-pulmonary TB. The production, quality control, screening and evaluation of antibody detection kits are very important for its clinical application.

Evaluation of screening of neonatal sepsis

Background: Neonatal sepsis is an important cause of neonatal mortality and morbidity. Early diagnosis of sepsis is difficult due to its non- specific clinical presentation. The gold standard for diagnosis is blood culture, which is obtained in only 25%-40% of cases and requires 48-72 hours. There is a need for a sepsis screen for early diagnosis of septicemia and identification of culture negative cases. The objective of the study was to study the role of sepsis for early diagnosis of septicaemia and identification of culture negative cases and to compare the rapid diagnostic tests with blood culture singly and in combinations for specificity and sensitivity.Methods: 60 cases of suspected septicemia were studied. Total leucocyte count, bandforms peripheral smear examination, C-reactive protein assay, micro-ESR, and blood culture study was investigated. Results: Study revealed that CRP had maximum sensitivity while band neutrophil ratio had balanced sensitivity and specificity. In the two tests, CRP with PS/BF had balanced sensitivity and specificity. In the three tests combination, CRP with TC with micro-ESR had balanced sensitivity and specificity in proven sepsis, While CRP with BF with micro-ESR had balanced sensitivity and specificity in most probable sepsis cases.Conclusions: Neonatal sepsis has vague signs and symptoms, so high index of suspicion helps in arriving early diagnosis and management. CRP had maximum sensitivity in the individual tests. Using either two tests (CRP + PS/BF) or three tests (CRP + Micro ESR + BF/TC) most of the sepsis cases could be identified and sepsis negative cases can be ruled out. Sepsis screen is helpful in avoiding overuse of antibiotics.

Interferon-Gamma Release Assay is Not Appropriate for the Diagnosis of Active Tuberculosis in High-Burden Tuberculosis Settings: A Retrospective Multicenter Investigation.

Background:Interferon-gamma release assay (IGRA) has been used in latent tuberculosis (TB) infection and TB diagnosis, but the results from different high TB-endemic countries are different. The aim of this study was to investigate the value of IGRA in the diagnosis of active pulmonary TB (PTB) in China.Methods:We conducted a large-scale retrospective multicenter investigation to further evaluate the role of IGRA in the diagnosis of active PTB in high TB-epidemic populations and the factors affecting the performance of the assay. All patients who underwent valid T-SPOT.TB assays from December 2012 to November 2015 in six large-scale specialized TB hospitals in China and met the study criteria were retrospectively evaluated. Patients were divided into three groups: Group 1, sputum culture-positive PTB patients, confirmed by positive Mycobacterium tuberculosis sputum culture; Group 2, sputum culture-negative PTB patients; and Group 3, non-TB respiratory diseases. The medical records of all patients were collected. Chi-square tests and Fisher's exact test were used to compare categorical data. Multivariable logistic analyses were performed to evaluate the relationship between the results of T-SPOT in TB patients and other factors.Results:A total of 3082 patients for whom complete information was available were included in the investigation, including 905 sputum culture-positive PTB cases, 914 sputum culture-negative PTB cases, and 1263 non-TB respiratory disease cases. The positive rate of T-SPOT.TB was 93.3% in the culture-positive PTB group and 86.1% in the culture-negative PTB group. In the non-PTB group, the positive rate of T-SPOT.TB was 43.6%. The positive rate of T-SPOT.TB in the culture-positive PTB group was significantly higher than that in the culture-negative PTB group (χ2 = 25.118, P < 0.01), which in turn was significantly higher than that in the non-TB group (χ2 = 566.116, P < 0.01). The overall results were as follows: sensitivity, 89.7%; specificity, 56.37%; positive predictive value, 74.75%; negative predictive value, 79.11%; and accuracy, 76.02%.Conclusions:High false-positive rates of T-SPOT.TB assays in the non-TB group limit the usefulness as a single test to diagnose active TB in China. We highly recommend that IGRAs not be used for the diagnosis of active TB in high-burden TB settings.

Evaluation of Xpert®Mycobacterium tuberculosis/rifampin in sputum-smear negative and sputum-scarce patients with pulmonary tuberculosis using bronchoalveolar lavage fluid.

Context:Sputum smear-negative and sputum-scarce pulmonary tuberculosis (PTB) is a diagnostic challenge. Xpert® Mycobacterium tuberculosis/rifampin (MTB/RIF) provides a rapid diagnosis on high-quality biological specimen obtained by bronchoscopy.Aims:The aim of this study is to evaluate Xpert® MTB/RIF on bronchoalveolar lavage (BAL) fluid in sputum smear-negative and sputum-scarce PTB patients.Settings:Tertiary care hospital in India.Design:This was prospective observational study.Materials and Methods:Between January 2015 and November 2016, we prospectively recruited sputum-smear negative and sputum-scarce patients under evaluation for PTB and performed BAL. Sensitivity, specificity, positive, and negative predictive values were calculated for the diagnosis of PTB on BAL fluid for acid-fast bacilli smear and Xpert® MTB/RIF using liquid culture as the reference standard and compared to the final diagnosis based on composite reference standard. Sensitivity, specificity, and predictive values were calculated with 95% class intervals. McNemar's test was used for comparison of sensitivities.Results:Of the 60 patients included, 52 (88.3%) had a final diagnosis of PTB and 16 (26.7%) were culture confirmed. Xpert® MTB/RIF had a sensitivity and specificity of 81% (54%–96%) and 73% (56%–85%) in culture confirmed cases; 46% (32%–60%) and 100% (63%–100%) for the final diagnosis; 32% (17%–51%) and 100% (54%–100%) in culture negative cases, respectively. Culture had a sensitivity of 32% (20%–47%) for the final diagnosis.Conclusions:In sputum smear-negative and sputum-scarce patients with clinico-radiological features of PTB Xpert® MTB/RIF has good sensitivity for diagnosis on BAL fluid. It is useful even when cultures are negative.

Aetiology of acute pyogenic meningitis in children in a tertiary care hospital, Kerala

INTRODUCTION: Acute bacterial meningitis is a medical emergency, and the diagnosis is by culture of cerebrospinal fluid (CSF). In this study, we looked at the changing aetiology of acute pyogenic meningitis in the context of the availability of several vaccines. MATERIALS AND METHODS: The aim of this study was to find out the aetiology and antibiotic susceptibility pattern of acute pyogenic meningitis in children between one month and 12 years in one year in a tertiary care centre. A cross-sectional study was conducted in the Department of Microbiology, Medical College Hospital, Trivandrum, in collaboration with Department of Paediatrics, SATH, Trivandrum and Christian Medical College, Vellore. CSF samples were processed by doing Gram-staining and culture in appropriate media. Latex agglutination test (LAT) and PCR were done in all cases for the primary pathogens. Blood culture and sensitivity were also done for all patients. RESULTS: Streptococcus pneumoniae (62.5%) was the most common aetiological agent for acute bacterial meningitis followed by Klebsiella pneumoniae subspp pneumoniae, Group B haemolytic Streptococci and Elizabethkingia meningosepticum. LAT could pick up one culture negative case of pneumococcal meningitis. Blood culture was positive in 37.5% of CSF culture positive cases.

Corrigendum: A randomized synbiotic trial to prevent sepsis among infants in rural India.

This corrects the article DOI: 10.1038/nature23480.

Asymptomatic bacteriuria and its associated factors in type II diabetes mellitus

Background: With the dawn of modern era the diabetes epidemic has spread over continents affecting the developed and the developing nations alike. Asymptomatic bacteriuria which is quite prevalent in the diabetic population is associated with increased diabetic complications and thereby increased morbidity. Thus, the aim of this study was to assess the proportion of cases with asymptomatic bacteriuria (ASB) among type II diabetes mellitus patients as compared to non-diabetic healthy controls and studying the factors associated with ASB.Methods: 93 eligible type II diabetes mellitus cases without genitourinary symptoms or abnormalities along with 93 non-diabetic healthy controls were recruited. Mid-stream urine was collected after taking informed consent and each sample tested using the dipstick, microscopy and culture techniques. Isolates were identified using standard biochemical tests.Results: Prevalence of ASB in our study was found to be 34.4% among cases of type II diabetes mellitus while it was 6.45% among non-diabetic healthy controls. Amongst the diabetics with ASB 71.9 % were females and 28.13 % were males. E. Coli was isolated in 21 subjects among diabetic cases (22.58%) and in 5 subjects among non-diabetic healthy controls (5.38%). Fasting blood sugar (FBS) in diabetic cases group was 151.08 ± 48.16 and in control group was found to be 98.57 ± 25.95 (p&lt;0.001). Mean duration of patients with DM who had ASB (32) was 8.46 ± 4.14 years and those who were culture negative was 4.59 ± 4.2 years (p &lt; 0.05). 24 subjects (75%) out of 32 diabetic ASB cases had glycosuria compared to 27 subjects (44.26%) out of 61 culture negative cases (p &lt; 0.05). 9 out of 32 (28.13%) diabetic ASB subjects had proteinuria compared to 4 out of 61 (6.56%) culture negative cases (p &lt; 0.05).Conclusions: Overall prevalence of ASB was significantly more in diabetic population as compared to non-diabetic healthy controls (34.4% vs 6.45%). Females have an increased likelihood of developing ASB as compared to males. E. Coli was the most common pathogen isolated in ASB cases. Longer duration of diabetes and poor glycemic control are independent predictors for the development of ASB. The risk of ASB is also significantly increased in those with glycosuria and proteinuria.

Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes

Pseudomonas aeruginosa (P. aeruginosa) is a menacing opportunistic, nosocomial pathogen; become a growing concern as conventional antimicrobial therapy is now futile against it. Multi-drug resistant P. aeruginosa (MDRPA) has distinctive resistance mechanisms such as production of β-lactamases, repression of porin genes and over-expression of efflux pumps. The focus of this study is to standardize and application of multiplex PCR (mPCR) to detect the presence of betalactamase genes encoding blaTem, blaOXA, blaCTX-M-15, blaVim, blaGes, blaVeb, blaDIM, AmpC and Efflux pump genes encoding Mex A,B-oprM, Mex C,D-oprJ, Mex X,Y-oprN, oprD, nfxB, MexR. A total of 200 clinical isolates of P. aeruginosa were tested for the presence of the above mentioned genes genotypically through mPCR and characterized by phenotypic methods for ESBL and MBL production. Out of 200 isolates, 163 (81.5%) nfxB regulator gene, 102 (51%) MexA, 96 (48%) MexC, 93 (46.5%) MexB, 86 (43%) MexD, 81 (40.5%) OprM, 74 (37%) OprJ, 72 (36%) OprD and MexR, 53 (26.5%) Mex X and OprN, 49 (24.5%) MexY gene. Betalactamase genes 145 (72.5%) blaTem, 67 (33.5%) blaOXA, 35 (17.5%) blaVim, 25(12.50%), 23 (11.50%) blaVeb, 21 (11.5%) blaGes, 14 (7%) Ctx-m and 10 (5%) AmpC and 5 (2.5%) blaDim-1 gene were tested positive by mPCR. Phenotypically 38 (19%) and 29 (14.5%) out of 200 tested positive for ESBL and MBL production. Application of this mPCR on clinical specimens is fast, accurate, specific and low-cost reliable tool for the screening, where culture negative Eubacterial PCR positive cases for an early molecular detection of drug resistance mechanism assisting the clinician to treat the disease with appropriate antibiotic selection.

Rapid diagnosis of new and relapse tuberculosis by quantification of a circulating antigen in HIV-infected adults in the Greater Houston metropolitan area

BackgroundHIV-associated immune defects inhibit tuberculosis (TB) diagnosis, promote development of extrapulmonary TB and paucibacillary pulmonary TB cases with atypical radiographic features, and increase TB relapse rates. We therefore assessed the diagnostic performance of a novel assay that directly quantitates serum levels of the Mycobacterium tuberculosis (Mtb) virulence factor 10-kDa culture filtrate protein (CFP-10) to overcome limitations associated with detecting Mtb bacilli in sputum or tissue biopsies.MethodsThis study analyzed HIV-positive adults enrolled in a large, population-based TB screening and surveillance project, the Houston Tuberculosis Initiative, between October 1995 and September 2004, and assigned case designations using standardized criteria. Serum samples were trypsin-digested and immunoprecipitated for an Mtb-specific peptide of CFP-10 that was quantified by liquid chromatography-mass spectrometry for rapid and sensitive TB diagnosis.ResultsAmong the 1053 enrolled patients, 110 met all inclusion criteria; they included 60 tuberculosis cases (12 culture-negative TB), including 9 relapse TB cases, and 50 non-TB controls, including 15 cases with history of TB. Serum CFP-10 levels diagnosed 89.6% (77.3–96.5) and 66.7% (34.9–90.1) of culture-positive and culture-negative TB cases, respectively, and exhibited 88% (75.7–95.5) diagnostic specificity in all non-TB controls. Serum antigen detection and culture, respectively, identified 85% (73.4–92.9) and 80.0% (67.3–88.8) of all 60 TB cases.ConclusionsQuantitation of the Mtb virulence factor CFP-10 in serum samples of HIV-infected subjects diagnosed active TB cases with high sensitivity and specificity and detected cases missed by the gold standard of Mtb culture. These results suggest that serum CFP-10 quantitation holds great promise for the rapid diagnosis of suspected TB cases in patients who are HIV-infected.

IP-10 Kinetics in the First Week of Therapy are Strongly Associated with Bacteriological Confirmation of Tuberculosis Diagnosis in HIV-Infected Patients

Simple effective tools to monitor the long treatment of tuberculosis (TB) are lacking. Easily measured host derived biomarkers have been identified but need to be validated in larger studies and different population groups. Here we investigate the early response in IP-10 levels (between day 0 and day 7 of TB therapy) to identify bacteriological status at diagnosis among 127 HIV-infected patients starting TB treatment. All participants were then classified as responding or not responding to treatment blindly using a previously described IP-10 kinetic algorithm. There were 77 bacteriologically confirmed cases and 41 Xpert MTB/RIF® and culture negative cases. Most participants had a measurable decline in IP-10 during the first 7 days of therapy. Bacteriologically confirmed cases were more likely to have high IP-10 levels at D0 and had a steeper decline than clinically diagnosed cases (mean decline difference 2231 pg/dl, 95% CI: 897–3566, p = 0.0013). Bacteriologically confirmed cases were more likely to have a measurable decline in IP-10 at day 7 than clinically diagnosed cases (48/77 (62.3%) vs 13/41 (31.7%), p < 0.001). This study confirms the association between a decrease in IP-10 levels during the first week of treatment and a bacteriological confirmation at diagnosis in a large cohort of HIV positive patients.

Direct Detection and Identification of Prosthetic Joint Pathogens in Synovial Fluid (SF) by Metagenomic Shotgun Sequencing

BackgroundDetection and identification of microorganism(s) involved in periprosthetic joint infection (PJI) can inform surgical management and directed antibiotic therapy. Metagenomic shotgun sequencing is a powerful tool with the potential to change how many PJIs are diagnosed as it allows direct detection and identification of pathogens in clinical specimens. In the largest series to date, we utilized a metagenomics-based approach applied to SF to define potential microbial etiologies of failed total knee arthroplasties (TKAs).MethodsSynovial fluid was collected from 112 failed TKAs [74 PJI and 38 aseptic implant failure (AF)] via preoperative arthrocentesis. Cell count and differential, standardized culture and DNA-based metagenomic shotgun sequencing were performed. Human DNA was depleted using the MolYsis basic kit prior to DNA extraction, whole genome amplification, and sequencing. Taxonomic assignment of reads and pathogen identification was achieved using a pipeline incorporating k-mer- and marker gene-based classification software. A scheme for analysis and filtration of false-positives was created and applied, incorporating cut-offs for the number of reads, quality scores, and coverage across a reference genome. Patients were classified as having PJI using the IDSA criteria and expert review. Analyses were recorded as percent agreement, with 95% confidence intervals (CI), of metagenomics to SF culture.ResultsMetagenomic analysis identified the known pathogen in 54 (90%) (CI, 79.5%–96.2%) of the 60 culture-positive PJIs analyzed and one (2%) (CI, 0.0%–8.9%) potential polymicrobial infection not detected by culture. For the 14 culture-negative PJIs tested, metagenomics showed 79% (CI, 49.2%–95.3%) agreement for negative findings; potential pathogens were identified in three (21%) (CI, 4.7%–50.8%) culture-negative PJI cases, with one being polymicrobial. Of the 37 culture-negative AF cases, metagenomics showed 97% (CI, 85.8%–99.9%) agreement with negative culture and identified one (3%) (CI, 0.0%–14.2%) potential pathogen. For the one culture-positive AF case, metagenomic results were negative, suggesting possible culture contamination.ConclusionMetagenomic shotgun sequencing performed on SF can be used to diagnose PJI and may be particularly useful for culture-negative PJI.Disclosures R. Patel, ASM: Board Member, None; CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Grant recipient; Curetis: Consultant, Monies paid to my employer; A patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Patents, Patents, any money is paid to my employer; Actelion: DSMB, Money paid to my employer; ASM and IDSA: Editor’s stipends, Editor’s stipends; NBME, Up-to-Date and the Infectious Diseases Board Review Course: NBME, Up-to-Date and the Infectious Diseases Board Review Course, Honoraria; Roche, ASM, and IDSA: Travel reimbursement, Travel reimbursement

Bacteriological diagnosis of childhood TB: a prospective observational study

Childhood TB diagnosis is challenging. Studies in adults suggest Microscopic Observation Drug Susceptibility (MODS) culture or the Xpert MTB/RIF assay might be used to expand bacteriological diagnosis. However data from children are more limited. We prospectively compared MODS and Xpert MTB/RIF with standard microscopy and culture using the BD MGIT 960 system among 1442 Kenyan children with suspected TB. 97 specimens from 54 children were TB culture-positive: 91 (94%) by MGIT and 74 (76%) by MODS (p = 0.002). 72 (74%) culture-positive and 7 culture-negative specimens were Xpert MTB/RIF positive. Xpert MTB/RIF specificity was 100% (99.7–100%) among 1164 specimens from 892 children in whom TB was excluded, strongly suggesting all Xpert MTB/RIF positives are true positives. The sensitivity of MGIT, MODS and Xpert MTB/RIF was 88%, 71% and 76%, respectively, among all 104 true positive (culture and/or Xpert MTB/RIF positive) specimens. MGIT, MODS and Xpert MTB/RIF on the initial specimen identified 40/51 (78%), 33/51 (65%) and 33/51 (65%) culture-confirmed pulmonary TB cases, respectively; Xpert MTB/RIF detected 5 additional culture-negative cases. The high sensitivity and very high specificity of the Xpert MTB/RIF assay supports its inclusion in the reference standard for bacteriological diagnosis of childhood TB in research and clinical practice.

Utility of a Direct 16S rDNA PCR and Sequencing for Etiological Diagnosis of Infective Endocarditis.

BackgroundCases of infective endocarditis (IE) require prompt etiological diagnosis for effective treatment. Molecular methods can aid in rapid and reliable diagnosis of culture-negative IE cases. We evaluated the utility of 16S rDNA PCR and sequencing in determining the causative agents of IE in valve tissues, especially when specimens were obtained after initiation of antimicrobial therapy.MethodsWe performed 16S rDNA PCR and sequencing in heart valve specimens and medical records review of 80 patients who underwent protocol-based cardiac surgery from 2013 to 2015. One patient did not meet the criteria for IE. Sixty-five (81.3%) and 14 pa-tients (17.5%) were diagnosed as having definite IE and possible IE, respectively. Blood and heart valve biopsy tissue were examined by using routine microbiological methods.ResultsBlood cultures in our hospital were IE-positive for 33 patients (41.8%), whereas 49 patients (62.0%) showed positive blood cultures when initial blood cultures performed at the referring hospital were included. Eighteen (22.8%) and 40 patients (50.6%) were IE-positive in valve tissue cultures and 16S rDNA PCR, respectively. Bacteria in the Streptococcus mitis group (n=26) were the most common etiological agents of IE. Eight (10.1%) culture-negative specimens tested positive by 16S rDNA PCR. In five of eight PCR-positive and culture-negative cases, fastidious or anaerobic organisms were the cause of IE.Conclusions Direct 16S rDNA PCR and sequencing can be used as a supplementary method to conventional blood and biopsy culture testing, especially in culture-negative IE cases that are negative for IE by culture.

Evaluation of Some Immunological Tests for Early Diagnosis of Bacterial Neonatal Sepsis

Neonatal sepsis is a major cause of death in newborns despite sophisticated neonatal intensive care. This crosssection study was done on 69 neonates with suspected sepsis who were admitted in Neonatal Intensive Care Unit (NICU) of Maternity and Child Teaching Hospital at AL-Diwaniya city, and 20 healthy neonates as a (control group) in the period from March to October 2009. This study was conducted to evaluate the diagnostic value of some immunological marker such as (CRP,IL-6, IL-8,TNF- α, in the early diagnosis of neonatal sepsis prior to the blood culture ( the golden standard test). The culture positivity rate 20 (28.9%) among suspected sepsis neonates, the male more affected than female among proven sepsis. it was found that the median concentration of CRP, TNF-α, IL-8, IL-6 decreased in order of definitive infection, clinical sepsis and healthy subjects respectively (P&lt;0.001), and found that the TNF-α (≥65.4pg/l) followed by IL-8 (≥60.9pg/l) were better than IL-6 and CRP with sensitivity of (80%) and (75%) respectively and negative predictive value of (97%) and (94.1%) respectively when used as a tests to predict culture positive sepsis cases differentiating them from culture negative sepsis cases .

Microbiological diagnosis in revision of infected knee arthroplasties in Denmark

Background: Revision of infected knee arthroplasties is associated with high failure rates (30–40%). An understanding of the microbiology is important to optimize treatment and outcome. We describe microbiological diagnostic practice and diagnosis in revision of infected knee arthroplasties.Methods: One hundred and two partial revisions (open debridement and exchange of tibial insert) and 213 two-stage procedures performed due to infection in 275 patients from 1 July 2011 to 30 June 2013 were included and analysed by linkage to data from a nationwide registry on microbiological test results.Results: 78 (24.8%) revisions were culture negative, 192 (60.9%) showed monomicrobial growth and 43 (14.3%) polymicrobial growth. Staphylococcus aureus was the most frequent isolate in mono-culture in 70 (22.2%) revisions and in polymicrobial culture in 15 revisions with a total frequency of 27.0%. Only one case (1.4%) of methicillin-resistance was registered. Coagulase-negative staphylococci (CoNS) were frequent, sole pathogen in 65 revisions and in polymicrobial cultures in 28 revisions with a total frequency of 29.5%. A pre-operative knee aspiration was performed in 50% and preoperative blood cultures were performed in 22% of cases. In 73% of the preoperative knee-aspirations bacteriological findings were fully or partially in accordance with intraoperative cultures. In 54% of the later re-revisions due to infection (treatment-failures) the cultured species differed from or was not detected at index revision surgery.Conclusions: The 25% culture negative cases in combination with only 50% preoperative knee aspirations and prevailing high failure rates confirm the need for improvement of diagnostic practice and treatment of infected knee arthroplasties.

Laboratory Diagnosis of Infective Endocarditis.

Infective endocarditis is life-threatening; identification of the underlying etiology informs optimized individual patient management. Changing epidemiology, advances in blood culture techniques, and new diagnostics guide the application of laboratory testing for diagnosis of endocarditis. Blood cultures remain the standard test for microbial diagnosis, with directed serological testing (i.e., Q fever serology, Bartonella serology) in culture-negative cases. Histopathology and molecular diagnostics (e.g., 16S rRNA gene PCR/sequencing, Tropheryma whipplei PCR) may be applied to resected valves to aid in diagnosis. Herein, we summarize recent knowledge in this area and propose a microbiologic and pathological algorithm for endocarditis diagnosis.

Procalcitonin, Interleukin-1, Interleukin-10, Interleukin-13 and Tumor necrosis factor-alpha predictive value in the diagnosis of sepsis

The present study is aimed to evaluate the role of some markers in the diagnosis of sepsis among SIRS (systemic inflammatory response syndrome) patients. The study included 180 patients with SIRS criteria within 24 hour of admission. They were admitted to different hospitals in Mosul city for the period from January 2013 to January 2014. From each patient two blood samples were collected, one for blood culture and the other separated by centrifugation and kept frozen for ELISA tests of PCT, IL-1, TNF-α, IL-10 and IL-13. IL-1, IL-10, IL-13 and PCT were having a significant difference in mean between culture positive and negative cases. PCT was significantly highly elevated in patients with sepsis (P=0.004) with high sensitivity (93.7%) and specificity (90.1%). Many combinations of markers were used in the current study to find out the best combination of markers. The results of using combined sensitivity and specificity of PCT together with 2 anti-inflammatory cytokines (IL-10 and IL-13) were the best. They were an excellent markers with 100% sensitivity and the highest specificity.

Viable bacteria persist on antibiotic spacers following two-stage revision for periprosthetic joint infection.

Treatment in periprosthetic joint infection (PJI) remains challenging. The failure rate of two-stage revision and irrigation and debridement with component retention in PJI suggests that biofilm cells have a high tolerance to antibiotic chemotherapy. Previous work has demonstrated that biofilm cells have high antibiotic tolerance in vitro, but there is little clinical evidence to support these observations. The aim of this study was to determine if retrieved antibiotic spacers from two-stage revision total knee arthroplasty for PJI have evidence of remaining viable bacteria. Antibiotic poly (methyl methacrylate) (PMMA) spacers from two-stage revision total knee arthroplasty for PJI were prospectively collected and analyzed for bacterial 16s rRNA using polymerase chain reaction (PCR), reverse transcription (RT)-PCR, quantitative RT-PCR (qRT-PCR), and single genome analysis (SGA). PCR and RT-PCR identified bacterial species on 53.8% (7/13) of these samples. When initial culture negative cases are excluded, 68% (6/9) samples were identified with bacterial species. A more rigorous qRT-PCR analysis showed a strong positive signal for bacterial contamination in 30.7% (4/13) of cases. These patients did not show any clinical evidence of PJI recurrence after 15 months of follow-up. Because the half-life of bacterial rRNA is approximately a few days, the identification of bacteria rRNA on antibiotic PMMA spacers suggests that viable bacteria were present after conclusion of antibiotic therapy. This study provides evidence for the high tolerance of biofilm cells to antibiotics in vivo and the important role of bacterial persisters in PJI. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:452-458, 2018.

Assessment of real-time PCR cycle threshold values in Microsporum canis culture-positive and culture-negative cats in an animal shelter: a field study.

Objectives Real-time PCR provides quantitative information, recorded as the cycle threshold (Ct) value, about the number of organisms detected in a diagnostic sample. The Ct value correlates with the number of copies of the target organism in an inversely proportional and exponential relationship. The aim of the study was to determine whether Ct values could be used to distinguish between culture-positive and culture-negative samples. Methods This was a retrospective analysis of Ct values from dermatophyte PCR results in cats with suspicious skin lesions or suspected exposure to dermatophytosis. Results One hundred and thirty-two samples were included. Using culture as the gold standard, 28 were true positives, 12 were false positives and 92 were true negatives. The area under the curve for the pretreatment time point was 96.8% (95% confidence interval [CI] 94.2-99.5) compared with 74.3% (95% CI 52.6-96.0) for pooled data during treatment. Before treatment, a Ct cut-off of <35.7 (approximate DNA count 300) provided a sensitivity of 92.3% and specificity of 95.2%. There was no reliable cut-off Ct value between culture-positive and culture-negative samples during treatment. Ct values prior to treatment differed significantly between the true-positive and false-positive groups ( P = 0.0056). There was a significant difference between the pretreatment and first and second negative culture time points ( P = 0.0002 and P <0.0001, respectively). However, there was substantial overlap between Ct values for true positives and true negatives, and for pre- and intra-treatment time points. Conclusions and relevance Ct values had limited usefulness for distinguishing between culture-positive and culture-negative cases when field study samples were analyzed. In addition, Ct values were less reliable than fungal culture for determining mycological cure.