Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet.

We retrospectively investigated cases of false-positive diagnoses using the BIOFIRE® FilmArray® meningitis/encephalitis (ME) panel to measure the impact of using a dedicated biosafety cabinet combined with preventive measures to reduce the prevalence of false-positive diagnoses due to pre-analytical in-laboratory contamination. False-positive results were identified by reviewing clinical data, biological parameters and cytology results of cerebrospinal fluid (CSF) samples showing discrepant results between the FilmArray ME panel and routine PCR assays. A total of 327 CSF were analysed over 16 weeks in point-of-care (POC) A and B, over two 8-week periods, periods 1 and 2. The analysis yielded 30 (9·17%) detection of at least one pathogen including 21/30 (70%) viruses and 9/30 (30%) bacteria. During period 1, POC-A and POC-B manipulated CSF under a non-dedicated hood featuring laminar flow, whereas during period 2, CSFs were manipulated under a dedicated biosafety cabinet without any airflow in POC-A. During period 1, false positives were detected in 3/114 CSF (2·63%) in POC-A and 1/36 (2·77%) in POC-B (P = 0·97); during period 2, false positives were detected in 0/139 CSF (0%) in POC-A and 1/38 (2·63%) in POC-B (P = 0·23). All false positives were bacterial. The use of a dedicated cabinet without ventilation along with preventive measures during period 2 in POC-A significantly reduced the number of false-positive results (P = 0·05). Preventive measures described in this study can mitigate false positives when using PCR-based multiplex assays such as the BIOFIRE FilmArray ME Panel for the diagnosis of meningitis and other infectious diseases.

Genome of a chronic osteitis-causing Clostridium tetani.

We sequenced the genome of a Clostridium tetani strain that caused chronic tibial osteitis without any clinical sign of tetanus in a 26-year-old man previously vaccinated against this disease. The genome contained a plasmid that harboured the tetX-tetR tetanospasmin operon, and was highly similar to that of a tetanus-causing strain.

Relation between nasal carriage of Staphylococcus aureus and surgical site infection in orthopedic surgery: the role of nasal contamination. A systematic literature review and meta-analysis.

UNLABELLED: Staphylococcus aureus is the pathogen most frequently implicated in infection on orthopedic hardware; various strategies are deployed to limit the risk of transmission and surgical infection. OBJECTIVES: The present study is based on a meta-analysis assessing firstly the relationship between nasal carriage of S. aureus and the development of osteo-articular infection and secondly current methods of decolonization. RESULTS: The meta-analysis showed increased risk of surgical site infection in case of nasal carriage of S. aureus: OR=5.92, 95% CI [1.15-30.39]; P=0.033. For cross-transmission, a scientifically proven reduction in surgical site S. aureus levels is ensured by associated mupirocin and 2% chlorhexidine antiseptic solution in subjects with positive nasal screening results for all surgical procedures taken together; the reduction was not, however, significant in the orthopedic surgery subgroup. The meta-analysis confirmed these findings: OR=0.60, 95% CI [0.34-1.06]; P=0.08. CONCLUSION: The literature review confirmed that nasal carriage of S. aureus is a major risk factor for surgical site infection. The efficacy of eradication could not be demonstrated for orthopedic surgery as samples were too small. The positive trend found, however, should encourage further studies with sufficient power and risk/benefit should meanwhile be assessed on a case-by-case basis. LEVEL OF EVIDENCE: Level 2. Meta-analysis.

The role of molecular diagnostics in implant-associated bone and joint infection.

Microbiological culture is the conventional method for establishing the diagnosis in implant-associated bone and joint infection, but it may lack both specificity and sensitivity. Molecular diagnosis has been an important step in the diagnosis of infectious diseases. We review the principles and the role of molecular diagnosis in improving the aetiological diagnosis of implant-associated bone and joint infection. Currently, molecular diagnosis mainly includes conventional broad-range PCR and specific PCR assays. These tools are efficient, but several pitfalls exist that necessitate rigour in all steps of the process. In implant-associated bone and joint infection, molecular assays have been shown to be useful in complementing culture techniques to identify microorganisms when patients have previously received antibiotics or in the presence of fastidious microorganisms. Broad-range PCR targeting the 16S rRNA sequence followed by sequencing must be performed in culture-negative specimens when infection is suspected on the basis of clinical signs and symptoms or inflammatory syndrome. This molecular tool has allowed not only increasing identification of anaerobic bacteria, such as Finegoldia magna, but also the discovery of the role of Tropheryma whipplei, an aetiological agent of implant-associated bone and joint infection in patients without Whipple's disease. Real-time pathogen-specific PCR assays performed in a closed system are more sensitive and specific than broad-range PCR, but each assay is typically able to detect only a single microorganism. These assays should be performed to confirm the identification provided by broad-spectrum PCR, and also when broad-range PCR fails to detect a microorganism despite efficient DNA extraction.

Comparison of the new InoDiag automated fluorescence multiplexed antigen microarray to the reference technique in the serodiagnosis of atypical bacterial pneumonia.

The aetiological diagnosis of pneumonia depends largely on culture-, antigen- or PCR-based tests. Atypical agents of pneumonia include Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis and Mycoplasma pneumoniae. In these cases, serological tests are commonly used for diagnosis. All of the above species were comparatively screened for by using the InoDiag multiplexed automatic immunofluorescence assay and established reference techniques. The InoDiag assay required 5 microL of serum, took 76 min per serum sample, and required an incubator, a fluorescence reader and interpretation software. In total, 248 single sera from patients were tested, for the diagnosis of pneumonia, and the results obtained with selected serum samples were compared with results obtained with the reference method. It was shown that, for the detection of Coxiella burnetii IgM, the automated assay had a sensitivity and specificity of 100%. For the detection of M. pneumoniae IgM, sensitivity was 100% and specificity was 98%. For the detection of Chlamydophila pneumoniae and Chlamydia psittaci IgG, sensitivity was 81% and specificity was 94%. For the detection of L. pneumoniae IgG, sensitivity was 63% and specificity was 98%. For the detection of F. tularensis IgG and IgM, sensitivity was 100% for both, and specificity was 95% and 100%, respectively. The performance of this serological assay was comparable to that of other assays reported in the literature. This preliminary study shows that the automatic InoDiag assay opens the way to immunofluorescence assay standardization.

Diagnosis of Coxiella burnetii pericarditis using a systematic prescription kit in case of pericardial effusion.

Coxiella burnetii, regarded as a potential agent of pericarditis, wa found to be responsible for almost 5% of the cases of idiopathic pericardial effusion reported in this series. Diagnosis was aided by use of a systematic kit described in this paper.

Pericardial effusion in a homeless man due to Bartonella quintana.

Bartonella quintana may cause trench fever, endocarditis, bacillary angiomatosis, and chronic bacteremia, and a reemergence among homeless populations in cities has been noted. Pericarditis from Rickettsia conorii and Coxiella burnetii infection has been described, but there have been no reports of pericarditis due to Bartonella spp. We report a case of pericardial effusion due to Bartonella quintana in a homeless man, diagnosed on the basis of PCR detection of Bartonella quintana in a pericardial biopsy sample and a fourfold rise in antibody titers. The patient recovered within 2 weeks with antibiotics active against bartonellae.

Coxiella burnetii pericarditis: report of 15 cases and review.

Q fever is characterized by its clinical polymorphism, and pericarditis associated with Q fever has occasionally been described. Herein we report 15 cases of Coxiella burnetii pericarditis, 9 from our data bank and 6 encountered within the past 12 months. Three patients presented with life-threatening tamponade. We compare our cases with the 18 previously reported and with 60 Q fever-matched controls at our center. This study showed that Q fever pericarditis can present as acute as well as chronic disease; we describe relapse after 6 months in association with a serological profile compatible with the chronic form of disease (phase I C. burnetii IgG titer of > or = 800). Discriminant factors among patients and controls are age of > 52 years (adjusted odds ratio [OR], 5.66), the occurrence of general symptoms such as arthralgias or myalgias (adjusted OR, 6.54), and a normal erythrocyte sedimentation rate (adjusted OR, 16.37). No specific symptoms or underlying cardiac predispositions are observed.

[Diagnosis of post-operative venous thrombosis using determination of plasma D-dimer]. / Diagnostic des thromboses veineuses post-chirurgicales par le dosage des D-dimères plasmatiques.

PURPOSE: The D-Dimer test has been shown to be highly sensitive for the diagnosis of deep vein thrombosis (DVT) and pulmonary embolism. Two automatic quantitative tests giving a rapid response within 10 and 30 minutes have been recently marketed. In the postsurgery situation however, the role of the D-Dimer test remains controversial and the optimal cutoff value remains open. The aim of this study was to determine the cutoff value during the postoperative period. PATIENTS AND METHODS: One hundred three consecutive patients admitted to surgery were included. In all patients, D-Dimer test was performed every 2 or 4 days from admission to hospital discharge. The Vidas D-Dimer (Biomerieux, Marcy l'Etoile, France) and the STA Liatest D-DI (Diagnostica Stago, Asnières, France) were performed in parallel in all cases. RESULTS: Thirty-five patients were excluded because the follow-up period was too short. Results suggest that a D-Dimer value below 2 micrograms/ml has a negative predictive value of 100%. A D-Dimer value over 4 micrograms/ml would indicate suspected deep vein thrombosis in half of the cases, even without clinical signs. Dividing the patients into three groups according to the D-Dimer value, the two tests correlated poorly (r = 0.36 and 0.57) in the middle group (between 2 and 3 and between 3 and 4) and correctly for values below 2 or over 4 micrograms/ml (r = 0.83 and 0.78 respectively). CONCLUSION: These two optimum cutoff values (< 2 micrograms/ml and > 4 micrograms/ml) are useful for determining the need for further explorations for DVT. By limiting need for ultrasonography and contrast venography, the cost-efficacy ratio for the detection of DVT during the postoperative period is greatly improved with the D-Dimer screening strategy.

[Evaluation, by flow cytometry, of activated platelet levels after coronarography and intraluminal angioplasty]. / Evaluation, par la cytométrie en flux, du taux de plaquettes activées après les coronarographies et les angioplasties intraluminales.

Coronarography and intraluminal angioplasty induce platelet activation. In this study, activated platelets were evaluated by measuring platelet-bound fibrinogen using a polyclonal fluorescent antibody in flow cytometry on whole blood. For normal subjects, the percentage of platelets binding fibrinogen was low (16.67%) and reached 81.0% in response to ADP. The percentages of platelets binding fibrinogen were evaluated 24 hours before and after coronarography. During intracoronary angioplasty, blood was collected from the catheter before and after the dilation and aspirin bolus (1 g). In both groups, the percentages of activated platelets were lower compared with those of the control group (respectively 3.96% and 5.59% versus 16.67%) following aggregation inhibitor, anticoagulant and calcium inhibitor therapies. Twenty-four hours after coronarography, platelet activation was noted (9.71% versus 3.96%). During angioplasty no significant activation was observed immediately after dilation (6.54% versus 5.59%). In both groups before the intervention, ADP stimulation was still responsible for platelet activation but to a lesser extent than in the control group (60.42% and 66.31% versus 81.0%). After coronarography, the platelet response to ADP was identical to that in the control group (81.01% versus 81.0%). Immediately after dilation, this activation was not observed in patients with an angioplasty. This study shows that platelet activation occurs 24 hours after coronarography, whereas after dilation and an aggregation inhibitor bolus this activation is not observed during angioplasty.

[The role of new molecules in surgical antibiotic prophylaxis]. / Place des nouvelles molécules en antibioprophylaxie chirurgicale.

The preoperative administration of a new antibiotic for antimicrobial prophylaxis is questionable because of the methodological difficulties to demonstrate its efficiency and benefits in decreasing the postoperative infectious complications. As their rate is very low, especially in clean surgery, the number of patients to be included in a comparative trial is very high. Most studies assessed only small groups and therefore any extrapolation for clinical practice is of limited value. Because of their therapeutic efficiency the fluoroquinolones are often recommended for antimicrobial prophylaxis. However, the rapid occurrence of resistances, directly related to their prescription should invite the prescribers to be cautions. They should be contra-indicated as long as an alternative of similar efficiency is existing, in case of bacteraemia, when an administration of more than 48 hours in required or when the intra-hospital resistance rate exceeds 10 p. 100.

Q fever and HIV infection.

OBJECTIVE: To study the frequency of Q fever in HIV-infected individuals. DESIGN: A seroprevalence study. SETTING: French National Reference Centre for Rickettsial Agents, Marseille, France. PATIENTS AND METHODS: Five out of the 68 hospitalized cases of Q fever diagnosed in 1987-1989 were also HIV-infected and are described here. Sera from a blood-donor bank (n = 925) and from HIV-positive individuals selected at random, irrespective of clinical or immunological status (n = 500) were tested for Q fever. RESULTS: Comparisons of the two groups showed a statistically significant difference (2.4 versus 0.8%; Fisher's exact test) at the diagnostic dilution 1:200 and at the dilution considered positive for seroprevalence study (1:1000). CONCLUSIONS: Using the estimated incidence of HIV infection in Marseille, the number of Q fever cases in 1987-1989 was 13 times higher and the clinical expression more frequently symptomatic in the HIV-positive population than in the general one. The prevalence:seroprevalence ratio for Q fever was 1.37% in the HIV-positive group and 0.36% in the blood-donor group. Sera positive for Q fever were confirmed by Western blot analysis in order to minimize cross-reaction. Transmission of Q fever appears to be more frequent in HIV-positive individuals than in the general population; this is not surprising, since Coxiella burnetii lives in the phagolysosome, like other micro-organisms described in immunocompromised hosts. Q fever should be added to the spectrum of diseases that occur more frequently during HIV infection.

[Eosinophilic meningitis. Review of the literature and a new case originating from Reunion Island]. / Méningite à éosinophiles. Revue de la littérature à propos d'un nouveau cas en provenance de la Réunion.

We report a case of eosinophilic meningitis occurring in a European patient living in the Reunion Island. We review the literature and discuss the diagnosis of angiostrongylosis.

[Monitoring after total hip prosthesis. Value of semi-quantitative D-dimer assay]. / Surveillance après prothèse totale de hanche. Intérêt du dosage semi-quantitatif du D-dimère.

Criteria for positive assay of the D-dimer were defined in order to establish its diagnostic value for phlebitis in the post-operative period. A retrospective study was carried out on the files of 94 patients who had received a total hip prosthesis in 1990. A semi-quantitative assay technique was used to measure the D-dimer because it is the only method giving immediate results. Three criteria were used to classify the results: criterium A: D-dimer greater than or equal to 2 micrograms/ml; criterium B: D-dimer greater than or equal to 4 times the preceding test; absence of both of these criteria. The results were compared to echo-doppler results and confirmed by phlebography when necessary. The incidence of proximal phlebitis was low (2 percent); criterium B showed a 100 percent negative predictability and a 29 percent positive predictability. None of the cases of phlebitis diagnosed with this test had been suspected clinically. This test provides a means of patient screening and spares the need for other explorations.

Comparison of different antibiotic regimens for therapy of 32 cases of Q fever endocarditis.

We studied 32 cases of Q fever endocarditis diagnosed in France between January 1985 and December 1989 to evaluate the efficacies of the different regimens of antibiotics used for treatment. Each patient was monitored during the treatment (range, 12 to 60 months), and clinical and biological information was computerized. Various treatments were prescribed, including doxycycline alone (9 cases) or in association with rifampin (4 cases), quinolones (16 cases), or sulfamethoxazole-trimethoprim (1 case). Two patients died before the beginning of the treatment. Nineteen patients had hemodynamic failure and subsequently underwent valve replacement. Nine valve tissue cultures were positive despite previous antibiotic treatment. In terms of their effects on mortality, the difference between doxycycline alone and doxycycline plus quinolones is statistically significant. We conclude that the addition of quinolones to doxycycline is beneficial. On the basis of clinical, serological, and valve tissue culture results, no treatment was able to cure Q fever endocarditis within 2 years, even with a combination of antibiotics. We advise a minimum duration of treatment of 3 years with therapy combining quinolones and doxycycline.

[Thrombocytopenia induced by low molecular weight heparin during hypotensive and diuretic treatment: coincidence or connection?]. / Thrombopénie induite par une héparine de bas poids moléculaire au cours d'un traitement hypotenseur et diurétique: coïncidence ou relation?

A case is reported of a 70-year-old female who presented with early thrombocytopaenia, apparently induced by a low molecular weight heparin. The patient was admitted with anasarca. The initial treatment consisted in digoxin, furosemide, potassium canrenoate and aldactone, and 0.3 ml of Fraxiparine daily, which replaced acenocoumarol. She had never been given any heparin previously. On the fourth day, her platelet count had dropped from 210 G.1-1 to 122 G.1-1, and to 15 G.1-1 on the sixth day. Heparin administration had been stopped on the fourth day, and the platelet count had returned to 141 G.1-1 five days later. Similar cases of such an early thrombocytopaenia have been reported in patients also taking diuretics and hypotensive drugs. However, a relationship between the two facts cannot yet be ascertained.