Feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) networking in university hospitals in Brussels.

The mutualisation of analytical platforms might be used to address rising healthcare costs. Our study aimed to evaluate the feasibility of networking a unique matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system for common use in several university hospitals in Brussels, Belgium. During a one-month period, 1,055 successive bacterial isolates from the Brugmann University Hospital were identified on-site using conventional techniques; these same isolates were also identified using a MALDI-TOF MS system at the Porte de Hal Laboratory by sending target plates and identification projects via transportation and the INFECTIO_MALDI software (Infopartner, Nancy, France), respectively. The occurrence of transmission problems (<2 %) and human errors (<1 %) suggested that the system was sufficiently robust to be implemented in a network. With a median time-to-identification of 5 h and 11 min (78 min, min-max: 154-547), MALDI-TOF MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p < 0.0001). However, the limited clinical benefits of the chromogenic culture media do not support their extra cost. Our financial analysis also suggested that MALDI-TOF MS networking could lead to substantial annual cost savings. MALDI-TOF MS networking presents many advantages, and few conventional techniques (optochin and oxidase tests) are required to ensure the same quality in patient care from the distant laboratory. Nevertheless, such networking should not be considered unless there is a reorganisation of workflow, efficient communication between teams, qualified technologists and a reliable IT department and helpdesk to manage potential connectivity problems.

Comparison of an in-house method and the commercial Sepsityper™ kit for bacterial identification directly from positive blood culture broths by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry.

The identification of bacteria directly from positive blood cultures using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a new challenge to microbiologists. However, the protocols previously described are often difficult to implement in routine and comparisons are not always possible due to the variability of interpretative criteria. This study evaluated the analytical and practical performances of an in-house (IH) method, adapted from previous protocols, and the Sepsityper™ kit (Bruker Daltonics, Bremen, Germany). Positive blood cultures from 63 different patients were prospectively evaluated by both methods. To enhance the sensitivity of these methods, lowered cut-offs were assessed and validated on 66 additional samples. The IH method produced 86.4% and 73.7% correct genus and species identifications, respectively, when using the lowered cut-offs of 1.4 and 1.6 for correct genus and species identifications. The Sepsityper™ kit showed similar results (78.0% and 68.4% correct genus and species identification, respectively). However, the IH method is ten-fold less expensive than the commercial option (0.72 vs. 7.45 /analysis) and its turnaround time is approximately 20 min versus the nearly 40 min required for the Sepsityper™ kit, which includes an extraction step. Finally, the IH method was introduced twice-daily in our routine practice.

Low levels of second-line drug resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Rwanda.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) has become a therapeutic problem in many parts of the world, necessitating the inclusion of second-line anti-tuberculosis drugs in specific treatment regimens. METHODS: We studied the susceptibility of 69 MDR Mycobacterium tuberculosis isolates from Rwanda to second-line drugs by the BACTEC 460 method. RESULTS: The results showed that 62 (89.9%) were resistant to rifabutin while a low rate (4.3%) of resistance was registered for ofloxacin; there was one case (1.4%) of resistance each for para-aminosalicylic acid, kanamycin, ethionamide, and clarithromycin. CONCLUSIONS: This information is important for devising an appropriate treatment regimen for MDR-TB patients in order to stop the spread of MDR strains and contain the acquisition of additional drug resistance in Rwanda.

Different presentations of ophthalmic aspergillosis.

PURPOSE: Aspergillus species is found worldwide and does not normally cause disease. However, when the immune system is compromised, it can invade many organs and be responsible for severe disease. The authors present cases with both classical and atypical features of ophthalmic aspergillosis. METHODS: Case series of three patients. RESULTS: All patients were female and had a long history of methylprednisolone use. The first two presented with endogenous endophthalmitis. One case was unilateral with a classical presentation of endophthalmitis. The other presented with a very severe bilateral acute retinal necrosis like syndrome. General work-up revealed disseminated disease in both cases. The diagnosis was made by serum immunologic testing in one case and after direct examination and culture from vitrectomy in the other. Despite intense antimycotic therapy, both patients died. The third patient presented with a unilateral progressive painful orbital apex syndrome. An orbital lesion was demonstrated by computed tomography scan and was unresponsive to methylprednisolone. Diagnosis of sino-orbital syndrome was made on biopsy. The lesion responded poorly to different antimycotic therapies, invaded the chiasma, and the patient lost all visual acuity. CONCLUSIONS: This case series illustrates that ophthalmic aspergillosis can present acutely with a devastating intraocular inflammation or more indolently in the setting of sino-orbital aspergillosis. Both forms have a poor visual prognosis and the systemic form is frequently associated with a fatal outcome.

Molecular investigation of recurrent tuberculosis in patients from Rwanda.

SETTING: Pulmonary tuberculosis (TB) patients enrolled in four provinces of Rwanda. OBJECTIVE: To determine the cause of recurrent TB. DESIGN: Serial Mycobacterium tuberculosis isolates obtained from patients with recurrent TB from January 2002 to September 2005 were genotyped by spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Drug resistance was determined by phenotypic susceptibility testing and sequencing of rpoB, katG, inhA and embB genes. RESULTS: Among 710 culture-positive TB patients enrolled in the study, initial drug susceptibility testing results were available for 638. Sixty-nine of these had multidrug-resistant (MDR) TB and 569 were non-MDR-TB. Among the MDR-TB patients, 22 had follow-up isolates after cure (n = 12) or chronic infection (n = 10). The DNA patterns of sequential isolates from 4 of the 12 previously cured MDR-TB patients were different, indicating re-infection. DNA patterns of isolates from the remaining 8 previously cured and 10 chronic MDR-TB patients were identical, suggesting reactivation and treatment failure, respectively. Among the non-MDR-TB patients, disease recurrence was observed in one case; this was determined to be due to reactivation after initial mixed infection. CONCLUSION: These results document a high treatment failure/reactivation rate for MDR-TB and suggest that re-infection within 2 years may not be a common cause of recurrent TB in this setting.

Evidence of 'amplifier effect' in pulmonary multidrug-resistant tuberculosis: report of three cases.

INTRODUCTION: A cluster of three related cases of tuberculosis (TB) with primary multidrug resistance was investigated at the Centre Hospitalier Universitaire of Kigali (CHUK) in Rwanda. The patients were HIV-1/2 seronegative. Patients 1 and 2 were hospitalized in the same room of CHUK for one month. Patient 3 was a younger sibling of patient 2. METHODS: Drug susceptibility of two consecutive Mycobacterium tuberculosis isolates from each patient was tested by the BACTEC 460 radiometric method. DNA fingerprinting was performed using spoligotyping and mycobacterial interspersed repetitive units of variable numbers of tandem repeats (MIRU-VNTR) analysis. All patients initially received the World Health Organization category I regimen. RESULTS: The isolates collected during the first TB episode were resistant to isoniazid, rifampin and ethambutol. After subsequent retreatment regimens with rifampin, isoniazid, streptomycin, pyrazinamide (8 months) and rifampin, isoniazid, streptomycin, pyrazinamide, ciprofloxacin (21 months), patients 1 and 2 developed additional resistance to streptomycin and quinolones. Patient 3 received only the category I regimen and consecutive isolates retained the initial drug susceptibility pattern. All isolates were genetically indistinguishable by spoligotyping and MIRU-VNTR, indicating the same origin. CONCLUSIONS: These observations highlight the risk of nosocomial transmission of multidrug-resistant (MDR) TB and the possible selection of secondary resistance to second-line drugs if a single new drug is added at the time of retreatment of MDR TB patients.

[Uterine cervical carcinoma and pericardial effusion]. / Cancer du col utérin et épanchement péricardique.

A 64-year-olf woman has been treated by chemotherapy for a uterine cervical carcinoma with known pathological lymph nodes in the abdomen and in the thorax. She is admitted in our Intensive Care Unit for fever and cardiac tamponade attributed to a large pericardial effusion. No diagnostic could be concluded from the analysis of the liquid or the pericardial biopsy. Complementary investigations are performed and the differential diagnosis of pericardial effusion is discussed in the context of a neoplastic disease.

Evaluation of the ProSpecT Microplate Assay for detection of Campylobacter: a routine laboratory perspective.

OBJECTIVES: To evaluate the use of the new enzyme-linked immunosorbent assay, the ProSpecT Campylobacter Microplate Assay (Alexon-Trend, Minneapolis, MN, USA), which allows 2-h detection of both Campylobacter jejuni and Campylobacter coli antigen directly in stool specimens. METHODS: Over 4 months, all stool samples preserved in Cary-Blair medium, or fresh specimens, from non-hospitalized children and HIV-infected patients (adults and children), submitted to our laboratory were evaluated with the ProSpecT Campylobacter Microplate Assay. Results were compared with those obtained by routine culture methods using both a specific medium and a filtration method for the recovery of Campylobacter spp. RESULTS: Of the 1205 stool specimens cultured, 101 were found to be positive for either C. jejuni or C. coli, giving an overall recovery rate of 8.38%. Ninety samples were positive by both culture and ProSpecT Campylobacter Microplate Assay, and 11 were positive by culture only, giving a sensitivity of 89.1%. In addition, of 1104 samples negative by culture, 25 were initially positive by ProSpecT Campylobacter Microplate Assay. We found no cross-reaction with other bacterial enteropathogens isolated from stool specimens. These results thus confirm a high specificity (97.7%) for both C. jejuni and C. coli. The positive and negative predictive values found were 78.3% and 99%, respectively. There was no statistically significant difference in sensitivity and specificity if the stool was fresh or preserved with Cary-Blair medium. CONCLUSION: These data suggest that the ProSpecT Campylobacter Microplate Assay is a rapid and easy-to-use test for the detection of both C. jejuni and C. coli in stool specimens. It could be used for patients for whom early antibiotic therapy is needed or for epidemiologic studies.

Atraumatic splenic rupture in the course of a pneumonia with Streptococcus pneumoniae. Case report and literature review.

Atraumatic splenic ruptures in the course of infectious diseases are rare but have been reported. Various germs of viruses can be at the origin of such rupture. The more often quoted viral disease is infectious mononucleosis. The more frequently involved bacteria are Streptococcus non pneumoniae, Pseudomonas, staphylococci and Clostridium. Rupture mechanism is not clearly elucidated; it can be connected with sepsis diffusion at spleen level via haematogenic way and consequently splenomegaly. Splenic rupture following septicaemia does not always entail major splenomegaly nor abscess formation but the attack of the splenic tissue itself is sometimes sufficient to bring about the rupture. The present case of atraumatic splenic rupture on spleen sepsis, no abscess, starting from a pulmonar infection with Streptococcus pneumoniae is, to our knowledge, the first case reported in literature.

Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.

For septic patients, delaying the initiation of antimicrobial therapy or choosing an inappropriate antibiotic can considerably worsen their prognosis. This study evaluated the impact of rapid microbial identification (RMI) from positive blood cultures on the management of patients with suspected sepsis. During a 6-month period, RMI by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for all new episodes of bacteraemia. For each patient, the infectious disease specialist was contacted and questioned about his therapeutic decisions made based on the Gram staining and the RMI. This information was collected to evaluate the number of RMIs that led to a therapeutic change or to a modification of the patient's general management (e.g. fast removal of infected catheters). During the study period, 277 new episodes of bacteraemia were recorded. In 71.12% of the cases, MALDI-TOF MS resulted in a successful RMI (197/277). For adult and paediatric patients, 13.38% (21/157) and 2.50% (1/40) of the RMIs, respectively, resulted in modification of the treatment regimen, according to the survey. In many other cases, the MALDI-TOF MS was a helpful tool for infectious disease specialists because it confirmed suspected cases of contamination, especially in the paediatric population (15/40 RMIs, 37.50%), or suggested complementary diagnostic testing. This study emphasizes the benefits of RMI from positive blood cultures. Although the use of this technique represents an extra cost for the laboratory, RMI using MALDI-TOF MS has been implemented in our daily practice.

Accuracy of the API Campy system, the Vitek 2 Neisseria-Haemophilus card and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Campylobacter and related organisms.

Biochemical identification of Campylobacter and related organisms is not always specific, and may lead to diagnostic errors. The API Campy, the Vitek 2 system and matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are commercially available methods that are routinely used for the identification of these microorganisms. In the present study, we used 224 clinical isolates and ten reference strains previously identified by multiple PCR assays, whole cell protein profiling and either DNA-DNA hybridization or sequencing analysis to compare the reliability of these three methods for the identification of Campylobacter and related pathogens. The API Campy accurately identified 94.4% of Campylobacter jejuni ssp. jejuni and 73.8% of Campylobacter coli, but failed to correctly identify 52.3% of other Epsilobacteria. The Vitek 2 Neisseria-Haemophilus card correctly identified most C. jejuni ssp.jejuni (89.6%) and C. coli (87.7%) strains, which account for the majority of campylobacterioses reported in humans, but it failed in the identification of all of the other species. Despite a good identification rate for both C. jejuni ssp. jejuni and C. coli, both methods showed poor sensitivity in the identification of related organisms, and additional tests were frequently needed. In contrast to API Campy and Vitek, MALDI-TOF MS correctly identified 100% of C. coli and C. jejuni strains tested. With an overall sensitivity of 98.3% and a short response time, this technology appears to be a reliable and promising method for the routine identification of Campylobacter and other Epsilobacteria.

Critical use of nucleic acid amplification techniques to test for Mycobacterium tuberculosis in respiratory tract samples.

The usefulness of employing Belgian selection criteria before performing nucleic acid amplification techniques (NAT) was evaluated. The results of this study show that for smear-negative patients with an abnormal chest radiology result in the absence of a respiratory tract infection by bacterial pathogens, testing with NAT is of major benefit.