False-positive rifampicin resistance on Xpert® MTB/RIF caused by a silent mutation in the rpoB gene.

The Xpert® MTB/RIF assay detects the presence of Mycobacterium tuberculosis and its resistance to rifampicin (RMP) directly in sputum samples. Discrepant results were observed in a case of smear-positive pulmonary tuberculosis that was Xpert-resistant but phenotypically susceptible to RMP. Complementary investigations (repeat Xpert, Genotype®MTBDRplus assay and sequencing of the rpoB gene) revealed the presence of a silent mutation in the rpoB gene, leading to the conclusion of a false-positive Xpert result. As misinterpretation of Xpert results may lead to inappropriate treatment, the presence of rpoB mutations should be confirmed by sequencing the rpoB gene.

Tuberculous adenitis: comparison of CT and MRI findings with histopathological features.

Our aim was to investigate the relationship between the various histopathological features and the CT and MRI findings in routinely submitted histopathological specimens for the diagnosis of tuberculous lymphadenopathy. Twelve formalin-fixed, paraffin-embedded tissue blocks from ten patients who were clinically suspected of having tuberculous lymphadenopathy were evaluated. We assessed the presence of histopathological features including granuloma formation, caseous necrosis, and presence of Langhans-type giant cells, calcifications, fibrosis or normal lymphoid tissue. We performed polymerase chain reaction (PCR)-based assay for mycobacterial DNA and Ziehl-Neelsen staining for acid-fast bacilli (AFB). Findings were compared with those of CT and MRI, including signal intensities on unenhanced MR images, lymph node homogeneity, attenuation values on contrast-enhanced CT and enhancement patterns on MRI. Based on CT and MRI findings, four lymph node types could be defined: (1) homogeneous nodes, visible on both pre- and post-contrast images and corresponding histopathologically to granulation tissue without or with minimal caseation necrosis (n = 2); (2) heterogeneous nodes, showing heterogeneous enhancement patterns with central non-enhancing areas and corresponding to minor or moderate intranodal caseation/liquefaction necrosis (n = 3); (3) nodes showing peripheral rim enhancement and corresponding to moderate or extensive intranodal caseation/liquefaction necrosis (n = 5); (4) heterogeneous nodes showing intranodal hyperdensities on CT and hypointense areas on T1- and T2-weighted images and corresponding to fibrosis and calcifications (n = 2). On CT and MRI, the findings reflect different stages of the tuberculous process. Imaging findings depend on the presence and the degree of granuloma formation, caseation/liquefaction necrosis, fibrosis and calcifications.

Surveillance of antibiotic resistance in non invasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winters 2003 and 2004.

A total of 391 and 424 non-invasive isolates of Streptococcus pneumoniae collected by 15 laboratories during the 2003 and 2004 survey were tested for their susceptibility by a microdilution technique following NCCLS recommendations. Insusceptibility rates (IR) in the two surveys (2003/2004) were as follows: penicillin 15.0/14.7% [8.4/6.4% Resistance (R)], ampicillin 17.4/14.6% (R 9.0/7.1%), amoxicillin +/- clavulanic acid 2.6/1.2 % (R 0/0%), cefaclor 14.3/14.1% (R 11.5/13.4%), cefuroxime 13.6/12.7% (R 10.5/11.8%), cefuroxime-axetil 10.5/11.8% (R 10.0/9.2%) (breakpoints based on 250 mg), cefotaxime 4.9/6.2% (R 1.3/2.4%), ceftazidime NotTested (NT)/6.4 (R NT/2.6%), cefepime NT/6.4 (R NT/2.6%), imipenem 7.7/8.9 % (R 1.8/1.4%), ertapenem 0.8/NT% (R O/NT%), ciprofloxacin 13.8/9.0% (R 4.3/2.4%), levofloxacin 3.3/2.8% (R 1.5/0.2%), moxifloxacin 0.6/0.2% (R 0.3/0%), ofloxacin 13.5/9.0% (R 4.3/2.4%), erythromycin 26.1/24.7% (R 25.3/24.5%), azithromycin 25.4/24.7% (R 24.6/24.5%), telithromycin 0.8/0.2% (R 0.5/0%), clindamycin 21.2/18.4% (R 19.2/17.7%) and tetracycline 32.3/22.1% (R 29.2/19.3%). There were only minor differences in resistance rates according to age, sample site, admission type (i.e. ambulatory, hospitalized or long-term care facility patients), gender and geographic origin. Overall, telithromycin (MIC50, MIC90 in 2003/2004: 0.015 microg/ml, 0.12 microg/ml/ 0.008,0.06 respectively), ertapenem (0.03; 0.25/NT), moxifloxacin (0.06; 0.25/0.06, 0.12), and amoxicillin +/- clavulanic acid (0.03; 0.25/0.015, 0.5) were the most active compounds in both surveys. In 2003, the most common resistance phenotype was isolated insusceptibility to tetracycline (10.5%) followed by combined insusceptibility to erythromycin and tetracycline (9.3%). Erythromycin-tetracycline resistance (10.4%) was the most common in 2004. Isolates showing resistance to an antibiotic were significantly more present in 2003 than in 2004 (50.4% versus 40.8%). In penicillin-insusceptible isolates, MICs of all beta-lactams were increased but cross-resistance between penicillin and other beta-lactams in the penicillin-insusceptible isolates was not complete. In the 2003 survey, most of these isolates remained fully susceptible to ertapenem (94.9%) and amoxicillin +/- clavulanic acid (83.1%). In the 2004 survey, 91.9% of the penicillin insusceptible isolates remained susceptible to amoxicillin +/- clavulanic acid. In both surveys, the most common serotypes in penicillin insusceptible isolates were 14, 23,19 and 9 (20.0%, 20.0%, 16.4% and 10.9% respectively in 2003; 41.6%, 11.7%, 15.0% and 18.3% respectively in 2004).

[The Lemierre syndrome: a complicated oropharyngeal infection]. / Het Syndroom van Lemierre: een gecompliceerde orofaryngeale infectie.

The Lemierre syndrome or 'necrobacillosis' is a post angina sepsis caused by an acute oropharyngeal infection with a secondary thrombophlebitis of the internal jugular vein. There are often septic emboli in the lungs, although intestinal organs can also be affected. This syndrome is caused by the strictly anaerobic gram-negative pathogen Fusobacterium necrophorum, sometimes in combination with other pathogens. The patient typically presents with high fever, pain in the neck, malaise and dyspnoea one week after the start of an angina. Plain chest radiograph shows bilateral nodular infiltrates, ultrasound reveals a thrombophlebitis of the internal jugular vein. CT scan can be useful to confirm the diagnosis and possible complications. In the beginning there is often a transient hyperbilirubinemia with toxic inflammatory blood results. Under the correct antibiotic regime complete recovery can be obtained.

Surveillance of antibiotic resistance in clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2000-2001.

A total of 314 isolates of Streptococcus pneumoniae collected by 10 different laboratories were tested for their susceptibility by using a microdilution technique following NCCLS recommendations. The following antibiotics were included: penicillin, ampicillin, amoxicillin, amoxicillin/clavulanate, cefaclor, cefuroxime, cefotaxime, imipenem, ciprofloxacin, gemifloxacin, levofloxacin, erythromycin, clarithromycin, azithromycin, miocamycin, clindamycin and tetracycline. The insusceptibility rate (IR) to penicillin was 21.0% [10.8% intermediate (> or = 0.12-1 microgram/mL) and 10.2% high-level (> or = 2 micrograms/mL)], to cefotaxime 7.3% [3.5% intermediate (> or = 1 microgram/mL) and 3.8% high-level (> or = 2 micrograms/mL)], to imipenem 3.8% [3.8% intermediate (> or = 0.25-0.5 microgram/mL) and 0% high-level (> or = 1 microgram/mL)], to ciprofloxacin 11.2% [8.3% intermediate (2 micrograms/mL) and 3.9% high-level (> or = 4 micrograms/mL)], to erythromycin 30.3% [3.5% intermediate (0.5 microgram/mL) and 26.8% high-level (> or = 1 microgram/mL)] and to tetracycline 38.5% [0.9% intermediate (4 micrograms/mL) and 37.6% high-level (> or = 8 micrograms/mL)]. No decreased susceptibility was found for gemifloxacin (> or = 0.5 microgram/mL). This compound was the most active with MIC50, MIC90 and an IR of 0.015 microgram/mL, 0.03 microgram/mL and 0% respectively, followed by amoxicillin/clavulanate, amoxicillin and imipenem (MIC50, MIC90 and IR: 0.015 microgram/mL, 1 microgram/mL, 1.6%/0.015 microgram/mL, 1 microgram/mL, 1.9%/0.008 microgram/mL, 0.12 microgram/mL, 3.8% respectively). Compared to the 1999 surveillance, penicillin and tetracycline-insusceptibility increased with 4.9% and 15.6% respectively, while cefotaxime, erythromycin and ciprofloxacin insusceptibility decreased with 5.4%, 5.8% and 4.4% respectively. MICs of all beta-lactams rose with those of penicillin for penicillin-insusceptible isolates. Imipenem, cefotaxime, amoxicillin and amoxicillin/clavulanate were generally 4, 2, 1 and 1 doubling dilutions respectively more potent than penicillin on these isolates while ampicillin, cefuroxime and cefactor were generally 1, 2 and 4 dilutions respectively [table: see text] less potent. Most penicillin-insusceptible isolates remained fully susceptible to amoxicillin/clavulanate (92.4%), amoxicillin (90.9%) and imipenem (81.8%). Erythromycin-tetracycline insusceptibility was the most common resistance phenotype (14.3%). Three- and four-fold resistance was found in 12.4% and 1.6% respectively of the isolates. Most penicillin-insusceptible isolates were of capsular types 14 (22.7%), 23 (21.2%), 6 (18.2%), 9 (13.6%) and 19 (12.1%).

Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay.

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5' untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.

Comparison of the reticulocyte mode of the Abx Pentra 120 Retic, Coulter General-S, Sysmex SE 9500, Abbott CD 4000 and Bayer Advia 120 haematology analysers in a simultaneous evaluation.

The Abx Pentra 120 Retic, Coulter General-S, Sysmex SE 9500, Abbott Cell Dyn 4000 and Bayer Advia 120 were evaluated simultaneously in a general hospital laboratory. Linearity, precision, sample stability, carry-over and comparability of the reticulocyte mode were determined following International Council for Standardization in Haematology guidelines for the evaluation of blood cell analysers. All analysers showed good results for dilution, stability and carry-over testing. The between-batch coefficient of variation of the General-S was high compared to the other analysers evaluated. Multiple correlation studies showed good agreement for all analysers in the normal and high reticulocyte range, with correlation coefficients above 0.7. Multiple correlation studies for reticulocytopenic samples (< 15.109/l) were less satisfactory, with a wider range of correlation coefficients (r-values 0.0-0.9). Overall, the General-S, SE 9500 and Advia 120 gave lower reticulocyte counts than the Pentra 120 Retic and CD 4000. Reagent costs were also evaluated. Reagent consumption was close to the manufacturers' specifications for the SE 9500 (Search reagent), CD 4000 (CD Retic) and Advia 120 (Retics) but was higher than stated for the Pentra 120 Retic (Retix), General-S (Retic kit) and SE 9500 (Sheath reagent). Our results show that these new generation haematology analysers will meet the needs of hospital laboratories for reliable and cost-effective reticulocyte counting.

In vitro study on the antimicrobial activity of various antibiotics against clinical isolates of Streptococcus pneumoniae from Belgium collected during winter 1998-1999.

A total of 205 isolates of Streptococcus pneumoniae obtained from 10 different centres were included in this study. The susceptibilities to penicillin, ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, cefotaxime, imipenem, ciprofloxacin, gemifloxacin, grepafloxacin, levofloxacin, trovafloxacin, erythromycin, clarithromycin, miocamycin, clindamycin and tetracycline were determined by a microdilution technique following NCCLS recommendations. Decreased susceptibility to penicillin was 16.1% [6.8% intermediate (0.12-1 microgram/mL) and 9.3% high-level (> or = 2 micrograms/mL)], cefotaxime insusceptibility (> or = 1 microgram/mL) 12.7%, ciprofloxacine insusceptibility (> or = 2 micrograms/mL) 15.6% with 1.5% of high level resistance (> or = 4 micrograms/mL), erythromycin insusceptibility (> or = 0.5 microgram/mL) 36.1% and tetracycline insusceptibility (> or = 4 micrograms/mL) 22.9%. Decreased susceptibility to cefotaxime was found in 78.8% of the penicillin-insusceptible isolates. No decreased susceptibility was found for gemifloxacin (> or = 0.5 microgram/mL) and trovafloxacin (> or = 1 microgram/mL). Compared to the 1996-1997 surveillance, penicillin, cefotaxime and erythromycin insusceptibility rose by 3.8%, 5.2% and 5.0% respectively, while tetracycline insusceptibility decreased with 8.2%. MICs of all beta-lactams rose with those of penicillin for penicillin-insusceptible isolates. Amoxicillin +/- clavulanate, cefotaxime and imipenem were generally 1, 1 and 5 doubling dilutions respectively more potent than penicillin on these isolates. Penicillin, ampicillin and cefuroxime were equally active while cefaclor was generally 5 dilutions less potent. Most penicillin-insusceptible isolates remained fully susceptible to amoxicillin +/- clavulanate and imipenem. The penicillin-insusceptible isolates were 36.4%, 27.3% and 3.0% co-insusceptible to erythromycin, erythromycin plus tetracycline and tetracycline respectively. A subpopulation of 52 isolates obtained from children aged < or = 3 years was also studied. Compared to the other isolates we found a statistically significant increase in insusceptibility for penicillin, cefaclor, cefuroxime, erythromycin, clarithromycin and tetracycline while a significant decrease was found for ciprofloxacin.

Surveillance of pneumococcal resistance in Belgium during winter 1996-1997.

This study tested 212 pneumococcal isolates from 9 institutions for their susceptibilities to penicillin, ampicillin, amoxycillin, amoxycillin/clavulanate, cefaclor, cefuroxime, cefotaxime, imipenem, tetracycline, erythromycin, and clarithromycin using NCCLS-standardized microdilution. Penicillin-insusceptibility was 12.3% [5.7% intermediate (0.12-1 microgram/ml) and 6.6% high-level (> or = 2 micrograms/ml)], tetracycline-insusceptibility (> or = 4 micrograms/ml) 31.1%, and erythromycin-insusceptibility (> or = 0.5 microgram/ml) 31.1% as well. Erythromycin-insusceptible isolates showed cross-insusceptibility to clarithromycin. Penicillin-susceptible isolates were susceptible to all beta-lactams. MICs of all beta-lactams rose with those of penicillin for penicillin-insusceptible isolates. Ampicillin and penicillin were equally potent against penicillin-insusceptible isolates, imipenem, cefotaxime, and amoxycillin +/- clavulanate were more potent (generally 5, 1, and 1 doubling dilution, respectively), and cefuroxime and cefaclor less potent (generally 1 and 6 doubling dilutions, respectively). Most penicillin-insusceptible isolates were high-level resistant to cefaclor (> or = 32 micrograms/ml). Although MICs of all beta-lactams rose with those of penicillin, resistance to penicillin was not absolute in terms of cross-resistance. Most penicillin-intermediate and high-level penicillin-resistant isolates remained fully susceptible and intermediate, respectively, to amoxycillin +/- clavulanate, cefotaxime, and imipenem, but not to cefuroxime. Penicillin-susceptible isolates were 76.9%, 42.3%, and 34.6% co-insusceptible to tetracycline, erythromycin, and tetracycline plus erythromycin, respectively. Most penicillin-, tetracycline-, and erythromycin-insusceptible isolates were of capsular types 23 >> 6 > 19 > 32, 19 > 6 > 28 > 23, and 19 > 6 > 14 > 23, respectively. Compared to winter 1994-1995, insusceptibility to penicillin, tetracycline, and erythromycin rose by some 4%, 4%, and 13%, respectively.

In vitro susceptibilities of 180 clinical isolates of Haemophilus influenzae to ampicillin, amoxycillin/clavulanate, cefaclor, cefuroxime, cefotaxime, clarithromycin, and azithromycin.

One hundred eighty consecutive, unduplicate isolates of Haemophilus influenzae from clinical specimens collected from November 1994 through February 1995 in nine general hospitals throughout Belgium were examined for beta-lactamase production using a nitrocefin-based test, and for their in vitro susceptibilities to ampicillin, amoxycillin/clavulanate, cefaclor, cefuroxime, cefotaxime, clarithromycin and azithromycin by means of the NCCLS agar dilution test. The isolates were all from respiratory tract specimens. The prevalence of capsular type b was 1.1%, and the overall rate of beta-lactamase production 16.7%. Rates of beta-lactamase production were higher in isolates from children (22.0%) than in those from adults (15.3%), and in isolates from upper respiratory tract specimens (22.0%) than in those from the lower respiratory tract (15.1%). Beta-lactamase-negative ampicillin resistance amounted to 1.1%. Cefotaxime had the highest activity on a weight basis [MIC (minimal inhibitory concentration) for 50% of the isolates tested (MIC50) < or = 0.06 microgram/ml], followed by ampicillin (MIC50 of 0.25 microgram/ml), amoxycillin/clavulanate and cefuroxime (MIC50 of 0.5 microgram/ml), azithromycin (MIC50 of 2 micrograms/ml), cefaclor (MIC50 of 4 micrograms/ml), and clarithromycin (MIC50 of 8 micrograms/ml). Cefotaxime was also the most active drug in terms of susceptibility rates of the isolates (100.0%), followed by amoxycillin/clavulanate and azithromycin (98.9%), cefuroxime (97.2%), cefaclor (89.4%), clarithromycin (82.8%), and ampicillin (82.2%). In conclusion, amoxycillin/clavulanate and cefuroxime retain an excellent activity against H. influenzae, while cefaclor lost some of its activity. The rate of susceptibility to azithromycin was markedly higher than that to clarithromycin; however, its ability to accumulate intracellularly while concentrations in serum and interstitial fluid remain low, should be considered, as it may represent a major drawback to its use in H. influenzae infections.

In vitro susceptibilities of 176 clinical isolates of Streptococcus pneumoniae to 11 beta-lactams, erythromycin, and tetracycline.

One hundred seventy six consecutive, non-duplicate pneumococcal isolates from clinical specimens collected from November 1994 through February 1995 in nine general hospitals throughout Belgium were tested for their in vitro susceptibilities to penicillin, ampicillin, amoxycillin with and without clavulanate, cefaclor, cefuroxime, cefonicid, cefprozil, cefpodoxime, cefotaxime, imipenem, tetracycline, and erythromycin by means of the NCCLS microdilution test. The overall rate of decreased susceptibility to penicillin was 12.5%, including 6.3% of intermediately and 6.3% of fully resistant isolates. Penicillin, ampicillin amoxycillin, amoxycillin/clavulanate, cefuroxime, cefotaxime and imipenem had the highest activity on a weight basis (MIC50 < or = 0.008 microgram/ml), followed by cefpodoxime and erythromycin (MIC50 of 0.015 microgram/ml), cefprozil and tetracycline (MIC50 of 0.12 microgram/ml), and eventually, cefaclor and cefonicid (MIC50 of 0.5 microgram/ml). Aggregate rates of susceptible plus intermediately resistant isolates at NCCLS-recommended breakpoints, i.e. overall percentages of isolates likely to respond to increased antibiotic doses in vivo (except for meningitis), were 100.0% for imipenem and cefotaxime, 98.9% for amoxycillin with and without clavulanate, 93.8% for penicillin, and 90.9% for cefuroxime. Overall rates of susceptibility to erythromycin and tetracycline amounted to 78.4% and 72.7%, respectively. MIC values of all beta-lactams increased with those of penicillin. Ampicillin was equally active as penicillin against isolates with reduced susceptibility to the latter (MIC90 of 2 micrograms/ml); imipenem, cefotaxime, and amoxycillin with and without clavulanate however, were more active (MIC90 3, 1, and 1 doubling dilution, respectively, below that of penicillin), while cefpodoxime, cefuroxime, cefprozil, cefonicid, and cefaclor on the other hand, were less active (MIC90, 1, 1, 2, 5, and 5 doubling dilutions, respectively, above that of penicillin). In conclusion, the present data confirm that pneumococcal resistance to penicillin has increased in Belgium, suggest that resistance to erythromycin may have stabilised, and reveal an unexpectedly high rate of resistance to tetracycline. Imipenem was the most active antibiotic tested overall, and amoxycillin with or without clavulanate the most active oral antibiotic, with activity almost similar to that of cefotaxime.