Stefan B. Svenson

Spread of Drug-Resistant Pulmonary Tuberculosis in Estonia

ABSTRACT Restriction fragment length polymorphism (RFLP) analysis of 209Mycobacterium tuberculosis clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS6110 banding pattern. These strains shared the majority of their IS6110 DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%). This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains. The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of M. tuberculosis belonged to family A. These strains were confirmed by spoligotyping as members of the Beijing genotype family. The spread of Beijing genotype MDR M. tuberculosis strains was also frequently seen in 1997 to 1999. The members of this homogenous group of drug-resistant M. tuberculosis strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.

Clinical Pyelonephritis and Focal Renal Scarring: A Selected Review of Pathogenesis, Prevention, and Prognosis

This article considers the pathogenesis of acute pyelonephritis; determinants of focal renal scarring; prevention of renal damage by early recognition of urinary tract infection in childhood; and renal growth patterns in kidneys damaged during early childhood.

Dynamics of Penicillin-Susceptible Clones in Invasive Pneumococcal Disease

In a 10-year period, 1987-1997, there was a >4-fold increase in the rate of pneumococcal bacteremia in Sweden. Invasive pneumococcal isolates (n=1136), which were obtained from 18 Swedish clinical microbiology laboratories from 1987 through 1997, and other national and international isolates were serotyped, and their clonal relationships were determined by molecular typing. The increase in invasive pneumococcal disease in Sweden during this period was associated particularly with an increase in isolates of serotypes 1 and 14. A 3-fold increase of type 14 was seen from 1987 through 1992, and a 10-fold increase of type 1 occurred from 1992 through 1997. One dominating penicillin-susceptible clone of type 14 was responsible for the increase of type 14 during the first 5 years. This clone also was found in Canada and the United States and was shown by multilocus sequence typing to correspond to a previously identified hyper-virulent clone. A novel penicillin-susceptible clone of type 1, which was not found among invasive isolates from 1987 or 1992, was responsible for the increase of serotype 1 during the last 5 years. These results illustrate the ability of virulent penicillin-susceptible pneumococcal clones to emerge and spread rapidly within a country.

Molecular Epidemiology of Streptococcus pneumoniae Causing Invasive Disease in 5 Countries

A multicenter study was done during 1993-1995 to investigate prospectively the influence of several prognostic factors for predicting the risk of death among patients with pneumococcal bacteremia. Five centers located in Canada, the United Kingdom, Spain, Sweden, and the United States participated. Clinical parameters were correlated to antibiotic susceptibility and serotyping of the 354 invasive pneumococcal isolates collected and to molecular typing of 173 isolates belonging to the 5 most common serotypes (14, 9V, 23F, 3, and 7F). Serotype 14 was the most common among all isolates, but serotype 3 dominated in fatal cases and in isolates from Spain and the United States, the countries with the highest case-fatality rates. Fewer different patterns were found among the type 3 isolates, which suggests a closer clonal relationship than that among isolates belonging to other serotypes. Of type 3 isolates from fatal cases, 1 clone predominated. Other penicillin-susceptible invasive clones were also shown to spread in and between countries.

Rapid diagnosis of tuberculosis by detection of mycobacterial lipoarabinomannan in urine

There is an urgent need for improved tools for laboratory diagnosis of active tuberculosis (TB). Here, we describe two methods, a catch-up ELISA and a dipstick test based on the detection in urine of lipoarabinomannan (LAM). LAM is a major and specific glycolipid component of the outer mycobacterial cell wall. Preliminary experiments showed that LAM is excreted in the urine of mice injected intraperitoneally with a crude cell wall preparation of Mycobacterium tuberculosis. Both methods were highly sensitive, detecting LAM at concentrations of 1 ng/ml and 5 pg/ml, respectively. Of 15 patients with active TB, all showed intermediate to high levels of LAM in their urine (absorbance values from 0.3 to 1.2, mean 0.74). Only one sample showed an absorbance value below the chosen cut off value of 0.4. All but one of the urine samples from 26 healthy nursing workers exhibited OD value below 0.4 cut off. These methods may prove valuable for rapid and simple diagnosis of TB in particular in developing countries lacking biosafety level 3 (BSL3) facilities.

Identification of a carbohydrate receptor recognized by uropathogenic Escherichia coli.

ZusammenfassungFrühere Untersuchungen haben gezeigt. daßEscherichia coli, die Erreger von Pyelonephritis sind, Kohlenhydratstrukturen, die mit den P-Blutgruppen-Antigenen in Beziehung stehen, spezifisch erkennen und sich an sie binden. Diese Ergebnisse werden in der vorliegenden Untersuchung bestätigt und erweitert. Zweiundzwanzig von 23 nicht selektiertenE. coli-Stämmen von Kindern mit akuter fieberhafter Pyelonephritis agglutinierten menschliche Erythrozyten nicht, bei denen Antigene im P-Blutgruppensystem fehlten. Nur eines von 32 Isolaten aus dem Stuhl zeigte diese spezifische agglutinierende Eigenschaft. Die neue Information aus dieser Arbeit ist, daß P2K-Erythrozyten, die das PK-Antigen enthielten, im selben Ausmaß vonE. coli-Stämmen, die Pyelonephritis-Erreger waren, agglutiniert wurden, womit sich die Vorstellung weiter bestärkt, daß das PK-Glykosphingolipid dem Rezeptor für PyelonephritisE. coli verwandt ist. Daneben wurde der Bedeutung der Oligosaccharid-verbindung des PK-Glykosphingolipids für die Bindung vonE. coli weiter nachgeforscht. Das synthetisierte Disaccharid α-D-Galp-(1–4)-β-D-Galp-1-O-Ø-NO2 hemmt die durch pyelonephritischeE. coli verursachte Agglutination von menschlichen Erythrozyten bei Konzentrationen von weniger als 1 mM. Folglich scheint die kleinste von diesenE. coli-Stämmen erkennbare Struktur das α-D-Galp-(1–4)-β-D-Galp zu sein. Wieweit diese Beobachtung allgemeine Bedeutung besitzt, bedarf weiterer Forschung. Die Ergebnisse können neue Möglichkeiten für die Diagnose und die Behandlung der Harnwegsinfektionen eröffnen.SummaryEarlier investigations have shown that pyelonephriticEscherichia coli specifically recognize and bind to carbohydrate structures correlated to the P blood group antigens. These findings are confirmed and extended in this study. Twenty-two of 23 nonselectedE. coli strains from children with acute febrile pyelonephritis failed to agglutinate human erythrocytes lacking the antigens within the P blood group system. Only one of 32 faecal isolates exhibited this specific agglutinating property. The new information in this paper is that P2k erythrocytes, containing only the Pk antigen, were agglutinated to the same extent by pyelonephriticE. coli strains, giving further support to the proposal that the Pk glycosphingolipid is related to the receptor for pyelonephriticE. coli. In addition, the importance of the oligosaccharide moiety of the Pk glycosphingolipid for the binding ofE. coli was further investigated. The synthesized disaccharide α-D-Galp-(1–4)-β-D-Galp-1-O-Ø-NO2 inhibited the agglutination of human erythrocytes caused by two pyelonephriticE. coli strains at concentrations of less than 1 mM. Hence, the minimal receptor structure recognized by theseE. coli strains appears to be the α-D-Galp-(1–4)-β-D-Galp structure. How generally valid this observation may be needs further investigation. The findings may open new possibilities for diagnosis and treatment of urinary tract infection.

Rapid identification of P-fimbriated Escherichia coli by a receptor-specific particle agglutination test

SummaryMost (>90%)Escherichia coli strains isolated from children with acute non-obstructive pyelonephritis exhibit a specific type of filamentous protein appendage known as P-fimbriae. These fimbriae enable the bacterium to adhere to human uroepithelial cells by the specific recognition of and binding to a particular class of glycosphingolipids correlated to the human P-blood group antigens. In this paper a new method for the rapid and reliable identification of such P-fimbriated pyelonephritogenic bacteria is described. The method is based on particles to which the minimal glycoside receptor structure recognized by P-fimbriae is attached. Mixing these receptor-containing particles with P-fimbriated bacteria results in a strong and immediate agglutination reaction. The specificity and sensitivity of this new particle agglutination test proved to be superior to the haemagglutination assay previously used.ZusammenfassungDie meisten (mehr als 90%) der von Kindern mit akuter, nicht obstruktiver Pyelonephritis isoliertenEscherichia coli-Stämme weisen einen spezifischen Typ von filamentösen Protein-Anhangsstrukturen auf, die als P-Fimbrien bekannt sind. Diese Fimbrien befähigen das Bakterium, sich an menschliche Urothelien anzuheften. Dabei erkennen die Fimbrien spezifisch Glykosphingolipide einer bestimmten Klasse, die mit den menschlichen P-Blutgruppenantigenen in Beziehung stehen, und binden sich an sie. In der vorliegenden Arbeit wird eine neue Methode zur raschen und sicheren Identifizierung von solchen P-Fimbrien-tragenden pyelonephritogenen Bakterien beschrieben. Die Methode basiert darauf, daß die kleinsten, von P-Fimbrien erkennbaren Strukturen des Glykosid-Rezeptors an Partikel gebunden werden. Die Mischung dieser Rezeptor-tragenden Partikel mit P-Fimbrien-tragenden Bakterien führt zu einer starken, sofortigen Agglutinationsreaktion. Dieser neue Partikelagglutinationstest erwies sich in Spezifität und Sensibilität als dem früher verwandten Hämagglutinationstest überlegen.Most (>90%)Escherichia coli strains isolated from children with acute non-obstructive pyelonephritis exhibit a specific type of filamentous protein appendage known as P-fimbriae. These fimbriae enable the bacterium to adhere to human uroepithelial cells by the specific recognition of and binding to a particular class of glycosphingolipids correlated to the human P-blood group antigens. In this paper a new method for the rapid and reliable identification of such P-fimbriated pyelonephritogenic bacteria is described. The method is based on particles to which the minimal glycoside receptor structure recognized by P-fimbriae is attached. Mixing these receptor-containing particles with P-fimbriated bacteria results in a strong and immediate agglutination reaction. The specificity and sensitivity of this new particle agglutination test proved to be superior to the haemagglutination assay previously used. Die meisten (mehr als 90%) der von Kindern mit akuter, nicht obstruktiver Pyelonephritis isoliertenEscherichia coli-Stämme weisen einen spezifischen Typ von filamentösen Protein-Anhangsstrukturen auf, die als P-Fimbrien bekannt sind. Diese Fimbrien befähigen das Bakterium, sich an menschliche Urothelien anzuheften. Dabei erkennen die Fimbrien spezifisch Glykosphingolipide einer bestimmten Klasse, die mit den menschlichen P-Blutgruppenantigenen in Beziehung stehen, und binden sich an sie. In der vorliegenden Arbeit wird eine neue Methode zur raschen und sicheren Identifizierung von solchen P-Fimbrien-tragenden pyelonephritogenen Bakterien beschrieben. Die Methode basiert darauf, daß die kleinsten, von P-Fimbrien erkennbaren Strukturen des Glykosid-Rezeptors an Partikel gebunden werden. Die Mischung dieser Rezeptor-tragenden Partikel mit P-Fimbrien-tragenden Bakterien führt zu einer starken, sofortigen Agglutinationsreaktion. Dieser neue Partikelagglutinationstest erwies sich in Spezifität und Sensibilität als dem früher verwandten Hämagglutinationstest überlegen.

Mycobacterium tuberculosis arabinomannan–protein conjugates protect against tuberculosis

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

Diagnostic evaluation of urinary lipoarabinomannan at an Ethiopian tuberculosis centre.

Direct capture enzyme-linked immunosorbent assay (ELISA) for lipoarabinomannan (LAM) was performed on urine samples from 200 tuberculosis (TB) patients and 800 non-TB patients routinely diagnosed among consecutive suspects in an Ethiopian TB centre. 50 healthy Ethiopians, 50 healthy individuals and 100 non-TB patients from Norway served as controls. Of the TB patients, 139 (69.5%) were positive for acid-fast bacilli (AFB). In the remaining cases the diagnosis was based on suggestive clinical findings. All Ethiopian non-TB patients were AFB negative and showed no clinical evidence of TB. In the Ethiopian groups, 148 (74%) of the TB patients, 105 (13.1%) of the non-TB patients and 5 (10%) of the healthy controls were positive by the LAM-ELISA. 113 (81.3%) of AFB positives and 35 (57.4%) of AFB-negative TB patients had positive LAM-ELISA. In the Norwegian groups all were LAM negative. The sensitivity and specificity of the LAM-ELISA for TB patients versus Ethiopian non-TB patients were 74% and 86.9%, respectively; the positive and negative predictive values were 58.5% and 93.0%. This study suggests that detection of LAM in the urine of TB patients may improve case finding and that diagnostic tests based on this principle may serve as valuable supplemental tools in TB control.Direct capture enzyme-linked immunosorbent assay (ELISA) for lipoarabinomannan (LAM) was performed on urine samples from 200 tuberculosis (TB) patients and 800 non-TB patients routinely diagnosed among consecutive suspects in an Ethiopian TB centre. 50 healthy Ethiopians, 50 healthy individuals and 100 non-TB patients from Norway served as controls. Of the TB patients, 139 (69.5%) were positive for acid-fast bacilli (AFB). In the remaining cases the diagnosis was based on suggestive clinical findings. All Ethiopian non-TB patients were AFB negative and showed no clinical evidence of TB. In the Ethiopian groups, 148 (74%) of the TB patients, 105 (13.1%) of the non-TB patients and 5 (10%) of the healthy controls were positive by the LAM-ELISA. 113 (81.3%) of AFB positives and 35 (57.4%) of AFB-negative TB patients had positive LAM-ELISA. In the Norwegian groups all were LAM negative. The sensitivity and specificity of the LAM-ELISA for TB patients versus Ethiopian non-TB patients were 74% and 86.9%, respectively; the positive and negative predictive values were 58.5% and 93.0%. This study suggests that detection of LAM in the urine of TB patients may improve case finding and that diagnostic tests based on this principle may serve as valuable supplemental tools in TB control.

Synergistic effects of antimycobacterial drag combinations onMycobacterium avium complex determined radiometrically in liquid medium

A technique to determine the effects of combining antimycobacterial drugs in liquid medium employing the radiometric growth readings in the Bactec system was tested using 20Mycobacterium aviumcomplex strains. Ten of the strains had been isolated from children with lymphadenopathy and ten from adults with pulmonary disease. All isolates were resistant to streptomycin, rifampicin, isoniazid and ethambutol when tested with a conventional resistant ratio technique on Löwenstein-Jensen medium. Synergistic interactions were shown for the two-drug combinations streptomycin + ethambutol and ethambutol + rifampicin against all 20 strains. Good efficacy was also found for all three-drug combinations containing ethambutol. Thus, although most isolates of theMycobacterium aviumcomplex are resistant to first-line antituberculous drugs when tested individually, they are susceptible in vitro to certain combinations of these drugs. This rapid radiometric assay is an efficient means for detecting such synergy.