Molecular characterisation of Mycobacterium bovis isolates from cattle carcases at a city slaughterhouse in Uganda.

During a period of eight months, the carcases of 16,800 slaughter cattle were inspected at a city abattoir in Uganda. Eighty-seven of them had tuberculosis-like lesions and tissue samples were cultured. Only 17 cultures yielded acid-fast bacilli; 11 of them were confirmed as Mycobacterium bovis and six as non-tuberculous mycobacteria (NTM). GenoType Mycobacterium assays on the six NTM identified two as Mycobacterium fortuitum and one as Mycobacterium intracellulare, but three were unidentified. Characterisation of the M bovis isolates by spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) revealed that five of the six spoligotype patterns observed in the 11 strains had not been previously reported, and seven of the nine isolates typed by RFLP had multicopy number IS6110 patterns. Six of the 11 infected carcases had multiple sites of infection, but none was condemned as unfit for human consumption.

Mycobacterium tuberculosis Uganda genotype is the predominant cause of TB in Kampala, Uganda.

SETTING: Rubaga Division, Kampala, Uganda. OBJECTIVE: To use polymerase chain reaction (PCR) based regions of difference (RD) analysis to study the species diversity of Mycobacterium tuberculosis complex isolates from a community-based sample of tuberculosis (TB) patients from Rubaga and to perform long sequence polymorphism (LSP) analysis to further characterise the M. tuberculosis Uganda genotype, a group of strains previously recognised by their characteristic spoligotype patterns. DESIGN: For the present study, 344 consecutive TB patients attending clinics in Rubaga Division were enrolled. Sample processing and culture were performed at the National Tuberculosis and Reference Laboratory and molecular assays at Makerere Medical School. Species identification was achieved by determining the RDs, while spoligotyping and LSP analysis were performed to characterise the M. tuberculosis Uganda genotype. RESULTS: Of the 344 isolates, 343 (99.7%) were M. tuberculosis sensu stricto, while one was classical M. bovis. The Uganda genotype strains characteristically lacked RD724, a locus that defines one of the major sub-lineages of M. tuberculosis, which suggested that this geographically constrained lineage is specifically adapted to a central African human host population. CONCLUSION: M. tuberculosis is the most prevalent species of the M. tuberculosis complex in Kampala, and the Uganda genotype is the predominant strain.

Extensive transmission of an isoniazid-resistant strain of Mycobacterium tuberculosis in Sweden.

SETTING: City of Stockholm, Sweden. BACKGROUND: The incidence of tuberculosis (TB) in Sweden increased by 40% between 2003 and 2005. The spread of a unique TB strain resistant to isoniazid (INH) contributed to this increase. OBJECTIVE: To describe outbreaks of TB caused by this single strain, elucidate possible causes for its extensive spread and identify shortcomings of the TB control programme in Sweden. RESULTS: We identified a cluster consisting of 102 culture-confirmed TB cases with identical DNA fingerprints and 26 epidemiologically related cases, not confirmed by culture, all diagnosed between 1996 and 2005. Five partly separate outbreaks of this strain were discovered. Epidemiological links were established for 56% of the culture-confirmed cases and for all cases not confirmed by culture. Three patients died while receiving treatment, four became failures and eight defaulted or were lost to follow-up. Only eight patients received directly observed treatment (DOT) up to a period of 3 months, although 40% had poor adherence. CONCLUSIONS: Shortcomings of the national TB programme were revealed. Improved contact tracing and case holding, including DOT, is crucial to reduce TB transmission in Sweden.

Outbreak of Mycobacterium tuberculosis infection among captive Asian elephants in a Swedish zoo.

Between 2001 and 2003, there was an outbreak of tuberculosis in a Swedish zoo which involved elephants, giraffes, rhinoceroses and buffaloes. Cultures of trunk lavages were used to detect infected elephants, tuberculin testing was used in the giraffes and buffaloes, and tracheal lavage and tuberculin testing were used in the rhinoceroses. The bacteria isolated were investigated by spoligotyping and restriction fragment length polymorphism. Five elephants and one giraffe were found to have been infected by four different strains of Mycobacterium tuberculosis.

Nasal boost with adjuvanted heat-killed BCG or arabinomannan-protein conjugate improves primary BCG-induced protection in C57BL/6 mice.

Today it is generally accepted that the Bacillus Calmette-Guerin (BCG) vaccine protects against childhood tuberculosis (TB) but this immunity wanes with age, resulting in insufficient protection against adult pulmonary TB. Hence, one possible strategy to improve the protective efficacy of the BCG vaccine would be to boost in adulthood. In this study, using the mouse model, we evaluated the ability of two new TB vaccine candidates, heat-killed BCG (H-kBCG) and arabinomannan-tetanus toxoid conjugate (AM-TT), given intransally in a novel Eurocine adjuvant, to boost a primary BCG-induced immune response and to improve protection. Young C57BL/6 mice were vaccinated with conventional BCG and, 6 months later, boosted intranasally with adjuvanted H-kBCG or AM-TT, or subcutaneously with BCG. Ten weeks after the booster, mice were challenged intravenously with M. tuberculosis (Mtb) strain H37Rv. In spleens, there was a significant reduction of cfu counts in mice boosted with either H-kBCG or AM-TT vaccines compared to the non-boosted BCG-vaccinated mice. None of the boosting regimens significantly reduced bacterial loads in lungs, compared to non-boosted BCG vaccination. However, the extent of granulomatous inflammation was significantly reduced in the lungs of mice that received two of the booster vaccines (AM-TT and conventional BCG), as compared with sham-vaccinated mice. All boosted groups, except for mice boosted with the AM-TT vaccine, responded with a proliferation of spleen T cells and gamma interferon production comparable to that induced by a single BCG vaccination.

The relationship between disease pattern and disease burden by chest radiography, M. tuberculosis Load, and HIV status in patients with pulmonary tuberculosis in Addis Ababa.

BACKGROUND: We evaluated the impact of HIV coinfection on the chest radiographic pattern and extent of disease and its relation to the load of Mycobacterium tuberculosis in Ethiopian out-patients with pulmonary tuberculosis. PATIENTS AND METHODS: A total of 168 patients with cultureverified pulmonary tuberculosis had their chest X-rays (CXR) reviewed for the site, pattern, and extent of disease and the findings were correlated to (a) the mycobacterial culture count and bacillus load after sputum concentration and (b) the HIV status of the patients. RESULTS: HIV-positive patients were less likely to have cavitary disease (p < 0.001) and more likely to have pleural effusion (p = 0.08), miliary (p < 0.05), and interstitial (p < 0.01) patterns. A total of 15 (9.2%) patients had normal chest X-rays. HIV-infected patients had a CXR classified as normal or with minimal involvement (p = 0.059) and a reduced mycobacterial colony count (p = 0.002) compared to HIV-negative patients. Middle and lower lung involvement were more common in HIV-positive patients. CONCLUSION: CXR findings in the setting of an underlying HIV infection tend to be more atypical and could present as either normal or with minimal involvement. In general, HIV-positive patients had lower colony count of M. tuberculosis than HIV-negative patients. Of particular interest is the finding of a large number of normal chest X-rays in HIV-infected patients. With the rising incidence of both tuberculosis and HIV infection in Ethiopia, the finding of a normal chest X-ray and a negative smear poses a challenge for the diagnosis of pulmonary tuberculosis.

A mycobacterial lipoarabinomannan specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis.

Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.

Immunization with heat-killed Mycobacterium bovis bacille Calmette-Guerin (BCG) in Eurocine L3 adjuvant protects against tuberculosis.

The current live attenuated vaccine against tuberculosis, BCG, poses a risk of disseminated infections in immunocompromised subjects. Therefore, in this study we compared the protective effect of a heat-killed bacille Calmette-Guerin (H-kBCG) vaccine given in a new adjuvant (Eurocine L3) with the protection provided by the conventional live attenuated BCG vaccine in mice (C57BL/6 and BALB/c) challenged with virulent Mycobacterium tuberculosis (strain Harlingen). The H-kBCG vaccine alone, in accordance with earlier studies, did not give any or only gave slight protection compared to sham-vaccinated controls. However, the same vaccine given with Eurocine L3 adjuvant, either formulated as a suspension or as an emulsion, afforded significant levels of protection. This protection was at least as good as that of the control live attenuated BCG vaccine. The Eurocine L3 adjuvant is approved for human use as a nasal vaccine adjuvant and a successful phase I trial with nasal immunization with diphtheria vaccine has recently been performed in Sweden. Here we show that, in mice, intranasal priming with H-kBCG in Eurocine L3 adjuvant followed by intranasal booster resulted in the same level of protection as subcutaneous priming followed by intranasal booster. All H-kBCG formulations in the Eurocine L3 adjuvant elicited mycobacterial antigen-specific serum IgG and IFN gamma responses. In general, among the different vaccine formulation(s) in the Eurocine L3 adjuvant those that produced a relatively high Th2 response, as measured by IgG1/IgG2a ratio and IFN gamma production in vitro, were the most protective. In conclusion, H-kBCG in Eurocine L3 adjuvant could represent a safe and a more stable alternative to the conventional live BCG vaccine.

RAPD-PCR and PFGE as tools in the investigation of an outbreak of beta-haemolytic Streptococcus group A in a Swedish hospital.

We evaluated the random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques for studying an outbreak of beta-haemolytic streptococci group A (GAS) occurred at two maternity wards at Danderyd hospital, Stockholm, Sweden. All the isolates were of T-type 8,25. The RAPD technique revealed that all RAPD-PCR profiles were identical. PFGE showed that all the patterns but one were identical. These patterns were compared with 10 different T-type GAS from the strain collection of the Swedish Institute for Infectious Disease Control (SMI) and T-type 8,25 from different years and locations. The SMI strains exhibited patterns different from each other and all different from the isolates from Danderyd hospital. Moreover, RAPD could not differentiate among the T-type 8,25 isolates from different years and locations but PFGE showed differences among the amplicons. Our results indicated that the RAPD and PFGE techniques could be efficient tools in epidemiological studies of GAS.

The mtp40 gene is not present in Mycobacterium bovis.

The mtp40 gene was initially reported to be lacking in classical Mycobacterium bovis strains, while being specific to classical M. tuberculosis strains. Later two clinical isolates reported to be M. bovis were shown to possess the mtp40 gene (A. Weil, B.B. Plikaytis, W.R. Butler, C.L. Woodley and T.M. Shinnik, J Clin Microbiol 1996; 34: 2309-2311). The two strains were, however, not fully characterized biochemically or genotypically. By PCR amplification of whole cell lysates and subsequent spoligotyping, which classifies isolates within the M. tuberculosis complex, the two strains were found to possess the spacers 40-43 which typically are lacking in classicalM. bovis, but had a spoligotyping pattern compatible with M. africanum. We conclude that the two strains, previously designated M. bovis, should be designated M. africanum. This reinvestigation has implications for the phylogenetic classification of M. bovis.

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.

Spread of drug-resistant pulmonary tuberculosis in Estonia.

Restriction fragment length polymorphism (RFLP) analysis of 209 Mycobacterium 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.

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.

Comparison of Mycobacterium avium complex (MAC) strains from pigs and humans in Sweden by random amplified polymorphic DNA (RAPD) using standardized reagents.

Infections with atypical mycobacteria belonging to the Mycobacterium avium/intracellulare complex (MAC) can cause infection in both animals and humans. Using a standardized reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis, 49 MAC strains isolated from 32 slaughter pigs and 17 humans in Sweden were identified and sorted out, yielding 6 RAPD types. By combining the results of RAPD primers 4 and 5 and the primer IS1245A, we found that pigs and humans may be infected with the same types of MAC strains, since 14 strains from humans and 8 strains from pigs were essentially identical and together, comprised RAPD type 2, the largest group of strains (44.8% of strains). With respect to grouping of strains, serotype and RAPD type were uncorrelated, except for serotype 20 and RAPD type 6. Using standardized beads, RAPD analysis is a reproducible technique for typing MAC strains, as the indistinguishable banding patterns obtained with repeated analyses of two isolates from each strain in this study demonstrate. However, primer selection and DNA purity were crucial for differentiating closely related strains.

Clinical isolates of Streptococcus pneumoniae that exhibit tolerance of vancomycin.

The ability of Streptococcus pneumoniae to escape lysis and killing by vancomycin, a property termed "tolerance," has recently been noted in a laboratory strain of the species. Vancomycin tolerance in clinical isolates represents a potential new health risk. We determined the prevalence of vancomycin and penicillin tolerance among 116 clinical isolates of pneumococci by monitoring lysis and viability after exposure to the respective antibiotic for 4 hours. Eight percent of the strains were tolerant to penicillin and 3% were tolerant to vancomycin. The 3 vancomycin-tolerant isolates also had a high ratio of minimum bactericidal concentration to minimum inhibitory concentration, in contrast to nontolerant strains. They were of serotype 9V and had reduced susceptibility to penicillin. Only 1 was also tolerant to penicillin. Growth rate and ability to divide were not affected in the 3 vancomycin-tolerant strains, and they all lysed with deoxycholate, which indicates autolysin production. Vancomycin tolerance among clinical isolates of pneumococci will necessitate tracking to determine the magnitude of the evolving health risk, since tolerance may contribute to treatment failure (in particular, cases of meningitis, in which bactericidal activity is critical for eradication) and since it may also be a favored background for acquisition of resistance of vancomycin.

Changes in serotype distribution may hamper efficacy of pneumococcal conjugate vaccines in children.

During the last 10 y we have observed an increased incidence of pneumococcal bacteremia in Sweden. In order to study the serotype distribution over time we collected 1136 invasive pneumococcal isolates from 1987, 1992 and 1997 from Swedish microbiological laboratories. Currently, new pneumococcal conjugate vaccines are being considered for introduction in the general childhood vaccination program in several countries, including Sweden. We studied the potential vaccine coverage rate for the new conjugate vaccines among our Swedish invasive isolates. We found that the serotype distribution fluctuated with time and observed a surprisingly low potential coverage rate for the 7-valent vaccine in Sweden, in contrast to other countries. Therefore we argue that pneumococcal conjugate vaccines have to be tailored to suit current, local serotype patterns and most likely will need to be changed over time.

Three-year follow-up of patients with pulmonary tuberculosis in Guinea-Bissau, West Africa.

SETTING: Raoul Follereau Hospital, Bissau, Guinea-Bissau. OBJECTIVE: To study the long-term outcome of patients with bacteriologically verified tuberculosis (TB), with or without human immunodeficiency virus (HIV) co-infection. DESIGN: Sputum samples were collected from all patients referred to the hospital with clinical symptoms of pulmonary tuberculosis. Direct microscopy and culture was performed at the Health Laboratory. Patients with a culture positive for Mycobacterium tuberculosis were followed for 3 years, and underlying factors were analysed regarding the outcome of treatment. A group of sex and age-matched HIV-negative individuals was used as controls. RESULTS: Of 206 bacteriologically verified pulmonary TB patients, 168 were followed up. Antibodies to HIV-2 were found in 33 patients (19.6%); eight patients (4.8%) had antibodies to HIV-1 or showed dual reactivity. Of 149 patients discharged to follow ambulatory treatment, the survival rate of HIV-2-positive patients was 42.3% (11/26) and for HIV-negative patients it was 81.9% (95/116). The difference in survival between HIV-2-positive and HIV-negative patients was highly significant (P < 0.00001). HIV-negative TB patients had a significantly higher mortality than their controls (mortality ratio 3.75, 95% confidence interval 1.58-8.90). Most patients who survived, regardless of HIV status, also became free from symptoms compatible wtih pulmonary TB. CONCLUSION: Although the mortality rate among HIV-positive TB patients was very much higher than among HIV-negative patients, there are weighty arguments for active contact tracing and effective treatment of all TB patients.

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.