Bacillus Calmette-Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control.

OBJECTIVE: Laboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette-Guérin (BCG)). METHODS: Seven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC90. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently. RESULTS: Our panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance. CONCLUSION: BCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.

Comparative genomics of BCG vaccines by whole-genome DNA microarray.

Bacille Calmette-Guérin (BCG) vaccines are live attenuated strains of Mycobacterium bovis administered to prevent tuberculosis. To better understand the differences between M. tuberculosis, M. bovis, and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent M. tuberculosis H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of M. bovis. Five additional regions representing 38 open reading frames were present in M. bovis but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.

Use of geographic and genotyping tools to characterise tuberculosis transmission in Montreal.

SETTING: In Canada, tuberculosis (TB) is increasingly an urban health problem. Montreal is Canada's second-largest city and the second most frequent destination for new immigrants and refugees. OBJECTIVES: To detect spatial aggregation of cases, areas of excess incidence and local 'hot spots' of transmission in Montreal. DESIGN: We used residential addresses to geocode active TB cases reported on the Island of Montreal in 1996-2000. After a hot spot analysis suggested two areas of overconcentration, we conducted a spatial scan, with census tracts (population 2500-8000) as the primary unit of analysis and stratification by birthplace. We linked these analyses with genotyping of all available Mycobacterium tuberculosis isolates, using IS6110-RFLP and spoligotyping. RESULTS: We identified four areas of excess incidence among the foreign-born (incidence rate ratios 1.3-4.1, relative to the entire Island) and one such area among the Canadian-born (incidence rate ratio 2.3). There was partial overlap with the two hot spots. Genotyping indicated ongoing transmission among the foreign-born within the largest high-incidence zone. While this zone overlapped the area of high incidence among Canadian-born, genotyping largely excluded transmission between the two groups. CONCLUSIONS: In a city with low overall incidence, spatial and molecular analyses highlighted ongoing local transmission.

Profound T-lymphocytopenia and cryptococcemia in a human immunodeficiency virus-seronegative patient with disseminated tuberculosis.

A 47-year-old human immunodeficiency virus-seronegative West African man who presented in extremis with cachexia, lymphadenopathy, multiple organ dysfunction, and marked T-lymphocytopenia received the diagnosis of disseminated tuberculosis, cryptococcal pneumonia, and cryptococcemia. His subsequent course and our review of the literature suggest that the profound T-lymphocytopenia and ensuing cryptococcal disease were likely attributable to disseminated tuberculosis.

Fever duration in hospitalized acute pyelonephritis patients.

PURPOSE: To study persistence of fever in treated pyelonephritis with respect to guidelines recommending investigation and modification of therapy after 2 to 3 days of fever. PATIENTS AND METHODS: A retrospective chart review was made of 70 patients hospitalized for febrile pyelonephritis at a community hospital in Canada. RESULTS: Median duration of fever was 34 hours; persistence of fever at 48 and 72 hours was 26% and 13%, respectively. No patients had complications such as intrarenal or perirenal abscess. Prolonged fever was independently associated with increasing baseline creatinine (P = 0.0001), younger age (P = 0.027), and increasing total leukocyte count (P = 0.026). Results of ultrasonography and intravenous urograms were not predictors of fever duration. CONCLUSION: Fever in treated pyelonephritis can take 4 days to resolve, and routine urologic investigation after 2 to 3 days of fever may be unwarranted.

Sauna-induced myocardial ischemia in patients with coronary artery disease.

PURPOSE: Sauna bathing is a popular recreational activity that is generally considered to be safe. However, there have been case reports of adverse cardiac events. We sought to determine whether sauna use caused myocardial ischemia in patients with coronary artery disease. METHODS: Sixteen patients with proven coronary artery disease were submitted to three conditions (rest, exercise, and sauna bathing) with continuous electrocardiographic (ECG) monitoring and regular blood pressure measurements. During each condition, patients were injected with Tc-99 sestamibi followed by nuclear scintigraphic imaging. Perfusion defect scores were calculated in 15 patients. RESULTS: Sauna bathing was well tolerated. There was a mean (+/- SD) increase in heart rate of 32% +/- 20% in the sauna (resting mean heart rate = 60 +/- 9 beats per minute vs sauna mean heart rate = 79 +/- 11 beats per minute, P <0.001) and a 13% +/- 6% drop in systolic blood pressure (resting mean systolic blood pressure = 142 +/- 14 mm Hg vs sauna mean systolic blood pressure = 123 +/- 15 mm Hg, P <0.001). There were no arrhythmias or ECG changes in the sauna. Compared with rest, there was significant ischemia during sauna bathing (average perfusion defect score at rest = -0.44 vs average sauna score = -0.93, P <0.001). The perfusion defect score in the sauna was worse than the resting score in 14 of the 15 patients. Sauna-associated perfusion defect scores were highly correlated with exercise-induced scores (R2 = 0.65, P <0.001). CONCLUSION: In patients with stable coronary artery disease, sauna use is clinically well tolerated but is associated with scintigraphically demonstrated myocardial ischemia.

Comparative genomics of BCG vaccines.

Bacille Calmette-Guérin (BCG) vaccines have been given to more people than any other vaccine. They have also probably resulted in as much controversy as any other vaccine. In clinical trials, the efficacy of BCG vaccination against pulmonary TB has been widely variable. At the same time, a number of investigators have observed phenotypic differences between BCG daughter strains, raising the possibility that differences between BCG products may in some way translate into different outcomes. With recent genomic analysis of BCG strains, it has become possible to piece together the molecular events that have resulted in current BCG vaccines. Between the derivation of BCG in 1921 and the lyophilization of BCG Pasteur 1173 in 1961, there have been at least seven genetic events, including deletions, duplications and a single nucleotide polymorphism. The phenotypic relevance of these changes in BCG vaccines remains to be explored.

Transmission of tuberculosis in San Francisco and its association with immigration and ethnicity.

OBJECTIVE: To determine tuberculosis transmission dynamics in San Francisco and its association with country of birth and ethnicity. METHODS: Restriction fragment length polymorphism (RFLP) typing was performed on Mycobacterium tuberculosis isolates from culture-positive pulmonary tuberculosis patients in San Francisco (1991 through 1996), using IS6110 as a probe. Patients were assigned to clusters based on mycobacterial isolates with identical DNA fingerprints. Clusters were assumed to have arisen from recent transmission. A transmission index was defined as the average number of culture-positive pulmonary tuberculosis cases generated by a single source case and calculated for subgroups. RESULTS: The transmission index was higher in US-born (0.59) than in foreign-born groups (0.21), and was highest in blacks, in particular those aged under 35 years. The increased transmission index among blacks was not explained by smear-positivity, human immunodeficiency virus infection, or increased susceptibility to disease progression. CONCLUSION: US-born tuberculosis cases generated more secondary cases than immigrants. Young blacks appear to be a high-risk group for tuberculosis transmission. These results suggest the need to develop interventions targeted towards this risk group.

Sex differences in the epidemiology of tuberculosis in San Francisco.

SETTING: Worldwide differences in sex-specific tuberculosis case rates remain fundamentally unexplained. OBJECTIVE: To explore various factors that may explain sex differences in tuberculosis incidence rates for San Francisco from 1991-1996. DESIGN: A retrospective epidemiologic analysis of sex-specific tuberculosis incidence rates in San Francisco from 1991-1996. Stratified analyses were performed on age at diagnosis, racial/ethnic group, human immunodeficiency virus (HIV) status, and place of birth. Molecular fingerprinting with IS6110 data was used to study sex differences in the incidence of disease for recently transmitted and reactivated cases of tuberculosis. RESULTS: In the study period, the male to female incidence rate ratio was 2.1 (95% CI 1.9-2.3). Stratified analyses revealed differences in sex-specific rates after the age of 14 and the highest male:female ratios were seen in the US-born, white, and black populations. High ratios were also observed for cases with clustered fingerprints, similar to those observed for the US-born population. In sub-populations with predominantly reactivated cases of tuberculosis, ratios were also above unity after adolescence, but the effect was less pronounced. CONCLUSION: The ongoing transmission of tuberculosis in the US-born population is one of the factors that explains the difference in sex-specific rates of disease in San Francisco. Observed differences in tuberculosis rates between the sexes may be due to a difference in transmission dynamics rather than diagnosis or reporting biases.

Use of multiple markers in population-based molecular epidemiologic studies of tuberculosis.

SETTING: Many epidemiologic studies of tuberculosis are being conducted worldwide. Fingerprinting with a secondary marker in strains with fewer than six IS6110-hybridizing bands enhances the tracking of strains, but its impact on population-level inferences has not been well studied. OBJECTIVE: To investigate the effects of secondary genotyping for low-copy Mycobacterium tuberculosis isolates with polymorphic guanine-cytosine-rich repetitive sequence (PGRS) on epidemiologic inferences in population-based research settings. DESIGN: For San Francisco tuberculosis cases (1991-1996), clusters were defined by IS6110 alone and by PGRS/IS6110 to 1) estimate recent transmission, 2) evaluate the theoretical influence of bacterial population parameters on these estimates, and 3) assess risk factors for recent transmission. RESULTS: Secondary typing on low-copy strains (20.3% of all isolates) decreased the estimate of recent transmission from 29.1% to 25.3% (P = 0.03). The most influential parameters in determining whether supplemental genotyping results in different estimates were the proportion of low-copy strains and the amount of clustering. Risk factors for recent transmission were identical for both definitions of clustering. CONCLUSION: The statistical and inferred effects of secondary genotyping of M. tuberculosis seem to depend on the proportion of low-copy strains in the population. When this proportion is low or when few secondary patterns match, supplemental genotyping may yield minimal insight into population-level investigations.

Comparative studies of antigens of erythrocytic and exoerythrocytic merozoites of Plasmodium lophurae: characterization of a surface glycoprotein from exoerythrocytic merozoites.

Rabbits were immunized with merozoite-enriched preparations of erythrocytic and exoerythrocytic Plasmodium lophurae. The antisera were used to compare antigens of the two types of merozoites. The indirect immunofluorescent antibody test showed the presence of common antigens. The growth of exoerythrocytic parasites was inhibited by the homologous antiserum and to a lesser extent by the antiserum prepared against erythrocytic forms. Cultures of exoerythrocytic parasites as well as their normal host cells were labeled metabolically with 35S-methionine, tritiated proline and glucosamine. Nonidet P-40 extracts of labeled merozoite-enriched preparations, infected cells, and normal cells were immunoprecipitated with the two types of antisera and the immunoprecipitates were analyzed on polyacrylamide gels. The results showed that erythrocytic and exoerythrocytic merozoites have several common proteins. A major difference was a glycoprotein with an approximate molecular weight of 110,000 daltons. This glycoprotein was associated with the surface of exoerythrocytic merozoites and was not recognized by antibodies prepared against erythrocytic forms.

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C.

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.

Laboratory diagnosis for Giardia lamblia infection: A comparison of microscopy, coprodiagnosis and serology.

OBJECTIVE: To evaluate newer techniques such as coproantigen detection and serology in the diagnosis of symptomatic Giardia lamblia infection. DESIGN: Blinded comparison of copro-antigen detection (by ELISA), serology (immunoglobulin IgG and IgM anti-G lamblia by ELISA, and IgG, IgM and IgA by immunoblot) and microscopy in clinical samples. Microscopic findings for three preserved stools were considered the gold standard. SETTING: Travel medicine clinic. POPULATION STUDIED: Adults, post-travel, with gastrointestinal symptomatology. MAIN RESULTS: For 152 previously collected stools, copro-antigen detection had a sensitivity of 73 of 74 (98.6%) and a specificity of 78 of 78 (100%). In clinical samples of 62 patients, eight of the 62 patients (13%) were diagnosed with G lamblia infection on microscopy. Copro-antigen diagnosis was accurate in symptomatic patients, with sensitivity of seven of eight (87.5%) and specificity of 52 of 54 (96.8%). Serology was less accurate. IgG response to G lamblia had sensitivity of four of seven and specificity of 24 of 50 (48%), and IgM response had sensitivity of three of six and specificity 27 of 48 (56%). Western blot had a sensitivity of five of seven and a specificity of 38 of 49 (78%). CONCLUSIONS: Copro-antigen diagnosis of G lamblia is highly accurate in patients with chronic gastrointestinal complaints, while serology is less accurate and appears to be less useful diagnostically.

Mycobacterium tuberculosis transmission over an 11-year period in a low-incidence, urban setting.

SETTING: Montreal, Canada, has a mean annual tuberculosis (TB) incidence of 9 per 100,000 population, 1996-2007. OBJECTIVE: To characterise potential Mycobacterium tuberculosis transmission by patient subgroups defined by age, sex, birthplace, smear and human immunodeficiency virus status, and to estimate the proportion of cases that resulted from transmission between these patient subgroups. DESIGN: Retrospective study using DNA fingerprinting techniques, with clinical and demographic information from the public health department. Among cases with matching fingerprints, a pulmonary index case was identified. The transmission index was defined as the average number of subsequent TB cases generated directly or indirectly from an index case, and was compared among subgroups, including Haitian immigrants. RESULTS: Compared to non-Haitian foreign-born index cases, Canadian-born index cases were associated with 2.38 times as many (95%CI 1.24-4.58) subsequent cases, while Haitian-born index cases were associated with 3.58 times as many (95%CI 1.74-7.36). Smear-positive index cases were not independently associated with increased transmission. However, middle-aged Canadian-born index patients were associated with a disproportionate number of subsequent cases. CONCLUSION: In Montreal, index patients from several high-risk groups are associated with subsequent transmission. This approach can be applied to other low-incidence settings to identify where targeted interventions could potentially further reduce transmission.

Divergence of immunologic and protective responses of different BCG strains in a murine model.

The ongoing evolution of BCG after its introduction in 1921 resulted in strains that differ genetically and phenotypically. Based on a genomic deletion (Region of Difference 2 or RD2) that occurred between 1927 and 1931, BCG strains can be sub-classified by the presence or absence of RD2. The existence of other mutations that distinguish BCG strains precludes simple comparison of RD2-positive and RD2-negative BCG strains to determine the importance, if any, of RD2 for vaccine protection. In this study, we have compared the RD2-containing BCG Russia, BCG Pasteur (which is a natural mutant for RD2), and BCG Pasteur complemented with RD2-genes Rv1979c-Rv1982 through various in vitro and in vivo assays of immunogenicity and protection. We determined that the presence of RD2 did not affect vaccine persistence, but lead to increased immunogenicity, as measured by ELISpot. Additionally, T-cells from animals immunized with BCG Russia and BCG Pasteur::Rv1979c-82 were more effective at killing Mycobacterium tuberculosis in macrophages than T-cells from animals immunized with BCG Pasteur. In a mouse vaccine-challenge model, the presence of RD2 had no effect on pulmonary TB, as measured by M. tuberculosis burden and degree of histopathology, at 4, 8 or 12 weeks post-infection. The presence of RD2 was however associated with decreased dissemination of M. tuberculosis to the spleen. Together, our data demonstrated that the loss of RD2 resulted in decreased immunogenicity but did not affect protection against pulmonary TB, indicating a dissociation between these phenotypes associated with BCG vaccination.

Use of simulated sputum specimens to estimate the specificity of laboratory-diagnosed tuberculosis.

SETTING: Cross-contamination is not uncommon in mycobacteriology laboratories of high-income countries, as documented by bacterial genotyping. The extent of this problem in low-income countries is largely unknown, where this method is impractical. OBJECTIVE: To estimate the rate of cross-contamination in a high-volume tuberculosis (TB) laboratory in South Africa. DESIGN: Simulated sputum specimens labelled with false names were sent from a TB clinic, interspersed with patient samples, and processed for culture and microscopy. Results were interpreted in the context of the observed proportion of samples with positive microscopy and culture results. RESULTS: With microscopy, 6/190 (3.2%) simulated specimens were positive (estimated specificity = 96.8%). Considering the 881 positive microscopy results in 6093 clinical samples, we extrapolate that 19.3% (95%CI 7.0-42.8) of positive smears were false-positives. On culture, 2/190 (1.1%) of the simulated specimens were positive for Mycobacterium tuberculosis (estimated specificity = 98.9%). Considering the 1862 positive cultures from 6093 clinical samples, we estimate that 2.4% (95%CI 0.3-8.8) of positive cultures were false-positives. CONCLUSION: Simulated specimens offer a simple means of estimating the proportion of false-positive results, providing information on all sources of potential error from the clinic, through the laboratory and to reporting of results.

Global tuberculosis trends: a reflection of changes in tuberculosis control or in population health?

BACKGROUND: Many international organizations are advocating for new funds for tuberculosis (TB) specific interventions. Although this approach should help reduce TB incidence, improvements in population health may also be important. We have analyzed the association between changes in population health and health service indicators, and concomitant changes in TB incidence between 1990 and 2005. METHODS: Country level data on population health and health services, economic and epidemiologic indicators were obtained for 165 countries. Regression methods were used to estimate the association of changes in potential predictors with changes in TB incidence. RESULTS: Improvements in population health and health services are associated with improvements in TB outcomes. In adjusted analyses, each 1 year increase in life expectancy was associated with a 7.8/100,000 decline in TB incidence. A 1/1000 decrease in mortality rate in children aged <5 years and a 1% increase in measles vaccination coverage (serving as a general health services indicator) was associated with approximately a 1/100,000 decrease in TB incidence. In countries with a lower prevalence of human immunodeficiency virus (HIV) infection, a 1% increase in TB treatment success rate was also associated with a 1/100,000 decrease in incidence. CONCLUSION: Investment in improving population health and health services may be as important as targeted strategies for controlling TB.

Correlation between BCG genomics and protective efficacy.

Between the derivation of bacille Calmette-Guérin (BCG) vaccine in 1921 and the lyophilization of BCG daughter strains in the 1960s, a number of clinical trials were performed looking at the protective efficacy of BCG vaccination against tuberculosis. These trials differed from one another in a number of ways: they employed different methodologies for delivering the vaccine and interpreting outcomes; they were performed on populations with different genetic backgrounds and different levels of exposure to environmental Mycobacteria; and, finally, they used different BCG vaccine strains. The results of these trials were estimates of protective efficacy against pulmonary tuberculosis ranging from about 80% to nil. Because of the differences in outcomes and confounding variables, it is difficult to conclude whether differences in interventions alone may have contributed to the remarkably variable results. Analysis of BCG vaccines used in clinical trials suggests a trend towards decreasing efficacy with increased passage in the laboratory; however, trials that used relatively "older" BCG strains were generally performed at different sites than trials which used "younger" BCG strains. Genomic analysis of BCG vaccines demonstrates that during the half-century of ongoing passage of BCG vaccines in vitro there have been numerous genetic changes, comprising single nucleotide polymorphisms, duplications and deletions. The impact of these changes in the BCG genome on the protective efficacy observed in field trials remains to be determined.

Molecular fingerprinting of Mycobacterium tuberculosis: how can it help the clinician?

In just a few years, molecular fingerprinting of Mycobacterium tuberculosis has provided clinicians with significant insight into the epidemiology of tuberculosis. This methodology has allowed for a new understanding of the extent of new transmission of tuberculosis among residents of various communities and within institutions. It has also allowed for differentiation between episodes of reinfection and relapse, a task hitherto almost impossible to accomplish. In addition, molecular fingerprinting has allowed assessment of situations where laboratory cross-contamination is suspected. Thus, this technology has in many ways made clinicians reexamine many of their long-held beliefs regarding tuberculosis. In this report, Drs. Behr and Small provide a lucid description of molecular fingerprinting of M. tuberculosis, its current uses, and its future potential value.