First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Genetic diversity of Mycobacterium tuberculosis in Madang, Papua New Guinea

Madang and surroundings, Papua New Guinea (PNG).

Tuberculosis and the risk of opportunistic infections and cancers in HIV-infected patients starting ART in Southern Africa

To investigate the incidence of selected opportunistic infections (OIs) and cancers and the role of a history of tuberculosis (TB) as a risk factor for developing these conditions in HIV-infected patients starting antiretroviral treatment (ART) in Southern Africa.

Humoral response to 2 inactivated bluetongue virus serotype-8 vaccines in South American camelids

BACKGROUND: Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. OBJECTIVE: To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. ANIMALS: Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. METHODS: The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. RESULTS: All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. CONCLUSIONS AND CLINICAL IMPORTANCE: The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.

Impact of antiretroviral therapy on tuberculosis incidence among HIV-positive patients in high-income countries

The lower tuberculosis incidence reported in human immunodeficiency virus (HIV)-positive individuals receiving combined antiretroviral therapy (cART) is difficult to interpret causally. Furthermore, the role of unmasking immune reconstitution inflammatory syndrome (IRIS) is unclear. We aim to estimate the effect of cART on tuberculosis incidence in HIV-positive individuals in high-income countries.

Immunogenicity and safety of seasonal and 2009 pandemic A/H1N1 influenza vaccines for patients with autoimmune diseases: a prospective, monocentre trial on 199 patients

The 2009 pandemic A/H1N1 influenza outbreak represented a theoretical risk for patients with autoimmune diseases (AID), especially those immunosuppressed. This study was undertaken to evaluate immunogenicity and tolerance of seasonal (SFV) and A/H1N1 flu vaccines (HFV) in AID patients.

Reducing tuberculosis incidence by tuberculin skin testing, preventive treatment, and antiretroviral therapy in an area of low tuberculosis transmission

BACKGROUND: Tuberculin skin testing (TST) and preventive treatment of tuberculosis (TB) are recommended for all persons with human immunodeficiency virus (HIV) infection. We aimed to assess the effect of TST and preventive treatment of TB on the incidence of TB in the era of combination antiretroviral therapy in an area with low rates of TB transmission. METHODS: We calculated the incidence of TB among participants who entered the Swiss HIV Cohort Study after 1995, and we studied the associations of TST results, epidemiological and laboratory markers, preventive TB treatment, and combination antiretroviral therapy with TB incidence. RESULTS: Of 6160 participants, 142 (2.3%) had a history of TB at study entry, and 56 (0.91%) developed TB during a total follow-up period of 25,462 person-years, corresponding to an incidence of 0.22 cases per 100 person-years. TST was performed for 69% of patients; 9.4% of patients tested had positive results (induration > or = 5 mm in diameter). Among patients with positive TST results, TB incidence was 1.6 cases per 100 person-years if preventive treatment was withheld, but none of the 193 patients who received preventive treatment developed TB. Positive TST results (adjusted hazard ratio [HR], 25; 95% confidence interval [CI], 11-57), missing TST results (HR, 12; 95% CI, 4.8-20), origin from sub-Saharan Africa (HR, 5.8; 95% CI, 2.7-12.5), low CD4+ cell counts, and high plasma HIV RNA levels were associated with an increased risk of TB, whereas the risk was reduced among persons receiving combination antiretroviral therapy (HR, 0.44; 95% CI, 0.2-0.8). CONCLUSION: Screening for latent TB using TST and administering preventive treatment for patients with positive TST results is an efficacious strategy to reduce TB incidence in areas with low rates of TB transmission. Combination antiretroviral therapy reduces the incidence of TB.

Hepatitis A vaccines and the elderly

Hepatitis A virus (HAV) exposure in unprotected adults may cause severe and serious symptoms, with risk of both morbidity and mortality increasing with age. As seroprevalence of HAV is low in industrialised countries, and an increasing number of people, with an increasing median age, travel from areas of low HAV endemicity to high endemicity, pre-travel vaccination is warranted. Vaccination of the elderly against HAV, however, may be associated with reduced seroprotection, since the immune response decreases with age. Studies with monovalent hepatitis A vaccine or combined hepatitis A and B vaccine show good efficacy in adults in general. Few studies have assessed the immune response in older adults. The only prospective study with monovalent hepatitis A vaccine in the elderly showed a reduced seroprotection of approximately 65% after a single primary dose in subjects over the age of 50 years, while seroprotection was 98% in this age group after receiving a booster dose. The only prospective study with combined hepatitis A and B vaccine in younger subjects or older than 40 years showed similar seroprotection (99-100%) against HAV compared to a monovalent vaccine after receiving three doses. As data on seroprotection for HAV in the elderly are limited, further studies are needed to elucidate how optimal protection in the elderly can be achieved. In the mean time, based on the available data, the suggestion is made to screen elderly travellers to areas endemic for HAV for the presence of naturally acquired immunity, and, if found susceptible, be immunised well in advance of their trip, to allow time for post-vaccination antibody testing and/or administration of a second dose of the vaccine.

CYP2E1 genotype and isoniazid-induced hepatotoxicity in patients treated for latent tuberculosis

OBJECTIVE: To determine whether pharmacogenetic tests such as N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1) genotyping are useful in identifying patients prone to antituberculosis drug-induced hepatotoxicity in a cosmopolite population. METHODS: In a prospective study we genotyped 89 patients treated with isoniazid (INH) for latent tuberculosis. INH-induced hepatitis (INH-H) or elevated liver enzymes including hepatitis (INH-ELE) was diagnosed based on the clinical diagnostic scale (CDS) designed for routine clinical practice. NAT2 genotypes were assessed by fluorescence resonance energy transfer probe after PCR analysis, and CYP2E1 genotypes were determined by PCR with restriction fragment length polymorphism analysis. RESULTS: Twenty-six patients (29%) had INH-ELE, while eight (9%) presented with INH-H leading to INH treatment interruption. We report no significant influence of NAT2 polymorphism, but we did find a significant association between the CYP2E1 *1A/*1A genotype and INH-ELE (OR: 3.4; 95% CI:1.1-12; p = 0.02) and a non significant trend for INH-H (OR: 5.9; 95% CI: 0.69-270; p = 0.13) compared with other CYP2E1 genotypes. This test for predicting INH-ELE had a positive predictive value (PPV) of 39% (95% CI: 26-54%) and a negative predictive value (NPV) of 84% (95% CI: 69-94%). CONCLUSION: The genotyping of CYP2E1 polymorphisms may be a useful predictive tool in the common setting of a highly heterogeneous population for predicting isoniazid-induced hepatic toxicity. Larger prospective randomized trials are needed to confirm these results.

Tuberculosis after initiation of antiretroviral therapy in low-income and high-income countries

We examined the incidence of and risk factors for tuberculosis during the first year of highly active antiretroviral therapy in low-income (4540 patients) and high-income (22,217 patients) countries. Although incidence was much higher in low-income countries, the reduction in the incidence of tuberculosis associated with highly active antiretroviral therapy was similar: the rate ratio for months 7-12 versus months 1-3 was 0.48 (95% confidence interval, 0.36-0.64) in low-income countries and 0.36 (95% confidence interval, 0.26-0.50) in high-income countries. A low CD4 cell count at the start of therapy was the most important risk factor in both settings.

Biological safety concepts of genetically modified live bacterial vaccines

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon gamma assay

OBJECTIVE: To analyse the performance of a new M. tuberculosis-specific interferon gamma (IFNgamma) assay in patients with chronic inflammatory diseases who receive immunosuppressive drugs, including tumour necrosis factor alpha (TNFalpha) inhibitors. METHODS: Cellular immune responses to the M. tuberculosis-specific antigens ESAT-6, CFP-10, TB7.7 were prospectively studied in 142 consecutive patients treated for inflammatory rheumatic conditions. Results were compared with tuberculin skin tests (TSTs). Association of both tests with risk factors for latent M. tuberculosis infection (LTBI) and BCG vaccination were determined and the influence of TNFalpha inhibitors, corticosteroids, and disease modifying antirheumatic drugs (DMARDs) on antigen-specific and mitogen-induced IFNgamma secretion was analysed. RESULTS: 126/142 (89%) patients received immunosuppressive therapy. The IFNgamma assay was more closely associated with the presence of risk factors (odds ratio (OR) = 23.8 (95% CI 5.14 to 110) vs OR = 2.77 (1.22 to 6.27), respectively; p = 0.009), but less associated with BCG vaccination than the TST (OR = 0.47 (95% CI 0.15 to 1.47) vs OR = 2.44 (0.74 to (8.01), respectively; p = 0.025). Agreement between the IFNgamma assay and TST results was low (kappa = 0.17; 95% CI 0.02 to 0.32). The odds for a positive IFNgamma assay strongly increased with increasing prognostic relevance of LTBI risk factors. Neither corticosteroids nor conventional DMARDs significantly affected IFNgamma responses, but the odds for a positive IFNgamma assay were decreased in patients treated with TNFalpha inhibitors (OR = 0.21 (95% CI 0.07 to 0.63), respectively; p = 0.006). CONCLUSIONS: These results demonstrate that the performance of the M. tuberculosis antigen-specific IFNgamma ELISA is better than the classic TST for detection of LTBI in patients receiving immunosuppressive therapy for treatment of systemic autoimmune disorders.

Validation of candidate smear microscopy quality indicators, extracted from tuberculosis laboratory registers

SETTING: Kinshasa Province, Democratic Republic of Congo. OBJECTIVE: To identify and validate register-based indicators of acid-fast bacilli (AFB) microscopy quality. DESIGN: Selection of laboratories based on reliability and variation in routine smear rechecking results. Calculation of relative sensitivity (RS) compared to recheckers and its correlation coefficient (R) with candidate indicators based on a fully probabilistic analysis incorporating vague prior information using WinBUGS. RESULTS: The proportion of positive follow-up smears correlated well (median R 0.81, 95% credibility interval [CI] 0.58-0.93), and the proportion of first smear-positive cases fairly (median R 0.70, 95% CI 0.38-0.89) with RS. The proportions of both positive suspect and low positive case smears showed poor correlations (median R 0.27 and -0.22, respectively, with ranges including zero). CONCLUSIONS: The proportion of positives in follow-up smears is the most promising indicator of AFB smear sensitivity, while the proportion of positive suspects may be more indicative of accessibility and suspect selection. Both can be obtained from simple reports, and should be used for internal and external monitoring and as guidance for supervision. As proportion of low positive suspect smears and consistency within case series are more difficult to interpret, they should be used only on-site by laboratory professionals. All indicators require more research to define their optimal range in various settings.

Demographic characteristics of patients with extrapulmonary tuberculosis in Germany

The aim of the present study was to determine the demographics of patients with extrapulmonary tuberculosis in Germany. Data on 26,302 tuberculosis cases from a national survey carried out during the period 1996-2000 were analysed. The crude proportion of tuberculosis patients with extrapulmonary manifestations was 21.6%. Extrapulmonary tuberculosis was most likely among females, children aged <15 yrs and persons originating from Africa and Asia. Females tended to be more likely to have any form of extrapulmonary tuberculosis than males, except pleural tuberculosis. The strength of this association was strongest in the age range 25-64 yrs and less pronounced amongst the oldest patients. Children were particularly prone to the development of lymphatic and meningeal tuberculosis, whereas the likelihood of genitourinary tuberculosis increased with increasing age. Asian and African patients were generally more likely than persons from other areas to have lymphatic, osteoarticular, meningeal and miliary tuberculosis. The analysis shows important differences, by age, sex and origin, in the likelihood of a tuberculosis patient presenting with extrapulmonary tuberculosis. Since the relative contribution of the foreign-born to tuberculosis in low-prevalence countries is rising, extrapulmonary tuberculosis must be taken into account more often in the differential diagnostic work-up of these patients, particularly among those originating from Asia and Africa.