Air pollution and airborne infection with mycobacterial bioaerosols: a potential attribution of soot.

Atmospheric pollutants are hypothesized to enhance the viability of airborne microbes by preventing them from degradation processes, thereby enhancing their atmospheric survival. In this study, Mycobacterium smegmatis is used as a model airborne bacteria, and different amounts of soot particles are employed as model air pollutants. The toxic effects of soot on aerosolized M. smegmatis are first evaluated and excluded by introducing them separately into a chamber, being sampled on a filter, and then cultured and counted. Secondly, the bacteria-soot mixture is exposed to UV with different durations and then cultured for bacterial viability evaluations. The results show that under UV exposure, the survival rates of the low-, medium-, and high-soot groups are 1.1 (±0.8) %, 70.9 (±4.3) %, and 61.0 (±17.6) %, respectively. This evidence significantly enhanced survival rates by soot at all UV exposures, though the combinations of UV exposure and soot amounts revealed a changing pattern of survival rates. The possible influence by direct and indirect effects of UV-damaging mechanisms is proposed. This study indicates the soot-induced survival rate enhancements of M. smegmatis under UV stress conditions, representing the possible relations between air pollution and the extended pathogenic viability and, therefore, increased airborne infection probability.

Rapid molecular testing for multi-resistant tuberculosis in Mongolia: A diagnostic accuracy study.

OBJECTIVE: The aim of this study was to assess the performance of a molecular line probe assay, GenoType® MTBDRplus, for rapid detection of rifampicin and isoniazid resistance in the Mongolian situation. The sensitivity and specificity of GenoType® MTBDRplus to detect rifampicin and isoniazid resistance-associated mutations in culture specimens and directly in smear-positive clinical specimens was examined. METHOD: 218 MDR-TB subjects aged between 14 and 75years old from eight districts in Ulaanbaatar city (between July 2009 and May 2010) were included in this study .The GenoType Mycobacterium tuberculosis drug resistance first line (MTBDR plus) assay (Hain Life-science, Nehren, Germany) was tested on 109 clinical isolates and directly on 41 sputum specimens for the ability to detect the resistances. Results were compared with conventional culture and drug susceptibility testing on solid medium. RESULTS: The high correlation of the results from GenoType® MTBDRplus and conventional drug susceptibility testing was obtained from this study. The results clearly showed a high performance of GenoType® MTBDRplus with almost 100% accuracy for all the important indicators, such as sensitivity, specificity, positive and negative predictive values and detection of rifampicin resistance. Discrepancies were obtained in comparison with DNA sequencing results. CONCLUSIONS: The Genotype® MTBDRplus assay was demonstrated as a rapid, reliable and highly accurate tool for early detection of MDR-TB through examining smear positive cases.

Nationwide survey of anti-tuberculosis drug resistance in Mongolia.

BACKGROUND: Drug-resistant tuberculosis (TB) has emerged as an obstacle to effective TB control. SETTING: The eight district and 21 province TB dispensaries and the two TB hospitals comprising the diagnostic centers for TB in Mongolia. METHODS: To investigate drug resistance levels among new and retreated TB cases. Specifically, we determined the prevalence of resistance to rifampin, streptomycin, isoniazid and ethambutol among TB patients with and without prior anti-tuberculosis treatment. RESULTS: A total of 850 patients (74.1% of eligible and 78.0% of patients with a specimen) had Mycobacterium tuberculosis isolated and are the subject of this analysis. Of these, 200 had a history of prior treatment and 650 did not. Any multidrug resistance was found in 7.5% (95%CI 5.9-9.5), with respectively 27.5% (95%CI 21.8-34.1) and 1.4% (95%CI 0.7-1.6) of patients with and without prior history of treatment. CONCLUSIONS: The most conspicuous finding in our survey was the relatively low prevalence of multidrug resistance among patients without a history of prior treatment, as compared to very high prevalence among previously treated patients. This suggests that retreatment is deficient and poses a threat to continued transmission, which has not yet manifested itself among new patients.

Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis.

OBJECTIVE: A new loop-mediated isothermal amplification (LAMP) test kit, including a simple DNA extraction device for the detection of Mycobacterium tuberculosis complex, was developed for commercial use and evaluated for its usefulness in diagnosing tuberculosis (TB). DESIGN: The LAMP test was performed using untreated and N-acetyl-L-cysteine (NALC) NaOH-treated sputum specimen. The efficiency of the kit was compared with other conventional laboratory examinations, including other nucleic acid amplification (NAA) tests. RESULTS: The sensitivity of LAMP using raw sputum (direct LAMP) in smear- and culture-positive specimens was 98.2% (95%CI 94.9-99.4), while the sensitivity in smear-negative, culture-positive specimens was 55.6% (95%CI 43.4-68.0). The diagnostic sensitivity of direct LAMP for the diagnosis of individuals with TB was 88.2% (95%CI 81.4-92.7). The sensitivity values of direct LAMP were slightly, but not statistically significantly lower than those of Cobas Amplicor MTB and TRC Rapid MTB, while the sensitivity of the LAMP test using NALC-NaOH treated sputum was significantly lower than other NAA tests (P < 0.05) for smear-negative, culture-positive specimens. The new commercial version of the LAMP kit was easy to handle and yielded results within 1 h of receiving sputum specimens. CONCLUSIONS: This test is considered a promising diagnostic tool for TB, even for peripheral laboratories with limited equipment, such as those in developing countries.

Currents connecting communities: nearshore community similarity and ocean circulation.

Understanding the mechanisms that create spatial heterogeneity in species distributions is fundamental to ecology. For nearshore marine systems, most species have a pelagic larval stage where dispersal is strongly influenced by patterns of ocean circulation. Concomitantly, nearshore habitats and the local environment are also influenced by ocean circulation. Because of the shared dependence on the seascape, distinguishing the relative importance of the local environment from regional patterns of dispersal for community structure remains a challenge. Here, we quantify the "oceanographic distance" and "oceanographic asymmetry" between nearshore sites using ocean circulation modeling results. These novel metrics quantify spatial separation based on realistic patterns of ocean circulation, and we explore their explanatory power for intertidal and subtidal community similarity in the Southern California Bight. We find that these metrics show significant correspondence with patterns of community similarity and that their combined explanatory power exceeds that of the thermal structure of the domain. Our approach identifies the unique influence of ocean circulation on community structure and provides evidence for oceanographically mediated dispersal limitation in nearshore marine communities.

Results of molecular genetic study of m. Tuberculosis strains isolated from specimens of mdr-tb suspects in Ulaanbaatar, Mongolia in 2009-2010 / Монголын Анагаах Ухаан

Bacground: DST by conventional methods takes several weeks, while early diagnosis of the disease and the rapid identification of resistant strains are essential for efficient treatment and control of the MDR strains. Rapid molecular testing of detecting MDR-TB is needed.Objective: The aim of this study was to assess performance of molecular line probe assay, Genotype16 MTBDRp/us, for rapid detection of RIF and INH resistance for M.Tuberculosis in Mongolia. The sensitivity and specificity of Genotype® MTBDRp/us to detect RIF and INH resistance-associated mutations in culture specimens and directly in smear-positive clinical specimens was examined and compared with conventional culture and drug susceptibility testing on solid medium.Material and Methods: The subjects of this study were 218 MDR-TB suspects aged 14-75 years from 8 districts in Ulaanbaatar city. The study was conducted from July 2009 to May 2010. The Genotype M. Tuberculosis drug resistance first line (MTBDR plus) assay (Hain Life-science, Nehren, Germany) was tested on directly on 41 sputum specimens and 109 clinical isolates.Results: The high correlation of the results from Genotype® MTBDRp/us and conventional drug susceptibility testing was obtained from this study. The results clearly show high performance of Genotype® MTBDRp/us with almost 100% accuracy for all the important indicators, such as sensitivity, specificity, positive and negative predictive values of detection of RIF and INH resistance. Some minor discrepancies were obtained in comparison with DNA sequencing results.Our study found that among high proportion for detection of RIF resistance, S531L mutation (MUT3 band) occurred the most commonly, with 80.0% of all RIF-resistant strains (83.6% of MDR) having the mutation. Other mutation in the 530-533 regions was common, as detected by the lack of binding to the WT8 probe in the absence of S531L mutation.In this study we observed that mutations in the promoter region of inhA gene played a major role (67.6 % (63.9% of MDR strains and 90% of INH-mono-resistant strains) had a mutation in the inhA.Conclusion: The Genotype® MTBDRp/us assay was demonstrated as a rapid, reliable and highly accurate tool for early detection of MDR-TB through examining smear positive cases enabling early start of appropriate therapeutic and public health measures to control of the spread of drug resistant M.tuberculosis in the population.

Beijing family Mycobacterium tuberculosis isolated from throughout Japan: phylogeny and genetic features.

To estimate the current population genetic structure of Mycobacterium tuberculosis in Japan, phylogenetic traits were analysed for 237 Beijing family strains isolated from tuberculosis patients throughout the country. Unlike previous reports from other countries, the ancient Beijing sublineage was predominant throughout Japan. Clustering analysis based on JATA-VNTR (Japan Anti-Tuberculosis Association variable numbers of tandem repeats), a specialised set of VNTR for the discrimination of Japanese M. tuberculosis strains, revealed high similarity of the modern Beijing sublineage strains, irrespective of their geographic origin. JATA-VNTR might be useful for the phylogenetic classification in populations where ancient Beijing strains are frequently isolated.

Pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis isolates in Japan.

Thirty-six multidrug-resistant (MDR) Mycobacterium tuberculosis isolates collected in Japan were examined for pyrazinamide susceptibility and pyrazinamidase activity, and analysed by pncA sequencing and a hybridization-based line probe assay (LiPA), which was used to detect pncA mutations for the rapid identification of pyrazinamide-resistant isolates. Pyrazinamide resistance was found in 19 (53%) of them. All pyrazinamide-resistant isolates had no pyrazinamidase activity and at least one mutation in pncA. Among the pncA mutations, 11 had not been previously reported. The results of the LiPA were fully consistent with the DNA sequencing results. A majority of MDR M. tuberculosis isolates in Japan were resistant to pyrazinamide.

The stochastic nature of larval connectivity among nearshore marine populations.

Many nearshore fish and invertebrate populations are overexploited even when apparently coherent management structures are in place. One potential cause of mismanagement may be a poor understanding and accounting of stochasticity, particularly for stock recruitment. Many of the fishes and invertebrates that comprise nearshore fisheries are relatively sedentary as adults but have an obligate larval pelagic stage that is dispersed by ocean currents. Here, we demonstrate that larval connectivity is inherently an intermittent and heterogeneous process on annual time scales. This stochasticity arises from the advection of pelagic larvae by chaotic coastal circulations. This result departs from typical assumptions where larvae simply diffuse from one site to another or where complex connectivity patterns are created by transport within spatially complicated environments. We derive a statistical model for the expected variability in larval settlement patterns and demonstrate how larval connectivity varies as a function of different biological and physical processes. The stochastic nature of larval connectivity creates an unavoidable uncertainty in the assessment of fish recruitment and the resulting forecasts of sustainable yields.

Performance of an interferon-gamma release assay for diagnosing latent tuberculosis infection in children.

Newly developed interferon-gamma release assays have become commercially available to detect tuberculosis (TB) infection in adults. However, little is known about their performance in children. We compared test results between the QuantiFERON-TB Gold test (QFT) and tuberculin skin test (TST) in young children living with pulmonary TB patients in Cambodia. Of 195 children tested with both QFT and TST, the TST-positive rate of 24% was significantly higher than the QFT-positive rate of 17%. The agreement between the test results was considerable (kappa-coefficient 0.63). Positive rates increased from 6% to 32% for QFT and from 15% to 43% for TST, according to the sputum smear grades of the index cases. The presence of Bacille Calmette-Guérin (BCG) scars did not significantly affect the results of TST or QFT in a logistic regression analysis. In conclusion, QFT can be a substitute for TST in detecting latent TB infection in childhood contacts aged

Nationwide survey on the prevalence of anti-tuberculosis drug resistance in the Republic of Yemen, 2004.

OBJECTIVES: To determine the prevalence of resistance to the four major anti-tuberculosis drugs, isoniazid, rifampicin, streptomycin and ethambutol, in Yemen. METHODS: Cluster sampling with probability proportionate to size was applied. Susceptibility to four major anti-tuberculosis drugs was examined. The proportion method using Löwenstein-Jensen medium or Ogawa medium was carried out. RESULTS: A total of 790 primary culture isolates from tuberculosis (TB) cases enrolled at the National Tuberculosis Institute, Yemen, were examined. In the confirmation culture at the supranational reference laboratory, 227 of them failed to grow on the secondary culture or were proved to be mycobacteria other than Mycobacterium tuberculosis and were excluded from further analysis. Among 563 cultures, 510 were obtained from new cases and 53 from previously treated cases. The prevalence of resistance to any four drugs was 9.8% (95%CI 7.0-12.5) among new cases and 17.4% (95%CI 12.0-33.5) among previously treated cases. The prevalence of multidrug-resistant TB was 3.0% (95%CI 1.5-4.5) among new cases and 9.4% (95%CI 0.2-18.7) among previously treated cases. CONCLUSION: The first nationwide prevalence survey on resistance to the four major anti-tuberculosis drugs in Yemen showed a relatively low prevalence of drug-resistant cases, but a high prevalence of multidrug resistance among new cases.

Anti-tuberculosis drug susceptibility testing of Mycobacterium bovis BCG Tokyo strain.

SETTING: The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine is the only vaccine against tuberculosis (TB), owing to its valuable protective effects and low virulence. However, it can occasionally cause systemic infection in immunocompromised hosts. Isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB) are known to be effective anti-tuberculosis drugs and are used for the treatment of BCG infections. Unfortunately, there are few studies of the susceptibility of BCG vaccine strains to these drugs. OBJECTIVE: To measure the minimum inhibitory concentrations (MICs) of BCG Tokyo vaccine products for anti-tuberculosis drugs and assess vaccine safety in terms of drug susceptibility. DESIGN: We measured the MIC for one seed and five product lots of BCG Tokyo strain for INH, RMP, SM and EMB using Middlebrook 7H11 agar plates. RESULTS: The MIC results for INH were 0.06 and 0.125 mg/ml for the product and seed lots, respectively. The MIC results for RMP, SM and EMB were 0.25-0.5, 0.25 and 2-4 microg/ml, respectively. CONCLUSION: Our results indicate that the BCG Tokyo strain was susceptible to the major anti-tuberculosis drugs and treatable even in cases of severe adverse events, including systemic infection.

Tuberculosis and multidrug resistance in Zambian prisons, 2000-2001.

BACKGROUND: Data on prevalence of tuberculosis (TB) and multidrug-resistant tuberculosis (MDR-TB) in Zambian prisons are lacking. METHODS: Between January 2000 and July 2001, a case-finding study was performed in 13 Zambian prisons for pulmonary TB. Prisoners were administered a questionnaire to obtain demographic information. Information regarding housing density and diet was also collected. Three consecutive first morning sputum specimens were cultured for Mycobacterium tuberculosis. Antimicrobial resistance testing was performed by the resistance ratio method. RESULTS: A total of 1080 prisoners were recruited: 1055 were males and 25 females. Sputum from 245 (22.7%) prisoners yielded M. tuberculosis, including 168 (15.6%) with smear-positive disease. Based on a total prison population of 6118, the minimal prevalence of TB was 4.0%. There was a linear relationship between the proportion of prisoners evaluated and the prevalence of TB (R(2) = 0.9366) across facilities, suggesting that the true prevalence of TB may approach 15-20%. Resistance to at least one anti-tuberculosis drug was detected for 40 (23.8%) isolates, while MDR-TB was identified for 16 (9.5%) isolates. CONCLUSION: There is a high rate of pulmonary TB in Zambian prisons, with significant rates of drug resistance and MDR-TB, highlighting the need for active surveillance and treatment programs.

Preparation of mycobacteria-containing artificial sputum for TB panel testing and microscopy of sputum smears.

SETTING: Panel testing, blinded cross rechecking and on-site evaluation are the three methods for external quality assessment (EQA) of acid-fast bacilli (AFB) smear microscopy. Panel testing can provide data on laboratory capabilities prior to implementing a rechecking programme, assess the current status of laboratory performance and detect problems associated with diagnostic performance. Thus far, two methods for preparing panel test slides have been reported: these use real AFB-positive and -negative sputum treated with sodium hydroxide (NaOH) or N-acetyl-L-cysteine (NALC). OBJECTIVE: To evaluate the above methods and to develop a new method to prepare panel test slides with artificial sputum. DESIGN: Panel test slides were prepared using the NaOH and NALC methods. New artificial sputum preparation methods were developed and examined using a cultured monocyte cell line, cultured avirulent mycobacteria and methylcellulose or polyacrylamide gel as substrate. Smears prepared by the four methods were compared. RESULTS: Panel test slides prepared with NaOH and NALC methods are not macroscopically or microscopically similar to real smears. Our new artificial sputum is similar to real sputum in viscosity and macroscopic and microscopic appearance; it is also consistent in panel positivity grades. CONCLUSION: The artificial sputum described here could contribute to the EQA and training in tuberculosis laboratories or microscopy centres.

Comparative study on the use of solid media: Löwenstein-Jensen and Ogawa in the determination of anti-tuberculosis drug susceptibility.

A total of 53 Mycobacterium tuberculosis strains were examined to evaluate the consistency of susceptibility tests using two solid egg-based culture media, Ogawa and Löwenstein-Jensen (L-J), in order to assess the inter-media differences in susceptibility tests for anti-tuberculosis drugs. Fifty-three M. tuberculosis strains were tested for resistance against isoniazid, rifampicin, ethambutol, streptomycin and other alternative drugs on L-J and Ogawa media. Data from isoniazid (INH) revealed 31 strains were susceptible and 11 strains were resistant in L-J medium. On the other hand, one strain showed resistance against INH only on Ogawa medium. This gave a consistency ratio of 97.7% for INH between these two media. Similarly, the methods had 100% compatibility with rifampicin, 86.0% with ethambutol and 88.4% with streptomycin. When 41 of these strains were tested against ethionamide, enviomycin, para -aminosalicylate, sparfloxacin, and levofloxacin, the same numbers of strains were shown to be resistant or susceptible to L-J and Ogawa media. Similarly, the consistency ratio was 75.6% for kanamycin and 61.0% for capreomycin. There are inconsistencies between results obtained with L-J and Ogawa media, Ogawa medium had a tendency to produce results that indicated more resistant strains. However, the differences were statistically negligible.

Evaluation of PCR-based methods for the diagnosis of tuberculosis by identification of mycobacterial DNA in urine samples.

SETTING: The Chest Clinic and the JICA (Japan International Cooperation Agency) Molecular Laboratories, University Teaching Hospital, Lusaka, Zambia, and the Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan. OBJECTIVE: To evaluate the polymerase chain reaction (PCR) as a laboratory test for the rapid diagnosis of pulmonary tuberculosis in the African situation by identifying mycobacterial DNA in urine samples using two commonly described molecular methods. DESIGN: Prospective collection and laboratory analysis of urine samples from adult Zambian patients with culture-confirmed pulmonary tuberculosis and healthy controls. METHODS: Urine was obtained from 63 patients with culture-confirmed active pulmonary tuberculosis and 63 'healthy' control patients with no active tuberculosis. DNA was isolated from urine sediment and subjected to analyses by two well-described PCR-based methods, 'the Sechi method' and 'the Githui method', for the identification of Mycobacterium tuberculosis DNA. The sensitivity and specificity of the two tests were determined. RESULTS: The sensitivity and specificity of the Githui method were 55.6% (35/63) and 98.4% (62/63), respectively. The sensitivity and specificity of the Sechi method were 28.6% (18/63) and 98.4% (62/63), respectively. Of the 63 patients, 50 (79%) were HIV sero-positive and the frequency of positive PCR urines using the Githui method was greater in HIV-positive patients than in HIV-negative patients (32/50 = 64% vs. 3/13 = 23%; P = 0.05). CONCLUSIONS: Neither the Githui method nor the Sechi method was sensitive enough to be recommended for routine use in clinical practice. PCR-based assays for the detection of M. tuberculosis DNA in urine will require further refinement before they can be recommended for use in clinical practice in Africa. The presence of mycobacterial DNA in urine samples of patients with pulmonary tuberculosis also requires further study.

Potential use of Amplicor PCR kit in diagnosing pulmonary tuberculosis from gastric aspirate.

Polymerase chain reaction (PCR) detection of mycobacteria from gastric aspirate for the diagnosis of tuberculosis is not fully evaluated up to now. A total of 116 gastric aspirate specimens were collected from patients with suspected pulmonary tuberculosis. The breakdown of diagnosis was 67 pulmonary tuberculosis, 16 nontuberculous mycobacterial infection, 5 extra pulmonary tuberculosis, and 28 other lung diseases. The conventional methods were shown to have a sensitivity of 47.8% and a specificity of 79.6%; on the other hand, Amplicor had 34.9% and 97.0%, respectively. The Amplicor provided a more rapid and specific method for diagnosing tuberculosis and was more useful than the conventional.

Amebiasis in Japanese homosexual men with human immunodeficiency virus infection.

We report cases of amebiasis in 6 human immunodeficiency virus (HIV)-positive male patients. Five were confirmed homosexuals while one was suspected. Three patients had liver abscess and 5 had colitis with duration of 10 days to months. The patients with liver abscess showed a lower incidence of abdominal pain but a higher incidence of concomitant diarrhea. Drainage therapy was effective for rapid afebrile results. Two invasive colitis cases died from perforation. This may have been due to delayed diagnosis. Invasive amebiasis is not common even in HIV-infected individuals. Among Japanese homosexual men, however, it may cause symptomatic diseases.