Genotyping of Mycobacterium leprae on the basis of the polymorphism of TTC repeats for analysis of leprosy transmission

The polymorphism of TTC repeats in Mycobacterium leprae was examined using the bacilli obtained from residents in villages at North Maluku where M. leprae infections are highly endemic (as well as from patients at North Sulawesi of Indonesia) to elucidate the possible mode of leprosy transmission. TTC genotypes are stable for several generations of passages in nude mice footpads and, hence, are feasible for the genotyping of isolates and epidemiological analysis of leprosy transmission. It was found that bacilli with different TTC genotypes were distributed among residents at the same dwelling in villages in which leprosy is endemic and that some household contacts harbored bacilli with a different genotype from that harbored by the patient. Investigations of a father-and-son pair of patients indicated that infections of bacilli with 10 and 18 copies, respectively, had occurred. Genotypes of TTC repeats were found to differ between a son under treatment and two brothers. These results reveal the possibility that in addition to exposure via the presence of a leprosy patient with a multibacillary infection who was living with family members, there might have been some infectious sources to which the residents had been commonly exposed outside the dwellings. A limited discriminative capacity of the TTC polymorphism in the epidemiological analysis implies the need of searching other useful polymorphic loci for detailed subdivision of clinical isolates.     

Development of a 32P-postlabelling method for the detection of 1,N 2-propanodeoxyguanosine adducts of crotonaldehyde in vivo

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.     

An epidemiological study on Mycobacterium leprae infection and prevalence of leprosy in endemic villages by molecular biological technique.

One of the most important unsolved questions in epidemiology of leprosy is the highly uneven geographic distribution of the disease. There are many hyperendemic "pockets" in endemic countries. Little is known about the reasons why leprosy is hyperendemic in these areas. We conducted, therefore, a series of epidemiological studies on Mycobacterium leprae infection and prevalence of leprosy in North Maluku district, Maluku Province, Indonesia where leprosy is highly endemic. It was found that considerable number of general inhabitants are seropositive to various mycobacterial antigens and 27% of the villagers were carrying leprosy bacilli on their surface of nasal cavity. These results suggested the importance of M. leprae in the residential environment in infection of the leprosy bacillus and the resulting transmission of the disease. Based on these observations, we conclude that new preventive measures are essential for global elimination of leprosy in addition to early diagnosis and multidrug therapy (MDT).     

Heterogeneity Analysis of 18F-FDG Uptake in Differentiating Between Metastatic and Inflammatory Lymph Nodes in Adenocarcinoma of the Lung: Comparison with Other Parameters and its Application in a Clinical Setting

Purpose

Lymph node (LN) characterization is crucial in determining the stage and treatment decisions in patient with lung cancer. Although ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) has a higher diagnostic accuracy in LN characterization than anatomical imaging, differentiating between metastatic and inflammatory LNs is still challenging because both could show high ¹⁸F-FDG uptake. The purpose of this study was to assess if the heterogeneity of the ¹⁸F-FDG uptake could help in differentiating between inflammatory and metastatic LNs in lung cancer, and to compare with other parameters.

Methods

A total of 44 patients with adenocarcinoma of the lung, who underwent preoperative ¹⁸F-FDG PET/CT without having any previous treatments and were revealed to have ¹⁸F-FDG-avid LNs, were enrolled. There were 52 pathology-proven metastatic lymph nodes in 26 subjects. The pathology-proven metastatic LNs were compared with 42 pathology-proven inflammatory/benign LNs in 18 subjects. The coefficient of variation (CV) was used to assess the heterogeneity of ¹⁸F-FDG uptake by dividing the standard deviation of standardized uptake value (SUV) by mean SUV. The volume of interest was manually drawn based on the combined CT images of ¹⁸F-FDG PET/CT (no threshold is used). Comparisons were made with the maximum standardized uptake values (SUVmax), visual assessment of ¹⁸F-FDG uptake, longest diameter, and maximum Hounsfield units (HUmax).

Results

Metastatic lymph nodes tended to have higher CVs than the inflammatory LNs. The mean CV of metastatic LNs (0.30 ± 0.08; range: 0.08–0.55) was higher than that of inflammatory LNs (0.17 + 0.06; range, 0.07–0.32; P < 0.0001). On receiver operating characteristic (ROC) curve analysis, the area under curve was 0.901, and using 0.20 as cut-off value, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 88.5 %, 76.2 %, 82.2 %, 84.3, and 83.0 % respectively. Accuracy of CV was slightly higher than SUVmax and diameter, but significantly higher than visual assessment and HUmax.

Conclusions

In patients with adenocarcinoma of the lung having no prior treatments, metastatic LNs showed more heterogeneous ¹⁸F-FDG uptake than inflammatory LNs. Measuring the CV of the SUV derived from a manual volume of interest (VOI) can be helpful in determining metastatic LN of adenocarcinoma of the lung. Including diagnostic criteria of CV into the diagnostic approach can increase the accuracy of mediastinal node status.     

The frequency of drug resistance mutations in Mycobacterium leprae isolates in untreated and relapsed leprosy patients from Myanmar, Indonesia and the Philippines

INTRODUCTION: The magnitude of drug resistance in Mycobacterium leprae to dapsone, rifampicin, and ofloxacin was studied in three Southeast Asian countries with a high prevalence of leprosy. METHODS: M. leprae from the skin of leprosy patients was collected in North Maluku and North Sulawesi in Indonesia, Yangon in Myanmar, and Cebu in the Philippines. Mutations in the drug resistance determining regions in the folP1, rpoB, and gyrA genes, which have been proven to confer resistance, were analysed. In addition, samples from 51 newly diagnosed cases and 13 patients with leprosy relapse in Cebu were submitted for susceptibility testing in the mouse footpad. RESULTS: Of 252 isolates obtained from new cases, 3% were dapsone resistant and 2% were rifampicin resistant. In samples taken from patients with relapsed leprosy (n = 53), significantly more resistance mutations were detected: 15% had dapsone resistance mutations, and 8% had rifampicin resistance mutations. Two patients with relapsed leprosy had mutations for both dapsone and rifampicin resistance. No mutations conferring quinolone resistance were detected. No mutations were detected in the folP1 gene of M. leprae isolates with a low degree of resistance to dapsone. DISCUSSION: Detection of drug-resistant cases by mutation detection in the drug resistance determining region of the genome is a practical method for monitoring resistance. A comparison of the results obtained in this study with previous data obtained prior to the use of multidrug therapy (MDT), does not indicate clearly whether the magnitude of drug resistance has changed. Larger studies of resistance mutations in M. leprae isolated from patients with relapsed leprosy are needed to confirm our results. CONCLUSION: We recommend monitoring the magnitude of drug resistance globally, by testing M. leprae DNA from relapse cases and a representative sample of new cases.     

Detection of 1,N(2)-propanodeoxyguanosine adducts in DNA of Fischer 344 rats by an adapted (32)P-post-labeling technique after per os application of crotonaldehyde

Crotonaldehyde is an important industrial chemical to which humans and animals are ubiquitously exposed. The main intake occurs via food, tobacco smoke and possibly also via beverages. Estimation of intake via the different routes is difficult since the data available on exposure are inconsistent. Crotonaldehyde is genotoxic, mutagenic and carcinogenic and forms 1,N(2)-propanodeoxyguanosine adducts as the main DNA adducts. We have developed a (32)P-post-labeling method for these adducts based on nuclease P1 enrichment and polyethyleneimine-cellulose TLC which allows reliable detection with a detection limit of 3 adducts/10(9) nucleotides, a labeling efficiency of 80-90% and a recovery of 38%. Using this method we found crotonaldehyde adducts in different organs of Fischer 344 rats after a single gavage of high doses of 300 and 200 mg/kg body wt in the range 0.3-3.2 +/- 0.4 adducts/10(8) nucleotides and after repeated gavage of low doses of 10 and 1 mg/kg body wt (five times a week for 6 weeks) 6.2 +/- 0.2 and 2.0 +/- 0.4 adducts/10(8)nucleotides, but not in untreated animals nor in calf thymus DNA not treated with crotonaldehyde. In contrast to our results, Chung and co-workers found adducts in tissue of untreated Fischer 344 rats. This discrepancy could depend on the different methods used but also on differences in exposure of the animals via food or due to animal housing, etc.     

Cancer risk assessment for crotonaldehyde and 2-hexenal: an approach

Crotonaldehyde and 2-hexenal are bifunctional compounds that form 1,N2-propanodeoxyguanosine adducts and are mutagenic and genotoxic; crotonaldehyde is carcinogenic. Analysis of the mutations resulting from crotonaldehyde-induced DNA damage revealed the importance of deoxyguanosine adducts. Humans are exposed ubiquitously to these compounds by various routes. The highest daily intake of crotonaldehyde is assumed to be derived from cigarette smoke (31-169 micrograms/kg body weight), and the highest intake of 2-hexenal is probably from fruit and vegetables (31-165 micrograms/kg body weight per day). Because these compounds are suspected to play on important role in carcinogenicity, we developed sensitive 32P-postlabelling techniques for DNA adducts of crotonaldehyde and hexenal, in order to improve estimates of cancer risk. The respective standards were also synthesized and characterized spectroscopically. We report here the results of the 32P-postlabelling, e.g. the stability of the adducts in respect of nuclease P1 treatment, their labelling efficiencies, thin-layer chromatography of adduct spots and the recoveries and detection limits. In untreated male Fischer 344 rats, neither crotonaldehyde nor 2-hexenal adducts were detected, but crotonaldehyde adducts were found in the tissues of rats given single doses of 200 or 300 mg/kg body weight and in the livers of rats after repeated doses of 1 or 10 mg/kg body weight. The adduct levels were higher 20 h after gavage than after 12 h. The adducts persist to a certain extent. 2-Hexenal adducts were detected in tissues of male Fischer 344 rats after gavage with single doses of 50, 200 or 500 mg/kg body weight. The highest adduct levels were measured 48 h after gavage, but no adducts were found 8 h after gavage. Two approaches for cancer risk estimation are discussed. One is based on the correlation between the covalent binding index, calculated from adduct levels, and the median toxic dose (TD50) (Lutz, 1986) and showed a cancer risk of 1 per 10(7) lives for hexenal, assuming dietary intakes of 31-165 micrograms/kg body weight per day. The other is based on a cancer incidence of 0.07 at a dose of crotonaldehyde of 4.2 mg/kg body weight per day assessed from the study of Chung et al. (1986), which can be interpreted as a risk of 5.8-18 new cases per 10(4) smokers, assuming a consumption of 30 cigarettes per day. The latter approach may, however, lead to an overestimate of the cancer risk associated with exposure to crotonaldehyde; the estimate based on our binding studies resulted in a 20-fold lower estimate of the carcinogenic risk of crotonaldehyde.     

Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.     

Diversity of potential short tandem repeats in Mycobacterium leprae and application for molecular typing

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multi-case households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.     

The Value of SPECT/CT in Localizing Pain Site and Prediction of Treatment Response in Patients with Chronic Low Back Pain

In many circumstances, causing sites of low back pain (LBP) cannot be determined only by anatomical imaging. Combined functional and morphological imaging such as bone scan with single-photon emission computed tomography/computed tomography (SPECT/CT) may be helpful in identifying active lesions. The purpose of this study was to evaluate the usefulness of bone SPECT/CT in localizing the pain site and the treatment of chronic LBP. One hundred seventy-five patients suffering from chronic LBP who underwent SPECT/CT were included, retrospectively. All of the patients received multiple general treatments according to the symptoms, and some of them underwent additional target-specific treatment based on SPECT/CT. Numerical rating scale (NRS) pain score was used to assess the pain intensity. Of 175 patients, 127 showed good response to the given therapies, while the rest did not. Overall, 79.4% of patients with definite active lesions showed good response. Patients with mild active or no lesions on SPECT/CT had relatively lower response rate of 63.0%. Good response was observed by the treatment with the guidance of active lesions identified on SPECT/CT. SPECT/CT could be useful in identifying active lesions in patients with chronic LBP and guiding the clinicians to use adequate treatment.

Graphical Abstract

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