A culture medium for cultivation of mycobacteria, probably Mycobacterium leprae from Mycobacterium leprae infected tissues

Mycobacterium leprae suspensions were prepared from infected armadillos. The M. leprae cells were inoculated into culture media containing KH2PO4 4.7. g. Na2HPO4 2 g, sodium thioglycolate 1 g, (NH4)2SO4 2 g, MgSO4 0.1 g, ferric ammonium citrate 0.05 g, and lipoic acid (thioctic acid) 0.1 g in one liter distilled water. The solution was enriched with heat killed, sonicated leprosy derived Mycobacterium X or crude mycobactin extract from M. phlei to contain + 0.2 micrograms mycobactin per 1 ml in the final medium. Twenty ml media was distributed into each of 25 ml screw cap tubes and autoclaved for 30 minutes. Positive growth was obtained from seven out of ten specimens when incubated at 34 degrees C. The cultures developed as a sediment in the liquid media, suggesting preference for microaerophylic conditions. No growth was seen on the surface of the semi-solid agar media containing the same ingredients. Latency period of growth was estimated as 10-16 days and time of division as 6 days. Subcultures were obtained. Cells were long, acid fast, arranged side by side or end to end, with a tendency to form long spiral cords or clumps when sedimented on siliconized slides. Pyridine extraction eliminated acid fastness, but not gram positivity. Cultures did not grow on Dubos, Lowenstein or 7H10 media. They produce the disease in the foot pads of mice characteristic of M. leprae. Subcultures remain dependent on the heat killed sonicated mycobacteria, or crude mycobactin extract, and reduced oxygen tension in the media. Results suggest that cultures might be identical to M. leprae.     

Evaluation of Mycobacterium leprae particle agglutination test, using eluates of filter paper blood spots.

A comparison of the ELISA test with the newly-developed MLPA test was carried out, using eluates of blood spots from filter paper for the detection of the anti-PGL-I antibody. A very good positive correlation was observed between these two tests. The concordance rate was found to be 92.6%, ranging from 71.4% to 100%. This nonconcordance was not found when freshly-collected samples were used. The MLPA test is simple and reliable. The use of eluates from blood spots collected on filter paper further simplifies the test in the collection and transportation of blood samples. This accurate and rapid method makes the MLPA test logistically feasible for large-scale screening. With our modification of MLPA with eluates more samples can be screened with the kit than with sera.     

云贵川三省麻风菌株基因分型的比较研究

目的:了解云南、四川、贵州三省麻风菌株基因型的地理分布。方法:从云南省丘北县、四川省凉山州、贵州省兴义市分别采集59、21、31例麻风患者皮损活检,提取麻风菌DNA,对6-7、AC9、GTA9 3个数目可变的串联重复(variable-number tandem repeat,VNTR)位点的PCR产物测序,确定各位点重复序列数进行分型。结果:三省在6-7位点上有5种基因型,云南以7个拷贝的基因型为主,其它两地区的菌株在该位点的等位基因型为7、8、9,其分布无显著性差异。AC9位点上有4种基因型,三省均以8个拷贝的基因型为主。GTA9位点有非常明显的多样性。云南GTA9为9个拷贝的菌株占总菌株数的18.18%。结论:本研究结果提示麻风菌的基因型与地理分布有关。但需要以更多的位点,了解以VNTR为主的基因分型能否应用于麻风病的传染源和传播链的流行病学研究。     

Transmission of viable Mycobacterium leprae by Aedes aegypti from lepromatous leprosy patients to the skin of mice through interrupted feeding

Female Aedes aegypti which took partial blood meals from the skin lesions of untreated lepromatous leprosy (LL) patients were then allowed to continue feeding on 72-96-hr-old Swiss albino suckling mice (Rockefeller strain). The bitten portion of skin was removed, divided into two parts and processed for the extraction of bacilli by two different methods using chloroform and petroleum ether. The proboscis of some of the fed mosquitoes was dissected out and examined for viable bacilli (stained by fluorescein diacetate and ethidium bromide) and acid-fast bacilli (AFB). Out of 50 probosces dissected 45 were found positive for AFB, with bacillary counts ranging up to 246 (average 40.20 +/- SD 41.80) per proboscis. The average percentage of viable bacilli (green solid) in the probosces immediately after feeding on LL patients was 43.90 and thereafter it decreased gradually to 3 on the seventh day. In the petroleum ether extract of mouse skin viable bacilli were observed in numbers up to 37 (average 15.25 +/- SD 10.25) per smear. The number of fluorescing bacilli (green and red) correlated with the total number of AFB.     

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.     

IgM and IgG antibodies to phenolic glycolipid I from Mycobacterium leprae in leprosy: insight into patient monitoring, erythema nodosum leprosum, and bacillary persistence

Serum IgM and IgG antibodies against Mycobacterium leprae-derived phenolic glycolipid I (PG) were determined in leprosy patients, contacts, and controls by enzyme-linked immunosorbent assay (ELISA). Anti-PG IgM levels increased from the tuberculoid (TT) to the lepromatous (LL) pole of the disease spectrum. There was a positive linear correlation between anti-PG IgM and bacillary index (BI). Patients with erythema nodosum leprosum (ENL) had lower levels of serum anti-PG IgM than non-ENL patients of comparable BI, suggesting that anti-PG IgM is involved in the pathogenesis of ENL. Initial observations indicate that high anti-PG IgM levels in bacillary-negative patients might reflect bacillary persistence. A study of 2 different substrate reagents in the ELISA [2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 0.1 mM H2O2, serum diluted 1:20, and o-phenylenediamine (OPD), 5 mM H2O2, serum diluted 1:300] showed generally good correlation in detection of anti-PG IgM. However the OPD system detected more paucibacillary disease (BT), while the ABTS system detected the significant effect of ENL on the relationship between BI and anti-PG IgM. Anti-PG IgM was clearly dominant over anti-PG IgG. However, certain patients, including several patients who had upgraded from LL and borderline lepromatous leprosy (BL), showed high levels of anti-PG IgG. Since studies have shown that LL patients are selectively deficient in cell-mediated immunity, T-cell products may be required for the IgM to IgG isotype switch. We conclude that anti-PG IgM is useful for monitoring the bacillary load in individual patients and should prove useful for leprosy control strategies.     

Study of cytokine response against panel of purified Mycobacterium leprae antigens by using whole blood assay in subjects residing in a resettlement village of cured leprosy patients

Mycobacterium leprae being an intracellular pathogen, cell mediated immunity is very important in the clinical outcome of leprosy. Manifestation of the disease is correlated with the level and type of cell mediated immune response. The main objective of this study was to analyse TNF-alpha and IFN-gamma production by T-cells when challenged with different M. leprae purified antigens in subjects with known exposure. 50 subjects residing in resettlement village of cured leprosy patients were included in the study. Whole Blood assay studies were undertaken in which the blood was placed in culture and was challenged with 35-kDa antigen, whole M. leprae cells, M. leprae cell wall antigen and M. leprae soluble antigen minus LAM. T-cell derived cytokines TNF-alpha and IFN-gamma were measured by ELISA. It was observed that challenging the lymphocytes with 35-kDa antigen, the cell wall antigen and M. leprae soluble antigen minus LAM resulted in increased levels of IFN-gamma whereas challenge with 35-kDa antigen and M. leprae cell wall antigen resulted in increased levels of TNF-alpha.     

Serological detection of leprosy employing Mycobacterium leprae derived serine-rich 45 kDa, ESAT-6, CFP-10 and PGL-I: a compilation of data from studies in Indian populations

This article is a compilation of our findings recorded in the recent past where we have investigated the serological performance of Mycobacterium leprae antigens like-serine-rich 45 kDa protein (45 kD), early secretary antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) and phenolic glycolipid-I (PGL-I) for detection (employing antibody detecting ELISA) of leprosy patients, particularly those belonging to the paucibacillary (PB) group. All of these antigens were capable of detecting, by themselves the majority (82-100%) of multibacillary (MB) patients. However, with respect to PB patients, only 18-47% (i.e. less than half) of the cases could be detected. Based on the results of serological assays for each of the four antigens separately a combinatorial approach was performed for these antigens, which increased the sensitivity for detection of PB patients to 73%, giving 36% improvement over conventional PGL-I based ELISA. Thus, the multi-antigenic serological approach is worthwhile for its establishment for detection of leprosy patients. Since ESAT-6 and CFP-10 are secreted proteins by nature, antibodies against them are worth exploring for detection of early infections and for monitoring of treatment efficiency. Nevertheless, efforts towards identification of more new antigens with serological potential are still desirable in order to further improve the detection rate of leprosy.     

Relationships between PGL-1 antigen in serum, tissue and viability of Mycobacterium leprae as determined by mouse footpad assay in multibacillary patients during short-term clinical trial

In connection with a 56-day controlled clinical trial for comparing the therapeutic effects between pefloxacin and ofloxacin in 21 lepromatous patients, we have studied the relationships between PGL-1 antigen level in serum and in skin and serum PGL-1 antibody titre on the one hand, and the viability of Mycobacterium leprae, as measured by serial mouse footpad inoculations, and other bactericidal parameters on the other. Before and during treatment, significant correlation was found between serum PGL-1 level and the morphological index (MI), and with the number of viable organisms per mg skin tissue. However, neither serum PGL-1 antibody titre nor skin PGL-1 antigen level showed significant change during the 56-day trial. Because the reduction of serum PGL-1 level was well correlated but less pronounced as compared with the evolution of viable organisms during treatment, the serum PGL-1 antigen assay may be useful as an early indicator of response to chemotherapy in short-term clinical trial, but it is unlikely to replace mouse footpad inoculation for the evaluation of viability of M. leprae.     

Isolation of mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique--a study of 209 cases

Using the mouse foot pad (MFP) system, isolation of Mycobacterium leprae was attempted in 209 skin biopsies obtained from 114 borderline tuberculoid (BT), 62 mid borderline (BB) and 33 indeterminate (1) untreated cases. Unequivocal growth in the foot pads of mice was seen in 100 (47.8%) cases. Of these 100 cases that showed growth in the mouse foot pad system, in 20 cases acid fast bacilli (AFB) were detected in small numbers (1 + ) in either smear or homogenate. The remaining 80 (42%) cases were negative for AFB in both smear and homogenate. The occurrence of viable bacilli and percentage take at 12 months was highest in BB (76 and 86%) followed by BT (38 and 75%) and I (30% and 52%) cases. In most of the BT (65%) and I (60%) cases, the first peak was seen only at 12 months. These results confirm that viable bacilli can be isolated and expanded from a good proportion of negative BT-BB cases using immunocompetent Swiss White mice.     

A study of knowledge, attitude and practices regarding leprosy among undergraduates and interns of a medical college and hospital from rural India

Leprosy is an Infectious disease caused by Mycobacterium leprae and acquired through droplet infection. India has been carrying the 2/3rd global leprosy burden. Inadequate or incorrect information and knowledge about the disease and its treatment are the root causes of many stigmas and inhibitions prevalent in the various sections of the community. The present study was undertaken to assess the knowledge, attitude and practices (KAP) regarding leprosy among undergraduates (final year medical students) and interns of Rural Medical College and Pravara Rural Hospital, Loni, Maharashtra, India. It is heartening to note that most of students and interns had good knowledge about regimens, counselling and were willing to work in leprosy. There were, however, misconceptions about several aspects of diseases which were more in case of final year students compared with interns. Significant improvementin the knowledge of interns in comparison offinal year MBBS students was mostly noted on the aspects like transmission of leprosy, involvement of ulnar nerve in the leprosy, immunological relevance, use of vaccine, treatment of leprosy affected person and leprosy associated stigma. This positive change in attitudes as well as knowledge highlight the requirement of proper training and clinical exposure of medical students and important role of internship. There is need to focus on important aspects (such as cardinal signs, public health aspects and definitions, infectivity, misconception about marriage in which insignificant changes were observed.     

AhR ligands, malassezin, and indolo[3,2-b]carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis

Malassezia yeasts are connected with seborrheic dermatitis (SD) whereas M. furfur pathogenicity is associated with the production of bioactive indoles. In this study, the production of indoles by M. furfur isolates from healthy and diseased skin was compared, the respective HPLC patterns were analyzed, and substances that are preferentially synthesized by strains isolated from SD lesions were isolated and characterized. Malassezin, pityriacitrin, indole-3-carbaldehyde, and indolo[3,2-b]carbazole (ICZ) were isolated by HPLC from extracts of M. furfur grown in L-tryptophan agar, and identified by nuclear magnetic resonance and mass spectroscopy. Of these, ICZ, a potent ligand of the aryl hydrocarbon receptor (AhR), is described for the first time to our knowledge as a M. furfur metabolite. HPLC-photodiode array detection analysis of strain extracts from 7 healthy subjects and 10 SD patients showed that M. furfur isolates from only SD patients consistently produce malassezin and ICZ. This discriminatory production of AhR agonists provides initial evidence for a previously unreported mechanism triggering development of SD and indicates that the variable pathogenicity patterns recorded for M. furfur-associated SD conditions may be attributed to selective production (P<0.001) of measurable bioactive indoles.     

Epidemiology and treatment of uncomplicated gonorrhoea caused by non-PPNG strains in Córdoba, Argentina: auxotypes, susceptibility profiles, and plasmid analyses of urethral isolates from men.

The official records of uncomplicated gonorrhoea for Córdoba state show that between 1975 and 1985, about one in 1000 sexually active people acquired gonorrhoea each year. A study was therefore undertaken to obtain information about treatment of uncomplicated gonorrhoea, as well as the nutritional requirements, plasmid analyses, and susceptibility profiles of gonococci in this geographical area. From August 1983 to April 1984, 219 men with uncomplicated gonorrhoea were treated with one of four antibiotic schedules, all of which were over 95% efficient. All 98 strains isolated and purified were non-penicillinase-producing Neisseria gonorrhoeae (non-PPNG). The minimum inhibitory concentrations (MICs) of benzylpenicillin, tetracycline, thiamphenicol, spectinomycin, kanamycin, and cefoxitin were assessed. The MIC of benzylpenicillin showed that 88% (86) of the strains were inhibited by 0.5 mg/l of the drug, and also showed a bimodal sensitivity pattern to that antibiotic. The nutritional requirements of the 62 strains tested showed that 53% (33) were of the non-requiring (wild type) auxotype, 42% (26) required proline (pro-) and 5% (3) required proline and arginine (pro- arg-). Resistance to antibiotics was more notable in the pro- than in the wild type strains.     

Alcohol use by men is a risk factor for the acquisition of sexually transmitted infections and human immunodeficiency virus from female sex workers in Mumbai, India

OBJECTIVE: We investigated whether men who were under the influence of alcohol when visiting female sex workers (FSW) were at greater risk for sexually transmitted infections (STI) and human immunodeficiency virus (HIV). STUDY: A cross-sectional analysis using baseline data from a randomized controlled trial of an HIV prevention intervention for high-risk men in Mumbai, India. RESULTS: The overall HIV prevalence among 1741 men sampled was 14%; 64% had either a confirmed STI or HIV; 92% reported sex with an FSW, of whom 66% reported having sex while under the influence of alcohol (SUI). SUI was associated with unprotected sex (odds ratio [OR]: 3.1; 95% confidence interval [CI], 2.3-4.1), anal sex (OR: 1.5; 1.1-2.0), and more than10 FSW partners (OR: 2.2; 1.8-2.7). SUI was independently associated with having either an STI or HIV (OR: 1.5; 1.2-1.9). CONCLUSION: Men who drink alcohol when visiting FSWs engage in riskier behavior and are more likely to have HIV and STIs. Prevention programs in India need to raise awareness of this relationship.     

Treatment assessment by monitoring parasite load in skin biopsies from patients with cutaneous leishmaniasis, using quantitative nucleic acid sequence-based amplification

Background.  Current diagnostic methods for cutaneous leishmaniasis (CL) have low sensitivity or are not useful for treatment follow-up. We previously described the quantitative nucleic acid sequence-based amplification (QT-NASBA) method as a sensitive and specific assay for detection and quantification of Leishmania parasites in skin biopsies. This assay could be a valuable instrument for monitoring response to treatment of CL and identifying treatment failures at an early stage.
Aim.  QT-NASBA results of skin biopsies at the end and 6 weeks after treatment from patients with proven CL on various treatment regimens were compared with clinical outcome.
Methods.  The QT-NASBA assay measured the parasite load in skin biopsies before, at the end and 6 weeks after treatment. The results were compared with treatment outcome (clinical cure, delayed healing response or treatment failure) up to 6 months after treatment.
Results.  In total, 137 skin biopsies were obtained from 53 patients. A positive QT-NASBA result 6 weeks after treatment was significantly associated with treatment failure/delayed healing up to 6 months ( P  < 0.001). The positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 92% (95% CI 82–100%). QT-NASBA results at the end of treatment and clinical outcome showed a less significant association ( P  < 0.05), with a PPV of 46% (95% CI 16–75% and an NPV of 89% (95% CI 79–99%).
Conclusions.  The QT-NASBA assay is a useful instrument to monitor parasite load in skin biopsies of patients with CL 6 weeks after treatment and can help to predict clinical outcome.