Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

Utility of multiplex PCR for early diagnosis and household contact surveillance for leprosy.

Early diagnosis of leprosy is important for limiting the severity of disease, which may lead to disabilities and deformities if not treated timely. Multiplex PCR employing more than one gene, specific to target DNA, is more efficient detection tool. In the present study, slit skin scrapings, blood, nasal swabs and saliva from Paucibacillary (PB) and Multibacillary (MB) cases as well as household contacts of PB cases were tested by multiplex PCR using three different gene targets namely RLEP, 16SrRNA and sodA. We found an increase in overall diagnostic positivity for M. leprae DNA detection by M-PCR as compared to individual PCR. In case of nasal swabs using M-PCR the PPV, NPV were 0.5454, 0.8333 respectively. There is remarkable increase in PPV in SSS of PB cases and nasal swabs of HHCs using M-PCR. Conclusively, our finding suggests the utility of M-PCR for early diagnosis and household contact surveillance for leprosy.

The National Tuberculosis Control Programme of Liberia Laboratory Programme Performance.

Background: Tuberculosis (TB) is a major public health problem in Liberia. Little is known about the TB laboratory performance of Liberia and the challenges after the 14 years of civil war which ended in 2003. The purpose of the study was to evaluate the TB laboratory performance of Liberia. Methods: A cross-sectional study was conducted from 2014 to 2015. The study was conducted using quantitative data of TB case findings, sputum microscopy proficiency testing, and on-site assessment of sputum microscopy laboratories in Liberia. 80 laboratories participated in the proficiency testing. Besides, four years' (2012-2015) TB case finding data obtained from the National Leprosy and Tuberculosis Control Programme (NLTCP) were used to complement the study. The data were analysed using descriptive statistics. Results: From the 80 TB sputum microscopy testing laboratories participating in proficiency testing, only 20 (25%) scored acceptable performance. 46 (58%) TB microscopy laboratories reported quantification errors for the proficiency panel slide 6 which was 3+. The national TB smear-positive cases notified were 4342 in 2012 but decreased to 3820 and 2448 in 2013 and 2014, respectively. The TB smear case detection rate showed an increase from 68% in 2010 to 78% in 2011 and a decrease to 60%, 57%, and 42% in 2012, 2013, and 2014, respectively. Conclusion: Between 2010 and 2013, the NLTCP succeeded in increasing the number of TB sputum microscopy laboratories. At most of the TB microscopy sites, the TB laboratory quality system was not implemented. The NLTCP of Liberia should develop strategies to overcome its challenges in TB laboratory testing.

Como é realizado o exame de baciloscopia para hanseníase?

Em pacientes com lesões cutâneas visíveis ou áreas com alteração de sensibilidade, a coleta deverá ser feita em lóbulo auricular direito (LD), lóbulo auricular esquerdo (LE), cotovelo direito (CD) e lesão (L), conforme a figura 1. Nas lesões planas, coletar no limite interno. Nos nódulos, tubérculos e placas eritematosas marginadas por microtubérculos, coletar no centro.

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia.

BACKGROUND: Diagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: In this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis. Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p<0.05). Sensitivity of PCR also increased (96.8%, p<0.05) when mechanical lysis was used during DNA extraction compared to enzymatic treatment alone (84.6%). CONCLUSIONS/SIGNIFICANCE: Our results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

Relationship between bacilloscopy and operational classification of Hansen's disease in patients with reactions.

Leprosy can be classified according to its operational form as paucibacillary or multibacillary. Bacilloscopy integrates its diagnostic armamentarium. Patients with the disease may present leprosy reactions. This study describes the association of bacilloscopy results and the type of operational classification of leprosy in patients with leprosy reactions. Medical records were analyzed at a reference center between 2010 and 2015. Reactions occurred in almost half of the patients, making their identification important. The bacilloscopic and operational characterization indicates a greater occurrence of leprosy reactions in patients with positive bacilloscopy and also in multibacillary.

Relationship between bacilloscopy and operational classification of Hansen's disease in patients with reactions

Abstract: Leprosy can be classified according to its operational form as paucibacillary or multibacillary. Bacilloscopy integrates its diagnostic armamentarium. Patients with the disease may present leprosy reactions. This study describes the association of bacilloscopy results and the type of operational classification of leprosy in patients with leprosy reactions. Medical records were analyzed at a reference center between 2010 and 2015. Reactions occurred in almost half of the patients, making their identification important. The bacilloscopic and operational characterization indicates a greater occurrence of leprosy reactions in patients with positive bacilloscopy and also in multibacillary.

Non-exponential growth of Mycobacterium leprae Thai-53 strain cultured in vitro.

In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell-associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells.

Paleomicrobiology of Leprosy.

The use of paleomicrobiological techniques in leprosy has the potential to assist paleopathologists in many important aspects of their studies on the bones of victims, solving at times diagnostic problems. With Mycobacterium leprae, because of the unique nature of the organism, these techniques can help solve problems of differential diagnosis. In cases of co-infection with Mycobacterium tuberculosis, they can also suggest a cause of death and possibly even trace the migratory patterns of people in antiquity, as well as explain changes in the rates and level of infection within populations in antiquity.

Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic.

UNLABELLED: This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCE: Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

Update on the epidemiology, diagnosis, and treatment of leprosy.

Leprosy is an infectious disease that has now been reported for more than 2000 years. The leprosy elimination goal set by the World Health Organization (WHO), i.e. a global prevalence rate <1 patient per 10,000 population, was achieved in the year 2000, but more than 200,000 new case patients are still reported each year, particularly in India, Brazil, and Indonesia. Leprosy is a specific infection: (i) it is a chronic infection primarily affecting the skin and peripheral nerves, (ii) Mycobacterium leprae is one of the last bacterial species of medical interest that cannot be cultured in vitro (mainly because of its reductive genome evolution), and (iii) transmission and pathophysiological data is still limited. The various presentations of the disease (Ridley-Jopling and WHO classifications) are correlated with the patient's immune response, bacillary load, and by the delay before diagnosis. Multidrug therapy (dapsone, rifampicin, with or without clofazimine) has been recommended since 1982 as the standard treatment of leprosy; 6 months for patients presenting with paucibacillary leprosy and 12 months for patients presenting with multibacillary leprosy. The worldwide use of leprosy drugs started in the 1980s and their free access since 1995 contributed to the drastic decline in the number of new case patients. Resistant strains are however emerging despite the use of multidrug therapy; identifying and monitoring resistance is still necessary.

Current and past strategies for bacterial culture in clinical microbiology.

A pure bacterial culture remains essential for the study of its virulence, its antibiotic susceptibility, and its genome sequence in order to facilitate the understanding and treatment of caused diseases. The first culture conditions empirically varied incubation time, nutrients, atmosphere, and temperature; culture was then gradually abandoned in favor of molecular methods. The rebirth of culture in clinical microbiology was prompted by microbiologists specializing in intracellular bacteria. The shell vial procedure allowed the culture of new species of Rickettsia. The design of axenic media for growing fastidious bacteria such as Tropheryma whipplei and Coxiella burnetii and the ability of amoebal coculture to discover new bacteria constituted major advances. Strong efforts associating optimized culture media, detection methods, and a microaerophilic atmosphere allowed a dramatic decrease of the time of Mycobacterium tuberculosis culture. The use of a new versatile medium allowed an extension of the repertoire of archaea. Finally, to optimize the culture of anaerobes in routine bacteriology laboratories, the addition of antioxidants in culture media under an aerobic atmosphere allowed the growth of strictly anaerobic species. Nevertheless, among usual bacterial pathogens, the development of axenic media for the culture of Treponema pallidum or Mycobacterium leprae remains an important challenge that the patience and innovations of cultivators will enable them to overcome.

Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator.

Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue.

The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3-10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice.

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized protocols for Mycobacterium leprae strain management: frozen stock preservation and maintenance in athymic nude mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

BACKGROUND: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. METHODOLOGY/PRINCIPLE FINDINGS: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. CONCLUSIONS: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.