Mycobacterium tuberculosis - the 10 years of epidemiological and diagnostics studies

Tuberculosis (ТВ) is the main bacterial pathogen that causes more deaths than AIDS, malaria and all infectious diseases. The unusual long doubling time (about 24h), highly hydrophobic cell envelope resistant to chemical lysis was the reason to delay the molecular study of this bacteria. Fifteen years ago, we did not have any molecular tools and methods for genetic manipulation or isolation and analysis of intracellular protein and nucleic acids. Today we have many useful shuttle or integration vectors for basic study of mycobacteria. The full sequence of M. tuberculosis genome is already known. At the present time the diagnosis of tuberculosis is supported with fast-culture system BACTEC and molecular techniques based on PCR and DNA hybridization. The mechanisms of resistance to antituberculosis drugs were described, and first identification of resistance profile is available by using PCR and sequencing or real time PCR methods. In our group in the Center for Microbiology and Virology Polish Academy of Sciences and in the Dept, of Genetics of Microorganisms, University Łódź we have characterized new insertion sequences from M. tuberculosis complex- 18990 and IS1607. In diagnostic studies we have proposed the DIG-PCR ELISA assay as a reliable, specific and sensitive test to identify M. tuberculosis directly in clinical samples. We have performed wide epidemiological studies of M. tuberculosis strains isolated from Polish ТВ patients including drug - and multidrug - resistant strains. Finally we identified the most frequently present mutations responsible for drug resistance of polish clinical isolates of M. tuberculosis.Zadanie pt. „Digitalizacja i udostępnienie w Cyfrowym Repozytorium Uniwersytetu Łódzkiego kolekcji czasopism naukowych wydawanych przez Uniwersytet Łódzki” nr 885/P-DUN/2014 dofinansowane zostało ze środków MNiSW w ramach działalności upowszechniającej naukę

Confirmation of the presence of Mycobacterium-tuberculosis complex-specific DNA in three archaeological specimens

This journal published the first reported identification of Mycobacterium tuberculosis complex (MTE) DNA in ancient human remains but CONCERNS were raised about the article two years after publication. These were based on methodology which, in the field of ancient DNA, was still developing. Here we present a re-examination of the 1993 research conducted on three specimens which exhibited palaeopathologies indicative of tuberculosis. The specimens were: an ulna from pre-European-contact Borneo, a spine from Byzantine Turkey, and a lumbar-sacral spine from 17th century Scotland. There was insufficient material to permit re-examination of all of the original samples. The earlier results were confirmed in two independent laboratories using different methodologies. MTB DNA complex-specific Dna amplicons were obtained, and sequenced in both laboratories, in a re-analysis of samples which supported the earlier findings

Enhanced heterogeneity of rpoB in Mycobacterium tuberculosis found at low pH.

OBJECTIVES: The aim of this study was to gain an insight into the molecular mechanisms of the evolution of rifampicin resistance in response to controlled changes in the environment. METHODS: We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2. RESULTS: The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10(-8) and 8.9 x 10(-8); however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12. CONCLUSIONS: The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive

Molecular Exploration of the First-Century Tomb of the Shroud in Akeldama, Jerusalem

The Tomb of the Shroud is a first-century C. E. tomb discovered in Akeldama, Jerusalem, Israel that had been illegally entered and looted. The investigation of this tomb by an interdisciplinary team of researchers began in 2000. More than twenty stone ossuaries for collecting human bones were found, along with textiles from a burial shroud, hair and skeletal remains. The research presented here focuses on genetic analysis of the bioarchaeological remains from the tomb using mitochondrial DNA to examine familial relationships of the individuals within the tomb and molecular screening for the presence of disease. There are three mitochondrial haplotypes shared between a number of the remains analyzed suggesting a possible family tomb. There were two pathogens genetically detected within the collection of osteological samples, these were Mycobacterium tuberculosis and Mycobacterium leprae. The Tomb of the Shroud is one of very few examples of a preserved shrouded human burial and the only example of a plaster sealed loculus with remains genetically confirmed to have belonged to a shrouded male individual that suffered from tuberculosis and leprosy dating to the first-century C.E. This is the earliest case of leprosy with a confirmed date in which M. leprae DNA was detected

New skeletal tuberculosis cases in past populations from Western Hungary (Transdanubia)

The distribution, antiquity and epidemiology of tuberculosis (TB) have previously been studied in osteoarchaeological material in the eastern part of Hungary, mainly on the Great Plain. The purpose of this study is to map the occurrence of skeletal TB in different centuries in the western part of Hungary, Transdanubia, and to present new cases we have found. Palaeopathological analysis was carried out using macroscopic observation supported by radiographic and molecular methods. A large human osteoarchaeological sample (n = 5684) from Transdanubian archaeological sites ranging from the 2nd to the 18th centuries served as a source of material. Spinal TB was observed in seven individuals (in three specimens with Pott's disease two of which also had cold abscess) and hip TB was assumed in one case. The results of DNA for Mycobacterium tuberculosis were positive in seven of the eight cases identified by paleopathology, and negative in the assumed case of hip TB. However, the molecular results are consistent with highly fragmented DNA, which limited further analysis. Based on the present study and previously published cases, osteotuberculosis was found in Transdanubia mainly during the 9th–13th centuries. However, there are no signs of TB in many other 9th–13th century sites, even in those that lie geographically close to those where osteotuberculous cases were found. This may be due to a true absence of TB caused by the different living conditions, way of life, or origin of these populations. An alternative explanation is that TB was present in some individuals with no typical paleopathology, but that death occurred before skeletal morphological features could develop

_Trichoderma pseudokoningii_ Rifai isolation from Egyptian immunocompromised cattle with _Mycobacterium bovis_ infection

Recently, _Trichoderma_ species have emerged as potent fungal pathogens in immunocompromised humans. We report the first three cases of _Trichoderma pseudokoningii_ Rifai pulmonary infection in the Egyptian tuberculous dairy cattle with _Mycobacterium bovis_, from the heart of a generalized bovine TB in a cow over 5 years old, a mediastinal lymph node of pulmonary TB in a cow of 3 years old, and a lung of mixed pulmonary and digestive BTB in a cow of 4 years old. We have also developed a pathogenisity test technique for _Trichoderma pseudokoningii_ Rifai infection in 3 G. pigs by intraperitoneal injection of 2 G. pigs with mixed infection of _Mycobacterium bovis_ and _Trichoderma pseudokoningii_ Rifai; death of both animals 14 days, thereafter, and by injection of 1 G. pig with single infection of _Trichoderma pseudokoningii_ Rifai; death of animal 21 days, thereafter. We did not report any animal case along review of literature

Analysis of the molecular diversity of Mycobacterium tuberculosis isolates from patients attending central referral hospital, East Sikkim based on the IS6110 element

Background: Using the marker IS6110, which is rated the gold standard for molecular research of Mycobacterium tuberculosis complex (MTB),this study seeks to determine the type of circulating strain of M tuberculosis complex (MTB). MTB genotype was determined from clinical samples of patients exhibiting pulmonary and extrapulmonary tuberculosis at the Central Referral Hospital, SMIMS, Sikkim.Methods: The PCR using IS6110 and 38kDa markers were used for identifying the isolates of M tuberculosis complex in LJ slants and restriction fragment length polymorphism methodology for identifying number of copies of IS6110 had been used to classify 100 M tuberculosis isolates (RFLP-PCR).Results: IS6110 comprised a number of bands ranging between 0 to 2, with the majority of isolates having a single copy, followed by two copies, and a few isolates bearing no copy.Conclusions: This study showed a low copy of IS6110 the most predominant circulating strain in the community of Sikkim with high frequency of 85% of single copy of IS6110.

Insertion Element IS6110 based characterisation of Nepalese tuberculosis strains into different genetic lineages

Nepal is geographically located between India and China, a region containing significant Tuberculosis (TB) and Multi-Drug Resistance (MDR-TB) burdens. However, limited information is available on the phylogenetic diversity of Mycobacterium tuberculosis (Mtb) in Nepal. To gain further insight into the diversity of Mtb in Nepal, consecutive clinical samples from 176 newly diagnosed pulmonary tuberculosis patients were collected from two hospitals in Nepal. Insertion Site IS6110 Fluorescent Amplified Fragment Length Polymorphism (FAFLP) PCR and rpoB sequence analysis were carried out on genomic DNA extracts of cultured strains to assign them to accepted genetic lineages and identify MDR-TB. In this study, the IS6110 based characterisation showed a prevalence of 36.36% Central Asian Strain (CAS), 18.75% Beijing, 7.95 % Haarlem, 3.97% X, 2.2% each of Latin American Mediterranean (LAM), T-Uganda and T, 1.7% S and 24.4% were unassigned. Further, 3.9% of total M. tuberculosis isolates were of rifampicin resistant genotypes thus indicating that the prevalence of MDR could be higher than the country wide prevalence of MDR among new TB cases (2.2%) as reported by the national drug resistance survey carried out in 2011/2012

Improved nucleic acid testing strategies to detect and discriminate veterinary relevant Mycobacterium tuberculosis complex members

Os membros do complexo Mycobacterium tuberculosis (MTC) são agentes causadores de tuberculose em humanos e animais. A tuberculose bovina tem sido sujeita nas últimas décadas a programas de erradicação bastante dispendiosos, na maioria dos países desenvolvidos, envolvendo a análise laboratorial de tecidos de animais suspeitos para a detecção dos membros do MTC, nomeadamente Mycobacterium bovis. O diagnóstico definitivo é obtido através da cultura bacteriológica, o que pode levar 6-12 semanas, período durante o qual a carcaça do animal suspeito e a exploração de origem permanecem sob embargo sanitário. Neste trabalho, descreve-se um protocolo de extracção de DNA de fácil utilização adaptado aos tecidos, o qual é acoplado a um semi-nested PCR em tempo real, utilizando como alvo a IS6110, por forma a melhorar a detecção directa de bactérias pertencentes ao MTC em animais, abreviando o período necessário ao diagnóstico. O ensaio foi avaliado num grupo de 128 amostras de tecido provenientes de bovinos, javalis, veados e raposas. O desempenho global do teste corresponde a uma sensibilidade e especificidade de diagnóstico de 98,2% e 88,7%, respectivamente. Foi observado um coeficiente kappa de 0,859 entre o ensaio de semi-nested PCR e a cultura bacteriológica. Este ensaio permite a detecção rápida de micobactérias tuberculosas em amostras de animais com alta sensibilidade e especificidade, sendo acessível e de baixo custo para uma utilização num laboratório de diagnóstico veterinário. As espécies do MTC são geneticamente muito semelhantes, mas podem divergir na sua epidemiologia, nomeadamente na distribuição geográfica e preferência pelo hospedeiro, factores de virulência e padrões de susceptibilidade antimicrobiana. No entanto, o diagnóstico laboratorial convencional não diferencia rotineiramente as espécies do MTC. Foi desenvolvido um algoritmo de identificação rápido e robusto, baseado em PCR em tempo real, dirigido para cinco alvos genómicos para a identificação das espécies do MTC vulgarmente associadas à tuberculose nos bovinos e outros animais. O primeiro passo permite a confirmação dos membros do MTC nas culturas, através da detecção da IS6110, ou como uma espécie micobacteriana, pela presença do 16S rDNA. Se uma espécie do MTC for identificada, o segundo passo do algoritmo permite avaliar a presença ou ausência das regiões genómicas RD1, RD4 e RD9. O padrão correspondente permite inferir a espécie do isolado como M. tuberculosis (se todas as RDs estiverem presentes), M. caprae (se apenas a RD1 e RD4 estiverem presentes) ou M. bovis (se apenas a RD1 estiver presente). O algoritmo de identificação desenvolvido demonstrou um coeficiente kappa de 0,970 com o resultado da análise bacteriológica. O ensaio pode ser implementado em laboratórios de diagnóstico veterinário, especialmente em laboratórios de referência. Tem-se registado uma procura crescente por métodos de diagnóstico de doenças infecciosas rápidos, de fácil utilização e acessíveis, passíveis de serem utilizados em pontos-de-decisão. A detecção dos membros do MTC é geralmente realizada por diversos métodos convencionais baseados na cultura, que normalmente necessitam de oito semanas. Foram também desenvolvidas estratégias de diagnóstico molecular, mas a maioria requer operadores qualificados e equipamentos e infra-estruturas sofisticadas. Recentemente, a técnica de Loop-Mediated Isothermal Amplification (LAMP) mostrou-se promissora para o desenvolvimento de testes rápidos, de baixo custo, sensíveis e específicos para a detecção de agentes patogénicos. Neste trabalho, foram optimizados dois sistemas LAMP em formato duplex (dLAMP) para a identificação do MTC e Mycobacterium tuberculosis, e do MTC e M. bovis, apresentando valores de sensibilidade e especificidade comparáveis a outras abordagens em que se utiliza o PCR convencional. Os resultados das amplificações são avaliados colorimetricamente utilizando dispositivos de fluxo lateral, simples e comercialmente disponíveis, para a detecção de ácidos nucleicos (NALF). Os resultados apresentados nesta dissertação contribuem para a melhoria das estratégias de diagnóstico molecular existentes no combate à tuberculose animal.Members ofMycobacterium tuberculosiscomplex (MTC)are causative agents of tuberculosis in both humans and animals.Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving thelaboratorial testing of tissue samples from allegedly infected animals for detection of MTCmembers, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6-12 weeks, during which the suspectanimal carcass and herd are under sanitary arrest.In this work we describea user-friendly DNA extraction protocol adapted for tissues andcoupled with an IS6110-targeted semi-nested real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis. The assay was evaluated with a group of 128 freshtissue specimens collected from bovines, wild boars, deer and foxes. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% and 88.7%, respectively. An observed kappa coefficient was estimated in 0.859 for the overall agreement between the semi-nested PCR assay and the bacteriological culture. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, beingamenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.MTC species are genetically very similar but may differ in their epidemiology, namely geographic distributionand host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species ofthe MTC. We developedarapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals.The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows to assess the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows to infer the species of the isolate as M. tuberculosis(if all RDs are present), M. caprae(if only RD1 and RD4 are present) and M. bovis(if only RD1 is present). The identification algorithmdevelopedpresented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970.The assay is able to be adaptable to automation and implementation in the routine diagnostics framework of veterinary diagnostics laboratories, with a particular focus for reference laboratories. Rapid, user-friendly and affordable diagnostictests for use in the point-of-decision or point-of-care settings are in high demand globally. Detection of MTC members is generally performedby cumbersome conventional culture-based methods,whichusually takesup to eight weeks.Molecular diagnosis strategies were also developed but most of these requires skilled operators and sophisticatedequipmentsand facilities. More recently, the Loop-Mediated Isothermal Amplification (LAMP) technique showed promise for the development of rapid, low-cost, sensitive and specific testsfor detecting pathogens.In this work we have optimized duplex LAMP (dLAMP) assays for the identification of MTC and Mycobacterium tuberculosis, and MTCand M. bovis, presenting similar sensitivities and specificitieswhen compared to standard PCRapproaches. The amplification results are assessed colorimetricallyby using simple and commercially available nucleic acid lateral flow (NALF) strips.With the work described in this dissertation we aim to modestly contributeto the improvement of the existing molecular diagnosis strategiesto combat animal tuberculosis