Taxonomic classification
1. Introduction
Tuberculosis (TB) is one of the infections with major impact worldwide and it is produced by members of the
In the last years the biology molecular methodologies have led many advances which allow the analyses of genetic material of different microorganisms such as
2. General aspects of disease and etiologic agent
2.1. Epidemiology
TB is a global disease. In 2012, the World Health Organization(WHO) estimated in its annual report about 8.6 million new cases of the disease (100,000 less than in 2011) which represents an incidence rate of 122 cases per 100,000 inhabitants in average worldwide: 5.7 million cases were diagnosed as new cases, and it is estimated that 1.1 million of the total cases showed co-infection infection with HIV; and 450,000 cases of multi-drug-resistant tuberculosis (MDR-TB), being South East Asia, Africa, and the Pacific region the most affected areas. Additionally, an estimate of 1.3 million deaths were caused by the disease, and despite of noticing a reduction in the mortality rate, the increase of deaths in populations such as children and women is remarkable.
On the other hand, approximately 2.9 million cases were lost, that is to say, they were not diagnosed or they were not notified to the national tuberculosis programmes. Regarding treatment, 56 million people were successfully treated from 1995 to 2012, saving 22 million lives among the countries which adopted the Directly Observed Treatment, Short-course (DOTS) strategy, proposed by the WHO.
According to the WHO report, the Americas and the Western Pacific regions are the only ones, out of the six regions, which have achieved goals with regards to the decrease of mortality, prevalence, and incidence by the year 2015.Prevalence of the disease was approximately 31 – 41 cases and incidence was 27 – 31 cases per 100,000 inhabitants in the Americas. Out of the 280,000 incident cases, about 31,000 showed co-infection with HIV; however only 219,349 cases were reported. Mortality of the disease reached a rate of 1.9 deaths per 100,000 inhabitants, that is to say 20,000 deaths among people suffering the disease.
In Colombia, 11990 confirmed cases of tuberculosis in all of its forms were confirmed for the same period. Out of these, 10956 corresponded to new cases for an incidence of 23.5 cases per 100,000 inhabitants. According to the clinical presentation of the disease, 80.4% of cases were pulmonary tuberculosis and 19.6% were extra pulmonary TB [3].
The departments with the largest number of cases in Colombia correspond to Antioquia, Cundinamarca concentrating its high number of cases in the city of Bogota, Valle del Cauca, Atlántico, Risaralda, and Santander.
2.2. Clinical forms
2.2.1. Pulmonary tuberculosis
It is the most common type since it is the manner in which the disease can be spread to other persons. The disease is directly transmitted from person to person; when a diseased person coughs, sneezes, or spits, bacilli are expelled to the air and they will be inhaled by people around the patient [4].
It is estimated that one third of the world population has latent TB; that is to say, they are infected by the bacillus but they have not gotten sick, nor they can spread the infection. People infected with the tuberculosis bacillus have a 10% risk of getting TB throughout their lives. However, this risk is higher for people who have a deficiency or compromise of their immune system, as in cases of infection with the human immunodeficiency virus (HIV), malnutrition, diabetes, or tobacco users [5].
When the disease appears, the symptoms (coughing, fever, night sweats, and weight loss, among others) may be mild for several months. As a result, patients delay seeking medical attention and they transmit the bacteria to other people. During one year, a tuberculosis diseased may infect approximately 10 to 15 people by close contact. Up to two thirds of tuberculosis patients die if they do not receive appropriate treatment [6].
2.2.2. Extra pulmonary tuberculosis
Even though it represents only a small proportion of TB cases, approximately 10 – 15%, patients develop the most severe forms of the disease. Since the disease may affect any organ, the diagnosis is often more difficult and delayed [7]. Populations most affected by extra pulmonary TB are children and persons with immunosuppression, such as those who have leukemia, diabetes and living with HIV.
In accordance with the WHO criteria, the forms of extra pulmonary TB are classified into severe and less severe. Meningitis, milliary, pericarditis, peritonitis, extensive or bilateral pleural, intestinal, spinal, genital urinary are considered to be severe forms. TB of lymph nodes, unilateral pleural, bones (except for the spine), and skin are considered to be the less severe [8].
2.3. Etiologic agent
The
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2.3.1. Microscopic characteristics
The dimensions of the bacillus are approximately 1-10 µm long (usually 3-5 µm) and 0.2 – 0.6µm wide (Figure 1) [11]. It has a complex cellular envelope composed of a cellular membrane and cellular wall; the latter provides mechanical support for the bacteria and gives its characteristic in acid-fast staining due to the large content of lipids [12]. The morphologic feature of presence of cord in the Zielh Neelsen staining is presumptive of mycobacteria of the
2.3.2. Morphologic characteristics in culture
Synthetic media use for the isolation of
3. Genome of Mycobacterium tuberculosis
With the complete genome sequencing of
Genome is rich in repetitive DNA, particularly in insertion sequences and in new multigene families; an example of these is the presence and distribution of insertion sequences (IS). IS6110, a sequence of the IS3 family, is of particular interest. It is widely used for typing of strains and molecular epidemiology due to its variation in the insertion site and the number of copies [19]. In the H37Rv
Recently another repetitive region highly preserved within the chromosome of
4. Molecular markers of M. tuberculosis genotyping
4.1. IS6110
Insertion element found within species of the
IS6110 is 1,361 bp long and contains 28-bp, imperfect inverted repeats at its extremities with three mismatches and 3-bp direct repeats that probably result from repetition of the target sequence [30] are present in different copy numbers and are integrated at various chromosomal site [31]. The number of IS6110 copies present in the genome is species- and strain-dependent. Most strains of
4.2. IS1081, direct repeat and major polymorphic tandem repeat
It is a 1324-bp insertion sequence found in
4.3. Polymorphic GC-rich repetitive sequence
It has numerous copies [35- 37] and consists of many tandem repeats of a 96 bp GC rich consensus sequence. PGRS elements are present in 26 sites of
4.4. DR locus
It contains multiple repeated DR regions of 36 pb, interspersed with non-repetitive spacer sequences of 34 to 41 pb which constitute DVR (Figure 3); the size of the DR locus varies from 6 DVR (06 kb) to 56 DVR (6 KB), and both the DR region and the spacers have shown little variation in the order of presentation among strains.
The DR region has been identified as an integration hot-spot of the IS6110 insertion element [40].
Deletion of one or more DVR [41] and duplication of DVR are the mechanisms which generate the variation among different strains. These deletions and duplications are likely to be mediated by the homologous recombination among neighboring or distant DRs, or by Splicing processes during DNA replication [42]. Nevertheless, the process in which the deletion of a DR occurs due to transposition of the IS6110 has also been described [43]. All of these changes generated in the DR locus are the foundation for the development of the Spoligotyping methodology [44].
4.5. VNTR
DNA segments containing “tandem repeated” sequences in which the number of copies of the repeated sequence varies among strains. Variable Number Tandem Repeat (VNTR) sequences have emerged as valuable markers for genotyping. MIRUs are a class oftandem repeated sequences. There are a total of 41 MIRU loci[45].
5. Genotyping methodologies
The following are the genotyping methodologies based on repetitive regions of the
IS6110-based restriction fragment length polymorphism (RFLP) analysis.
Spoligotyping.
Mycobacterial interspersed repetitive units (MIRU) analysis
5.1. IS6110-RFLP methodology
Identification of IS6110 was a great break through for epidemiology of tuberculosis [46]. IS6110 is an insertion sequence made up by 1361 bp, capable of making copies of themselves and then inserting the copies in a genome locus in a process known as transposition [47]. IS6110-based restriction fragment length polymorphism analysis has been considered the golden standard method for
The first step in conducting RFLP is purification of DNA from a culture of
There is a small number of
5.2. Spoligotyping
It is a PCR-based method for detection and typing of the
The amplification of the DR locus is carried out by means of PCR, using primers, one of which is marked with biotin. The PCR products are hybridized perpendicularly to the membrane that contains 43 oligonucleotides of known sequence. The membrane is incubated with streptavidine-peroxydase conjugate which links to biotin of the PCR products. The detection of hybridization signals is made by means of a chemiluminiscence system (ECL). When DR regions of several strains are compared, the order of the spacers is almost the same in all of the strains, but deletions of the spacers may be found which generate the differences among each one of the strains [54].
In order to compare the patterns obtained with the results published, it is necessary to make the conversion of the obtained bands pattern. Each one of the 43 spacers produces either a dark box (indicating the presence of the spacer) or a clear box (indicating the absence of the spacer), and a binary numeric code 1 -0 is assigned respectively.
To simplify this numbering, the 43-digit binary code is converted to a 15-digit octal code (i.e. digits 0 to 7). Each 3-digit binary set is converted to its octal equivalent, and ultimately the remaining digit shall be either 1 or 0. The translation of the binary numbers to octal numbers is as follows: 000 = 0; 001 = 1; 010 = 2; 011 = 3; 100 = 4; 001 = 5; 110 = 6; 111 = 7. Each octal designation is unique, thus representing a specific brands pattern [55]. Finally, this code is entered in the SITVIT WEB international database which presents information on the diverse lineages and its world distribution. It contains information about the genetic diversity of the
Spoligotyping offers great advantages such as its usefulness in genotyping strains of
By using spoligotyping, species and subspecies comprising the
5.3. MIRU-VNTR methodology
Mycobacterial interspersed repetitive units are loci in the genome of
It is a PCR-based method that uses these 12 different interspersed units for genotyping. The estimation to determine the number of repetitions is based on the size of the amplicon. The results are reported as 12 numbers, each one corresponding to the number of repetitions. The power of discrimination of the 12 MIRU-VNTR regions is much greater than the one of spoligotyping and close to IS6110 RFLP for typing of
6. Species and lineages of M. tuberculosis
6.1. M. tuberculosis
It was the first member of the complex to be described by Doctor Robert Koch in 1882. It is the species most frequently involved in the development of pulmonary TB in humans [65].
6.2. M. bovis
It causes TB in a wide range of wild and domestic animals.
6.3. M. africanum
It is the species that causes more TB in humans in Western Africa [67]. There are two large variants of
6.4. M. bovis BCG or Bacillus Calmette-Guerin (BCG)
It is the vaccine’s strain, and it is a live attenuated variant of
6.5. M. microti
It causes the disease in voles,wood mice and shrews. Its isolation is rare in human clinical samples, but recently, isolations from humans have been characterized. The spoligotypes analysis in isolates reveals the single presence of spacers 37 and 38 [53].
6.6. M. canetti
It was added to the list of
6.7. M. caprae
This subspecies has been isolated mainly from goats in Spain [44], but it has also been found in boars, pigs, and in some cases of persons related to goat breeding [54]. The genetic footprint obtained by spoligotyping is characterized by the absence of spacers 1, 3 to 16, 10 to 33, and 39 to 43 [54].
6.8. M. pinnipedii
It was originally isolated from TB cases in pinnipeds such as sea lions and seals. Recently, cases have been described in terrestrial animals such as the Brazilian tapir. Its spoligotype has only the spacers 25 to 38 [55].
6.9. M. mungi
Identified in 2010 as a pathogen of the
The spoligotype pattern of
6.10. M. orygis
Species described in 2012 by Van Ingen and collaborators. It has been isolated from members of the Bovidae family such as oryx, gazelles, antelopes, cows, rhesus monkeys and waterbucks, although their exact host range remains unsettled, however cases have been described in humans [57]. Its spoligotype pattern is characterized by the absence of spacers 4-9, 14-24. 35, 36, and 39.The most common spoligotype (ST587) is present in the SITVIT WEB database and labeled
6.11. M. tuberculosis lineages (Fig. 4)
The lineages of
6.12. Haarlem (H)
Characterized by a pattern with absence of spacer 31 and the presence of at least one spacer between 1 and 30. It is highly prevalent in Northern Europe, while it is less extended in the Caribbean and Central Africa, where it is thought to be introduced by the European colonization [59].
6.13. Latin America and Mediterranean (LAM)
Characterized by the absence of spacers 21 to 24, 33 to 36, and the presence of at least one spacer between 1 and 30. It is frequent in Mediterranean and Latin American countries. Some genotypes have shown strong geographic associations, for instance LAM10-Cameroon or LAM7-Turkey which were initially catalogued as LAM although there has not been phylogenetic association with other spoligotypes [60, 61].
6.14. T Lineage
Comprised by modern strains of TB. This lineage is characterized “by default” and it includes strains which are difficult to classify into other groups [11] It has absence of spacers 33 to 36 and presence of at least one spacer between 1 to 30 besides the presence of the spacers 9 or 10, and 31; there is also presence of one spacer between 21 to 24 [62].
6.15. X lineage
Its pattern has absence of spacers 18 and 33 to 36. It was identified in Anglo-Saxon cities, and it is highly prevalent in South Africa, less in Latin America. However, there is high presence of this genotype in Mexico, which can be explained by its closeness to the United States. The X lineage was the first group identified in Guadeloupe [63] and the French Polynesia [64].
6.16. East African-Indian (EAI)
Absence of spacers 29 to 32 and 34; and presence of at least one spacer between 1 and 30. It is frequent in South East Asia, India, and Western Africa [62].
6.17. Central Asian (CAS)
Absence of spacers 4 to 27, and 23 to 34. It is highly prevalent in sub-Saharan countries and Pakistan. This linage has also proved to be endemic in Sudan, sub-Saharan countries, and Pakistan. This spoligotype has numerous variants and subgroups such as CAS1-Kili (Kilimanjaro), CAS-Dar (Dar-es-Salaam), and CAS-Delhi [62].
6.18. Beijing
This genotype has absence of spacers 1 to 33 and presence of spacers 34 to 43; in terms of public health it continues to be a serious problem for TB control due to its high virulence and association with multi-drug resistance [11].
6.19. MANU
It may be the ancestral clone of the strains in genetic group 1. It was subdivided into Manu 1 (absence of spacer 34), Manu 2 (absence of spacers 33-34), and Manu 3 (absence of spacers 34 to 36). It is a new family from India [62].
7. Molecular epidemiology support in controlling tuberculosis
Molecular epidemiology has become in recent years an essential tool in the study of cases of tuberculosis, together with classical epidemiology, thus making the analysis of situations such as:
Early detection and rapid control of outbreaks.
Set transmission cases restricted communities
Establish the geographic origin of the strains
Monitoring of cross-contamination in the laboratories of TB
Detection of genotypes associated with drug resistance
Differentiation of cases of relapse and reinfection
In the analysis of data obtained by molecular epidemiology is essential to know definitions created from the development of molecular epidemiology, as they are
Cluster: A genotyping cluster is two or more
Matching genotypes: two or more
Nonmatching genotype: an isolate that has a unique genotype (i.e., a genotype pattern that does not match the pattern of any other isolate in a TB program’s database) [38].
7.1. Early detection of outbreaks
Analysis of clinical isolates of certain areas can help determine if isolates share the same genotype, and they form a cluster. But additionally be analyzed factors such as place of residence, work and time spent on them [38].
Genotypic guide Atlanta CDC studies raises three criteria for an outbreak [38].
An increase in the expected number of TB cases.
Transmission continues despite adequate control efforts by the TB program.
The contact investigation has grown to a size that requires additional outside help.
7.2. Transmission between cases of restricted communities
In conducting the study epidemiologically linked contacts and TB patients can be established [38]:
Epidemiologic links between two TB cases that were identified during contact investigations and later confirmed by subsequent cluster investigations.
New epidemiologic links that were identified during cluster investigations but not discovered during previous contact investigations.
7.3. Establishment of the geographical origin of the isolates
The analysis of several clinical isolates obtained worldwide, has established partnerships lineages with specific geographical areas, and in the case of LAM lineage or Latin American and Mediterranean, which as its name suggests occurs at high frequency in these geographic areas. The EIA or East African Indian [41].
7.4. Detecting genotypes associated drug resistance
The main genotype associated with drug resistance is the Beijing genotype, these strains exhibit high virulence and ease of propagation, but additionally the majority of clinical isolates belonging to this lineage with mutations that confer drug resistance, so its eradication much more difficult in the population, was first identified in China in 1995 [65], but has quickly spread to other countries, and has been responsible for outbreaks of MDR-TB [66, 67]:
7.5. Differentiation of relapses and reinfections
Initially, we must take into account in terms of TB programs, it only considers the situation of relapse which is defined as a patient previously treated for TB who has been declared cured or completed treatment and is newly diagnosed with bacteriologically positive tuberculosis. But the genotypic analysis classifies this situation relapse and reinfection, which are defined as, a case of relapsed TB represents a worsening of an infection after a period of improvement and is caused by the same strain of
7.6. Cross contamination in the laboratories of TB
With the genotypic analysis of clinical isolates processed in a laboratory during the same time period can be set if cross-contamination occurred between crops.
Genotyping can help identify instances of incorrect TB diagnoses that are based on false positive cultures. Incorrect diagnoses can result from laboratory cross-contamination of cultures during batch processing, pipetting, transfer of bacilli from a broth-culture system, work in a faulty exhaust hood, and species-identification procedures, mislabeling of patient specimens, clinical equipment contaminated [38].
The identification of cross-contamination in a laboratory setting allows control measures to prevent patients are misdiagnosed.
7.7. Incorporating genotyping methodologies in TB diagnostic laboratories
Since the development of the genotyping methodologies, especially those that are based on nucleic acid amplification, it has been observed their advantage for use with isolated genetic material directly from clinical samples. Examples of these methodologies is spoligotyping, described by Kameberck, which allows simultaneous detection and typing of
8. Conclusion
The methods most commonly used in developing countries via bacteriological diagnoses were smear and culture show the presence of the causative agent of the disease. Currently being implemented new, more sensitive diagnostic technologies based on the detection of deoxyribonucleic acid (DNA) mycobacterial [68] and with which results are obtained in less time. Some of these methods provide the additional advantage of performing genotyping processes allowing the identification of family or families of
9. Application exercise
9.1. Exercise 1: analyses of cases
Patient presented an episode of TB in 2010, he completed his treatment but with some disruption, then in 2013, he presents a new case of TB. The following are spoligotypes patterns identified for Spoligotyping in each of the isolated (table 2).
Patient presented an episode of TB in 2012, he completed his treatment in June of this year. In October 10th 2013, the patient was diagnosed with TB. The analyses for spoligotyping of the strains showed the following patterns (table 3).
Define the situation of each case, justified your response
Which other diagnosis methodologies you can use for confirmation the case.
9.2. Exercise 2
Complete the gaps on the table 4
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001 | 1101111111110111111100001111111100001111111 | |||
002 | 1111111111111111111111111111111100001111111 | |||
003 | 1111111111111111111100001111111100001111111 | |||
004 | 273 | |||
005 | 1111111111111111111111111000000100001110111 | 777777774020731 | 62 | |
006 | 91 | |||
007 | 1111111111111111111100001111111100001111111 | |||
008 | 1111111111111111111111111000000100001110111 | |||
009 | 777777777720771 | |||
010 | 0000000000000000000000000000000000111111111 | 000000000003771 | 1 | |
011 | 1111111111111111111111111111110100001111111 | |||
012 | 447 | |||
013 | 1111111111111111111111101111111100001111111 | |||
014 | 1111111111111111111111111111111100001111111 | |||
015 | 1101111111110111111100001111111100001111111 | |||
016 | 1110000111111111111111000000000000111111111 | |||
017 | 1001111111111111111111111111000010110001111 | 11 | ||
018 | 777777607760771 | |||
019 | 1111111111111101111111111111111100001111111 | |||
020 | 1111111111111111111100001111111100001111111 | |||
021 | 373 | |||
022 | 42 | |||
023 | 64 | |||
024 | 777777477760771 | |||
025 | 1111111100111111111111110000000000001110111 | |||
026 | 0000111111111111111000001111011100001110111 | |||
027 | 1111111111111111111101111111111100001110111 | |||
028 | 1111111111111111111100001111111100001111111 | |||
029 | 33 | |||
030 | 53 |
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