Relevance of Genotyping for the Clinical Management , Transmission and Control Programs of Tuberculosis

Tuberculosis (TB) has re-emerged over the past three decades worldwide. Molecular epidemiology (ME) is very usefulin thestudy of the phenotypic variation among strains, tracking the geographic distribution and clonal expansion of specifi c strains, to evaluate the transmissibility, virulence, and immunogenicity of different lineages of the organism. ME studies over the last three decades have contributed by answering long standing questions, such as the proportion of cases attributable to recent transmission, risk factors for recent transmission, the occurrence of infection with multiple strains of Mycobacterium tuberculosis (Mtb), the proportion of recurrent TB cases attributable to re-infection and a more precise quantifi cation of mixed infection. In the laboratory, ME can be used to identify cross contamination.Many new DNA typing methods have been proposed after the initialintroduction of restriction fragment length polymorphism (RFLP) in 1993 which is still in use. Most of the newer DNA typing methods are polymerase chain reaction (PCR) based.Since genotyping continues to unravel the biology of mycobacteria,it offers enormous promise in the fi ght against and prevention of the diseases caused by this pathogen.


INTRODUCTION
More than 130 years after the discovery of its causative agent, TB still produces nearly 9 million new infections and 1.5 million deaths every year (one-quarter among them are patients co-infected with human immunodefi ciency virus -HIV) 1 .Th e incidence of TB ranges from less than 10 per 100.000 in North America to 100 -300 per 100.000 in Asia and Western Russia, to over 300 per 100.000 in Southern and Central Africa.In several regions of the world, the rise of incidence is linked to the increasing impact of HIV epidemics, drug resistant TB and defi ciencies of current TB control programs.Th ese reasons determined WHO to declare TB a global emergency in 1993 2 .Despite the availability ofeff ectiveanti-TB chemotherapy for over 65 years, TB remains a major global health problem.As such, there is a need for new therapeutics,diagnostics, and vaccines in conjunction with improvedoperational guide lines to enhance current TB control strategies.
By the early 1990's, when molecular fi ngerprinting fi rst appeared, many questions in TB epidemiology remained unresolved.Th ey included the relative contributions of reactivation and primary disease in areas of high and low prevalence, risk factors for recent infection and/or primary disease, and the occurrence and frequency of exogenous re-infection.More recent i ssues include the impact of HIV co-infection on transmission, the infectivity of smear-negative TB, the relative transmissibility of diff erent strains, and other phenotypic diff erences among strains of Mtb 3 .In recent years, changes in the socio-epidemiological scenarios generated by immigration have revealed new challen ges in ME.Th e key questions include comparison of the role of recent transmission with that of reactivation/importation in immigrant TB cases, the impact of potential importation of previously unidentifi ed Mtb strains and cross-transmission between cases from diff erent nationalities.Without the support of ME, these questions would prove diffi cult to answer 4 .

GENOME OF MYCOBACTERIUM TUBERCULOSIS
Th e complete genome of MtbH37Rv was published in 1998.Th is revealed a circular genome with 4.4 million base pairs (bp) containing approximately 4000 genessee Figure 1.Th e genome of Mtbis guanine-cytosine (G+C) rich (65.6%), a feature that has been associated more often with aerobic prokaryotes.It was demonstrated that Mtbpossesses all the genes necessary to synthesize essential amino-acids, vitamins and enzyme co-factors.It was noted that there isa high proportion of genes encoding enzymes involved in lipogenesis and lipolysis.Th e tuberculous bacillus possesses enzymes used in energy cycles, in aerobic, microaerophylic and anoxic electron transfer and is capable of surviving in a number of diff erent environments including the oxygen rich lung, the macrophage and at the centre of caseating granuloma (Cole ST et  al. 1998).
Although the mycobacterial species of the Mtb are highly similar to each other on deoxyribonucleic acid (DNA) level, Mtb members diff er widely in terms of host tropism, phenotype and pathogenity.Genotyping methods have enlarged our understanding of phylogenetic relations and evolutionary origin of the members of the Mtb 6 .

GENOTYPING METHODOLOGIES
Genotyping methods are based on the analysis of chromosomal DNA of the Mtb.An ideal typing method should be highly discriminatory, easy toperform, fast and inexpensive and should generate results that arereproducible within a laboratory and between laboratories.It should be able to analyze very small quantities of bacterial material,preferablywithout the need for culturing, at the very earlystages of diagnostic and epidemiologic investigations.Th e following are the genotyping methodologies based on repetitive regions of the Mtbchromosome mostly used worldwide 7 : • IS6110-based restriction fragment length polymorphism (RFLP) analysis.• Spoligotyping.

IS6110-RFLP methodology
IS6110 was initially described in 1989.Insertion sequences (IS) are small mobile genetic elements, with 1355 bp, that are widely distributed in most bacterial genomes 8 .It is exclusively present in the Mtb human TBand is also involved in drug resistance mechanisms 3,9 .
Mtbstrains with identical IS6110-RFLP profi les isolated from diff erent patients are generally considered to correspond to recent transmission events.Consistently, strains identifi ed in outbreaks with the same IS6110-RFLP patterns have revealed that clustering is associated with identical risk for transmission 1 .IS6110-based typing is the most widely applied genotyping method in the molecular epidemiology of Mtb and is the gold standard to which other methods are currently compared 9 .
An internationally accepted, standardized protocolfor RFLP typing of the Mtb using IS6110 was published in 1990 and is still used today.Between 0-25 copies of IS6110 are found in almostall strains of MTBC and it is notknown to be present in other organisms.Strains of Mtbdiff er in their position and number of IS6110 copies and their distribution is highly variable in the genome.Th isvariability is exploited to distinguish between strains 10 .Applications include: phylogeny studies, detection of laboratory error/cross contamination, detection of mixed infection and provides best resolution for the analysis of W-Beijing isolates 8 .
Th ough IS6110 RFLP typing is the gold standardfor typingstrains of Mtb, it has severaldisadvantages: requires an amount of high quality DNA that can only be extracted from a large number of bacteria grown from clinical material, slow turnaround time (30-40 days), process is laborious, cannot be used to reliably type isolates with ≤ 6 IS6110 insertions.Strains with fewer than six IS6110 insertion sites have a limited degree of polymorphism, and supplementary methods of genotyping are used in these cases 10,11 .

Spoligotyping
Th e Mtb genome contains multiple direct repeated (DR) regions of 36 bp, interspersed with non-repetitive spacer sequences of 34 to 41 bp which constitute DVR (direct variable repeat); both the DR region and the spacers have shown little variation among strains.Deletion of one or more DVR and duplication of DVR are the mechanisms which generate the variation among diff erent strains.All of these changes generated in the DR locus are the foundation for the development of the spoligotyping methodology, a PCR-based method 7 .
It is the simplest technique for Mtb strain genotyping, data are presented in binary format (positive or negative), allowing inter-and intra-laboratory comparisons, can be performed directly on cell lysate, no DNA purifi cation is required, can be performed on nonviable bacteria 12 .It is economical, easy to perform and a rapid method of typing the Mtb.Th is characteristics makes it a candidate for use in poor resource situations.Th e ability to perform spoligotyping directly on sputum samples makes it applicable in acute clinical situations 9 .It is ideal for a fi rst-step analysis ofMtb, particularly in regions with diverse populations, for ME studies and detection of laboratory error/cross contamination.Nonetheless, the discriminatory powerof this method is less than that of IS6110 typing, itcannot recognize mixed infections and it is less informative in regions with predominant or endemic strains; e.g., W-Beijing in China, Southeast Asia, and Russia 11 .
Spoligotyping is the backbone of the largest public available database, SpolDB4, which, on its release in 2006, described a total of 1939 clustered patterns representing 39.295 strains from 122 countries.Developed and housed at Pasteur Instituteof Guadeloupe, SpolDB4 has recently evolved to a multimarker database (SITVIT2) that contains genotyping information on nearly 75.000Mtb isolates from 160 countries, including MIRU-VNTRs on about 15.000 isolates.

MIRU-VNTR methodology
DNA segments contain "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.Mycobacterial interspersed repetitive units (MIRU) are a class of tandem repeated sequences.Th ere are a total of 41 MIRU loci.Of the 41 diff erent MIRU loci, 12 loci were identifi ed as hyper variable repetitive units 13 .
MIRU-VNTR is a PCR-based method that uses "tandem repeated" sequences in which the number of copies of the repeated sequence varies among strains.Th eresults are reported as 12 numbers, each one corresponding to the number of repetitions.Recently a systemwas proposed which includes typing of 24-loci ducted in San Francisco and Baltimore)that have indicated that limitedor casual contact is suffi cient for Mtbtransmission.
When a second case is recognized as part of a contact investigation, it is generally assumed that the two cases show the same strain of Mtb.ME studies have established that this is not always true.Of 211 contacts evaluated in San Francisco, the contact cases were infected with the same strain of Mtb in 70%, but had unrelated strains in the rest of 30%.Unrelated infections were more common among abroad-born, particularly Asian, contacts.Similarly, in a study from Denmark, among 22 cases in contact with one or more persons with TB, six (27%) cases among the contacts had diff erent genotype patterns from the presumed source case; in two instances the case index and thecontact lived in the same household 15 .

Identifi cation of unsuspected transmission
Studies of the ME of TB have elucidated both suspected and unsuspected transmission.Use of genotyping has identifi ed contacts that were not found via traditional contact investigations, as well as networks and places of transmission that were not originally suspected.In addition, genotyping has distinguished between true secondary cases and coincidental, active TB among contacts 15 .ME studies have identifi ed many unsuspected community outbreaks of TB, resulting in focused public health interventions.For example, in Los Angeles, homelessshelters were identifi ed as major sites of TB transmission andin San Francisco, the transmission index(defi ned as the number of cases of TB generated by a single source case), was seven times higher among U.S.-born African-Americans as among U.S.-born whites or Hispanics.Th isdiff erencewas not due to an increased prevalence ofHIV infection or of acid-fastpositive sputum smears among African-Americans but may havebeen related to delays in seeking medical attentionor to social factors such as over-crowding that facilitates the transmission of disease 11 .
Th e identifi cation of secondary cases of TB among those previously unsuspected demonstrates the limitationsof traditional contact investigation.In one of the fi rst genotyping studies, investigators in Switzerland described the transmission of TB among members of a defi ned social group (drug addicts, homeless persons, and alcoholics) and documented its spread to the general population.Th ese studies demonstrate that once a connection is suspected or identifi ed, valuable information can be obtained by re-evaluating contacts 15 .
MIRU-VNTR combining multiplex PCRanalysis or a DNA analyzer based on fl uorescence with the computer automation of genotyping 7 .
Th e discriminatory power of MIRU genotyping is almost as great as that of IS6110-based genotyping.Unlike IS6110-based genotyping, MIRU analysis can be automated and thus can be used to evaluate large numbers of strains, yielding intrinsically digital results that can be easily catalogued on a computer database.A web-site has been set up so that a worldwide database of MIRU patterns can be created.MIRU genotyping istechnically simpler than IS6110-based genotyping and can be applied directly to cell lysatecultures without DNA purifi cation, although is less discriminatory than IS6110RFLP genotyping11.Applications include population genetic/evolutionary investigations, the potential for real-time genotyping and it can be used to identify mixed infections 8,14 .

Study of M. tuberculosis transmission dynamics
ME investigations have beenvery useful in providing understanding of the transmission dynamic of TB within a community.Th ese studies are based upon the premisethat patients infected with strains showing identical fi ngerprints, termed "clustered cases", are the resultof recent transmission, whereas those infected withisolates with unique RFLP patterns are presumed torepresent remote transmission and thus reactivationof strains acquired in the more distant past 9,15 .
A basic principle was that the strains of Mtb who infected the contactsare likely to have the samedrug susceptibility pattern as the index case.Th ishypothesis was tested in a fi ngerprint study of index and contact cases, which were bothdiagnosed with TB.Th irty percent of pairs had diff erent fi ngerprints,demonstrating that the contact was infectedby an unidentifi ed third person.Th is illustrates thattransmission links are often more complex than thoseassumed by conventional epidemiology.Similarly, acontact investigation among fi ve large clusters inthe Netherlands showed that transmission occurred afteronly transient contact, contrary to the conventionalview that TB is usually acquired followingprolonged exposure to an infectious case.
Th e imprecision of contact-tracing investigations has been highlighted by several reports (studies con-se.Individuals with latent Mtbinfection who contract HIV are at risk to develop active TB at a rate of 7 to 10 per cent per year, compared to approximately 8 per cent per lifetime for HIV-negative individuals.
ME has also documented thepotential for spread of MDR strains amonghospitalized patients with HIVinfection.During one 43-month period, New York City accounted for almostone-quarter of all cases of MDR-TB in the USA.Most of these patients were infected with HIV and found to have acquired their MDR-TB duringtheirhospital-stay.

Early detection of outbreaks
Analysis of clinical isolates in certain areas can help determine if isolates share the same genotype, and they form a cluster.Additionally, there shouldbe analyzed factors such as place of residence, work, as well as the time spent in each of them.
Atlanta CDC genotypic guide studies propose three criteria for an TB outbreak 7 : • An increase in the expected number of TB cases; • Transmission continues despite adequate control eff orts by the TB program; • Th e contact investigation has grown to a size that requires additional outside help.If epidemiologic data suggest the occurrence of an outbreak of TB, genotyping of the isolates, in combination with an epidemiologic investigation, can help determine whether an outbreak has occurred or whether there is a coincidental occurrence of a large number of cases.Th is strategy can value the extent of the outbreak and guide public health measures to reduce disease transmission 11 .

Identifi cation of risk factors and groups at risk of M. tuberculosis infection
Risk factors for TB attributable to recent transmission include male sex, being a young adult, being native (vs.foreign-born), urban residence, alcohol and drug abuse, being homeless, exposure to crowded settings, including prisons, and having pulmonary TB.HIV and MDR-TB were found to be risk factors in some settings, but not in others.On the contrary, the elderly in low-incidence countries have a much higher risk of TB attributable to endogenous infection than the young, so the proportion of TB in that age group attributable to recentinfection may be expected to be smaller than among the young.However, young age is also, in absolute terms, a risk factor for recently transmitted TB 12 .Patterns of TB transmission diff er greatly between settings, as they refl ect an interplay between various Th e increasing heterogeneity of the population, now accounting for more than 50 countries, from the African continent to Latin America, from Asia to East Europe is represented primarily by the wide heterogeneity of the Mtb strains detected in the years 2004-2012 with the identifi cation of 11 lineages and 35 families-subfamilies, comparing with that of the years 1994-2000, when only seven lineages could be identifi ed after reanalyzing the old spoligotyping dataset.Not only was the recent number of lineages higher, but the distribution of isolates was signifi cantly diff erent between the two consecutive periods mirroring the increasing contribution of isolates from foreign-born TB patients 16 .

Identifi cation of transmission in a given setting
Increased surveillance with prompt diagnosis and appropriate therapy in settings in which there are many HIV-infected persons, in hospitals, prisons, schools and homeless shelters is now resulting in an overall decrease in TB transmission.Analysis of isolates by IS6110 RFLP demonstratedthat newly acquired TB infection in HIV-infectedpatients spread rapidly and progressed within3 months of exposure to disease, demonstrating theparticular vulnerability of HIV-infected individual-stoexogenousTBinfection.Also, HIV infection exerts immense infl uence on the natural course of TB disea-and heavily dependent by a number of factors, including the annual rate of TB infection, the molecular method used, eff ective TB control programs, the size of the infected pool of individuals, age cohort eff ects, immigration history, population susceptibility (e.g., genetic susceptibility, HIVprevalence, BCG vaccination).

Detection of laboratory error/crosscontamination
Approximately 3 percent of patientsfromwhomMtbis apparently isolated inclinical laboratories do not have TB; thepositive cultures are due to cross-contamination.Th e occurrence of cross-contamination is mostlikely when acid-fast smears are negative and onlyone specimen is culture-positive.WhenMtbis isolated from a specimen, without an epidemiological or clinical explanation,laboratory cross-contamination should also be suspected.Th e isolates should be analyzed by reliable molecular typing techniques, and compared with specimens that were originally processed during the same time period.Many investigators used IS6110 RFLP typing, VNTR typing or spoligotyping to detect and evaluate laboratory cross-contamination 10,11 .

Determination of geographic spread of strains
Th ere is increasing evidence that the genetic diff erence of Mtb is strongly associated with specifi c geographical locations.Th us,ME studies in a high TB incidence country may provide unique insights into dissemination dynamics and virulence of the pathogen 2 .Th e lineages of Mtbare distributed worldwide, as shown below: • Haarlem (H) -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.• Latin America and Mediterranean (LAM) -It is frequent in Mediterranean and Latin American countries.• T Lineage -Comprised by modern strains of TB.Th is lineage is characterized "by default" and it includes strains which are diffi cult to classify into other groups.• X lineage -It was identifi ed 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 proximity to the United States.Th e X lineage was the fi rst group identifi ed in Guadeloupe and the French Polynesia.
factors, including existing TB control strategies, endemicity (the rate of clustering is lower in settings of low endemicity than in settings of higher endemicity), population density, subpopulations with lower TB immunity, migration patterns, geography, demography, and transmissibility of locally relevant Mtbcomplex strains.Genotyping data have given important clues about risk factors for TB transmission and can help to inform health authoritiesto plan adequate action to curb local TB transmission.
A ME study conductedover the period 2010-2013 on 945 positive culture TB patients living in Brusselsidentifi ed classic risk factors for TB transmission such as being underprivileged (41.4%), having a recent contact with a TB patient (23.2%) or being asylum-seekers or undocumented immigrants (24.9%).Having a previous history of TB was also confi rmed to be a MDR-TB predictor.Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and native patientsof East-Europe (40.7%) 17 .

Discriminating recurrent TB due to exogenous reinfection and reactivation
Before 1990, it was generally believed that, in low-incidence countries, most TB cases were attributable to endogenous reactivation of latent infection, and only a small proportion, about 10%, would derive from recent transmission.Molecular techniques can help distinguish endogenous from exogenous infection.
Studies conducted in countries with diff erent rates of TB incidence have demonstrated various levels of recurrent diseaseattributable to exogenous re-infection.
Ninety percent of TB cases in industrialized nations were sometime believed to result from a reactivation of infection acquired in the distant past.However, population-based genotyping indicatesthat recent transmission causes 20 to 50 percent of cases in urban areas.Continuedtransmission results from two major factors.First,there is a substantial rate of transmission amongcasual contacts in workplaces and other social settings even if contact was often limited.Second, in most cases, transmissionprobably precedes anti-TB therapy.Th erefore, even after many years of global, directlyobserved therapy and high rates of treatment completion, studies have shown that one third of TB casesare still due to recent transmission.Furthermore, a large proportion of TB patients,generally request medical care long after symptomsdevelop, contributing to the spread of disease 11 .
Th e precise proportion of disease due to recent transmission or endogenous reactivation is variable XDR-TB can give rise to potentially untreatable forms of disease.In addition mortality is signifi cantly higher among persons infected with MDR and XDR strains than among those infected with sensitive strains.Moreover, patients with MDR and XDR-TB remain infectious for longer time thus increasing the risk of disease transmission.
Genotyping permits the evaluation of isolates with diff erent patterns of drug susceptibility.Suchan evaluation may be helpful in cases in which the original organism developed drug resistance during or after anti-TB therapy, the patient was re-infected with a diff erent Mtbstrain, or cross-contamination is suspected.Genotyping the isolates fromthe patient and other isolates processed at the same time, can distinguish among these possibilities 11 .
Th e mechanisms of drug resistance for Mtb are chromosomal, caused by accumulation of one or more mutations in independent genes.Detection of drug resistance is performed by culturing Mtb in the presence of antibiotics.More recent methods are based on liquid media including the BACTEC radiometric and the Mycobacterial Growth Indicator Tube methods (MGIT).However, due to the long time period necessary to obtain results and laboriousness of these methods, molecular approaches have been proposed.Th ese methods have an important role in identifying prevalent drug resistance mutations amongst Mtb population in a particular geographical location, make timely diagnosis of Mtb drug resistance and an adequate anti-TB therapy possible 2 .
Th ere is no evidence of a lower risk of infectionamong contacts exposed to pulmonary MDR-TB patients as demonstrated by studies in Mexico, among South African gold miners 20 and inthe Netherlands.Except in localized areas withpoor cure rates, and a high prevalence of HIV, it isunlikely that drug resistance strains spread fast.
It is possible that in patients with mixed infections, the drug resistance profi le may be composed of strains with diff erent susceptibilities (e.g., simultaneous infection with mono-INH-and mono-RIF-resistantstrains), leading to incorrect MDR resistance profi les.Th erefore, genetic heterogeneity may require therapeutic targeting of both drug-resistant and drug-susceptible phenotypes, especially with fi rst-line agents 8 .

Detection of mixed infections among TB patients
It has been understood from the recent reports that a single patient could be infected with more than one- Th e most commonly cited andreviewed example of the geographical disseminationof a particular Mtb clone is that ofW-Beijing strains which is a MDRstrain, responsible for causingmany cases of TB and deaths attributable to TBamong patients and health care workers in nosocomialoutbreaks and other institutional settings in New YorkCity during the 1990s.Th is "Beijing family"strains were also detected in high proportions in Asia, the former Russian Federation and Estonia, Latin America.
Th e key fi nding of the study conducted in Cape Town -South Africa, which bears 28% of the global burden of HIV-related TB, was the associationbetween W-Beijingand HIV infection.Th is association persistedafter the control of a number of clinical factors and could bedue to either an increased pathogenicity or virulence of thestrain or an increased susceptibility of HIV-infected patients tothese strains 18 .
At the Muñiz Hospital in Buenos Aires, Argentina, spoligotyping identifi ed predominance of the Haarlem family among the MDR-TB cases (Mtb family responsible for the 1990s outbreak) as well as the LAM and T families 19 .

Monitoring of transmission of drug-resistant strains
Despite the implementation of multiple anti-TB therapies, a steady increase in the frequency of TB with single and multiple drug resistant Mtb strains has been reported throughout the world.In the early 1990s outbreaks of MDR-TB received global attention.Nosocomial outbreaks of MDR-TB have been reported in the USA, France and other countries.MDR and -negativepatients suggested that AFB smear-negative patientswere responsible for 21% of TB transmittedin the city of San Francisco.Th us intensifying TB control measures for smearnegativecases could signifi cantly reduce the transmission of TB.

CONCLUSIONS
Molecular methods could enhance understanding of tuberculosis transmission dynamics and could be used to improve current control programs locally and globally.Genotyping information of Mtb strains, together with epidemiologic investigations, may provide important information about the spread of Mtb strains by identifying factors related to transmission and progression to TB disease.Th is in turn could greatly assist in formulating strategies for control of TB.Moreover, rapid detection of drug resistance using molecular methods in Mtb isolates of TB patients, in conjunction with routine susceptibility testing, could further assist in timely and adequate use of anti-TB therapy thusplaying a pivotal role in treatment and containment of sensitive as well as drug resistant TB patients.
strain of Mtb 10 .Mixed infection with diff erent strains has also been identifi ed with molecular techniques.Whereas, in low-incidence countries, the probability of multiple infection is expected to be low, in high-incidence countries this risk may be high.Evidence of multiple strains involved in TB disease has emerged in recent years.From TB patients in Cape Town, 19% were infected with both a Beijing strain and a non-Beijing strain.Two studies in Taiwan found that, among TB patients, 3% and 11%, respectively, were infected with a Beijing strain and a non-Beijing strain.In Malawi, 3% of patients were infected with strains of the LAM and non-LAM lineages.

Quantifi cation of the level of infectiousness amongsmear-negative patients
It is generally believed that patients with TB whose sputum microscopic examination failsto detect acidfast bacilli (AFB) are signifi cantly lessinfectious than those with positive smears.However,a ME study that comparedtransmission from AFB smear-positive and