The International Journal of Mycobacteriology

ARTICLE
Year
: 2012  |  Volume : 1  |  Issue : 2  |  Page : 87--93

Testing for the presence of Mycobacterium tuberculosis Beijing genotype strains in Syrian samples


Abdulkader Rahmo1, Buthainah Alsalamah1, Maya Habous2,  
1 National Commission for Biotechnology, Damascus, Syria
2 Reference Tuberculosis Laboratory, Ministry of Health, Damascus, Syria

Correspondence Address:
Abdulkader Rahmo
National Commission for Biotechnology, Damascus
Syria

Abstract

The Beijing family of Mycobacterium tuberculosis (MTB) has been reported to have an exceptional capacity to spread tuberculosis (TB) and induce multi-drug resistance. A method has been developed to distinguish this family from the rest of the MTB families through real-time DNA amplification and subsequent analysis of the melting point of an amplicon. Two pools of multi-drug resistant (MDR) MTB samples collected at two different time periods from various regions in Syria have been selected. This preliminary screening indicated a complete absence of the Beijing family in all samples. This research presents an effective differentiation of bacterial Beijing strains, with minimal effort and cost through analysis of differential amplicon melting points.



How to cite this article:
Rahmo A, Alsalamah B, Habous M. Testing for the presence of Mycobacterium tuberculosis Beijing genotype strains in Syrian samples.Int J Mycobacteriol 2012;1:87-93


How to cite this URL:
Rahmo A, Alsalamah B, Habous M. Testing for the presence of Mycobacterium tuberculosis Beijing genotype strains in Syrian samples. Int J Mycobacteriol [serial online] 2012 [cited 2021 Jun 18 ];1:87-93
Available from: https://www.ijmyco.org/text.asp?2012/1/2/87/201213


Full Text

 Introduction



Tuberculosis (TB) is one of the main infectious causes of death worldwide, with more than 9 million new cases of active disease every year and nearly 2 million deaths [1]. TB is an endemic disease in Syria and accounts for thousands of notified cases (new and relapse incidence rate: 21/100,000 pop/year) [2]. Mycobacterium tuberculosis (MTB) is the causative agent of most TB cases; its ability to spread and the outcome of infection depend on epidemiological, host and bacterial factors [1]. Cases of drug resistant (DR) and multi-drug resistant (MDR) TB (i.e., at least resistant to rifampicin [RIF] and isoniazid [INH]) are increasing worldwide. In some parts of the world 10% or more of new TB cases are MDR [3]. In the Baltic region and Russia a large increase in DR–TB has occurred during the last two decades [4]. During this period many Russians of Jewish descent immigrated to the Middle East (Palestine).

Strains with changed biological properties belonging to the Beijing family of strains have been implicated in many outbreaks of TB suggesting a more efficient transmission or an enhanced ability to progress to active disease compared with other TB strains [5]. Beijing strains, in vitro and in vivo models, show more virulent phenotypes than other MTB lineages. They are often associated strongly with primary drug resistance, and cause in some regions clusters ten times as big as “traditional” strains [6]. Beijing strains appear better adapted to intracellular growth, and drug resistance. They have specific properties in terms of protein and lipid structures, which allow the immune-modulation of the host response to the pathogen [7].

Beijing strains represent about 50% of the strains in East Asia and at least 13% of the strains worldwide [8]. They appear to spread further and establish themselves in new regions. In Cape Town during the period 2000–2003, the Beijing clade spread and became the cause of disease in children; the number of cases rose to 33% outnumbering the earlier value of 13% [9]. It has been suggested that the emergence of the Beijing genotype family represents an evolutionary response of MTB to THE BCG vaccination or/and antibiotic treatment, resulting in a significant impact on TB control [10].

Active disease caused by Beijing strains was associated, in some populations, with exceptional clinical phenotypes. In a study in Russia, Beijing strains were associated with more severe radiographic manifestations of pulmonary TB. Other studies associated the Beijing family with extra-pulmonary TB, a more likely poor outcome in elderly patients, and in many areas an increased risk for relapse [5].

Recent studies have shown associations among MTB strains, geographic regions, and human populations. These indicate that specific strains of MTB coevolved with human subpopulations [5]. The clinical presentations of patients with tuberculosis caused by a Beijing strain were found to vary between different geographical settings [8]. Currently, the exact origin of observed variability in a clinical presentation is still unknown [8]. The prevalence of Beijing strains has not yet been studied in Syria despite the reported presence of these strains in neighboring countries and the influx of workers from the Far East.

The real-time polymerase chain reaction (PCR) method used by the authors is an earlier modification of the assay presented by Hillemann et al. [11]; it targets the same TB genome loci, but detection was based on SYBR Green amplicon detection, followed by profiling of the characteristic melting point of the amplicon. This research applies a quantitatively efficient, qualitatively unequivocal and sensitive assay for the detection of a virulent Beijing strain. Samples collected were from two different periods and from a pool of MDR MTB. The research was entirely performed at the National Commission for Biotechnology in Damascus, Syria.

 Materials and methods



MTB samples

The first pool (22 samples) was collected between 2003 and 2005. The second pool (29 samples) was collected between 2010 and 2011 from various regions in Syria ([Table 1]). All samples represent MDR MTB. Drug resistance was established following the guidelines by the National Committee for Clinical Laboratory Standards (NCCLS) [12]. Control DNA of Beijing and non-Beijing strains were obtained from Azm Center for Biotechnology at the Lebanese university [13]. Association with the Beijing family strains had been established earlier through the standard method of spoligotyping. The research has been approved by the institutional bioethical committee.{Table 1}

DNA extraction

DNA extraction was applied with maximum precautions under a biosafety class II hood [13]. Isolates (about 10mg of colonies) are incubated in a water bath at 80 °C for about 30min to kill the bacteria and centrifuged for 10min at 8000rpm. TE buffer containing 1% Triton X-100, 0.5% Tween 20, 10mM Tris–HCl pH 8.0 and 1mM EDTA was added to the pellet. The rest of the procedure was according to instructions provided by Qiagen DNA Blood Mini Kit (Qiagen Germany), with the following minor modification: the period of incubation at 37 °C was for 2h instead of 90min.

Real-time PCR assay

Real-time PCR experiments were run using the Stratagene Mx3005174;. Primers were synthesized according to the sequence published in Hillemann et al. [11]. Synthesis was performed by VBC Biotech (HPLC grade). PCR polymerase mix – DyNAmo174; flash SYBR Green qPCR kit – was purchased from Finnzymes. The primer concentrations used were 0.5μM. Final reaction volume was 50μl, and MTB genome DNA amount added was 5μl after 1000 × dilution. The thermal profile was: 95° for 10min, and 40–50 two steps cycle; consisting of a denaturation step: 92 °C for 15s, and an annealing/extension step: 62 °C for one min. Measuring the derivative of fluorescence emission with respect to temperature and plotting it against temperature unveiled the presence of a peak reflecting the melting temperature of the amplicon that is diagnostic of the Beijing family (Tm: 87.23 °C, SE: 0.01), and one that is specific to the non-Beijing family strains (Tm: 82.67 °C, SE: 0.11) [14].

Amplicon melting point temperature

Determination of the dissociation curve, and the melting point temperature (Tm), which is based on maximal fluorescence emission change at varying temperatures multiplied by −1; (−dF/dT) was performed at the temperature range {60-94} °C], using the Fam filter (λex: 492nm, λem: 516nm) suitable for the SYBR Green flourophore. The applied software was: MxPro from Stratagene.

Statistical calculations

Standard error (SE) was calculated using Microsoft Office Excel software.

 Results



Based on the analysis of BLASTN of the TB genome sequence, the expected non-Beijing and Beijing family of strains amplicon length produced should be 129bp for the Beijing strains, and 95bp for the non-Beijing strains [11].

Testing non-Beijing and Beijing primers on non-Beijing and Beijing samples resulted in Tm peaks and associated emissions as described in [Figure 1].{Figure 1}

Testing non-Beijing primers on non-Beijing samples resulted in one Tm peak (~82.67 °C, SE: 0.11). Using the non-Beijing primers with a Beijing sample resulted in only one Tm peak (~77 °C).

Testing Beijing primers on Beijing samples at 62 °C annealing temperature resulted in two Tm peaks (~78 °C, ~87.23 °C). Using Beijing primers on non-Beijing samples at 62 °C annealing temperature resulted in several Tm peaks (~78 °C, ~86 °C, ~90 °C). The Tm peak at ~87.23 °C (SE: 0.01) is the diagnostic peak used for the Beijing strains [14].

Exploring non-Beijing and Beijing strains on collected samples resulted in Tm peaks and associated emissions as described in [Table 2].{Table 2}

 Discussion



The results of the applied method were based on its clear ability to discriminate between the Beijing family strain and the rest of the TB family strains. The distinction is based on the amplified region near RV 2820 uniquely present in Beijing strains, providing the specific fragment of 129bp, and the amplified region in Rv 2819 present solely in non-Beijing strains; providing the specific fragment of 95bp.

The difference in diagnostic melting temperature (Tm values) obtained for Beijing and non-Beijing families (~87.23 °C, ~82.67 °C resp.) is consistent with the specific amplicon size of Beijing strains being 36% larger than that of non-Beijing strains.

Beijing family strains have been implicated in fast transmission, which emphasizes the importance of a fast, simple and economic detection method. The use of rapid diagnostics would reduce the time for implementation of appropriate treatment.

The major advantages of this method are its rapidity, simplicity and sensitivity. The strains are quantifiable. The modified method presented does not require any further specific probes or modified primers. The detection is carried out, unlike the method by Hillemann et al. [11], based on just SYBR Green selective binding to double stranded DNA. The use of amplicon melting analysis is an established technique in differential detection of microorganisms [15].

The observed variations in melting point of amplified DNA in the various strains tested suggest significant genetic variations among strains. This is expected since non-Beijing samples likely represent a heterogeneous family of strains.

The prevalence of Beijing strains in the Middle East and its association with drug resistance has been unfortunately scarcely studied with some exceptions. In Iran reports indicate that the overall prevalence of the Beijing genotype is 3.2%, of which 21.7% are MDR [16]. An extensive study in Saudi Arabia revealed a prevalence value of 4.5% [17].

The testing of all multi-resistant samples obtained from a recent pool and from an earlier one indicated the absence of any Beijing strain in these samples. Such absence if further confirmed through larger sampling pools is striking given the proven presence of Beijing strains in neighboring countries (Turkey, Palestine, Saudi Arabia). On the other hand, it is possible that the Syrian population is less prone to infection by the Beijing family of strains [18]. This has been earlier hypothesized for other cases of limited spread of the Beijing genotype [10],[19],[20].

 Conclusion and recommendation



Using the present method allows for a fast assessment of epidemiological distribution and dynamics of the Beijing family of strains; this may be of exceptional value in regions where their contribution to TB is still unknown. Simplifying and economizing the detection of the Beijing family provides for a fast and widespread real-time surveillance of their epidemiologic contributions.

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