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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 3  |  Page : 211-217

Spoligotyping with pncA sequencing strategy conferring the transmission of multidrug-resistant tuberculosis in Egypt


1 Department of Biotechnology, Animal Health Research Institute, Giza, Egypt
2 Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt
3 Mycobacteriology Unit, Central Labs of Ministry of Health and Population, Cairo, Egypt

Date of Web Publication12-Sep-2019

Correspondence Address:
Dr Suzan Ismail
Department of Biotechnology, Animal Health Research Institute, Dokki, Giza
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_111_19

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  Abstract 


Background: This study explored the genetic diversity of Mycobacterium tuberculosis isolates in Egypt by spoligotyping in combination with pncA gene sequencing, spoNC. Methods: First, isolates were selected from 400 isolates positive for M. tuberculosis that referred to Central Labs Ministry of Health and then were subjected to the study analyses. Results: Twenty one isolates were found to be multidrug resistant (MDR) and 29 isolates were sensitive for isonizide (INH) and rifampicine (RIF) after testing by phenotypic drug susceptibility testing (DST) and Mycobacteria Growth Indicator Tube (MGIT). Spoligotyping yielded 45 patterns belonging to seven families that previously reported in neighboring countries such as Iraq, Syria, Iran, and Turkey. While four isolates were orphans. Conclusion: Application of spoNC on obtained spoligotype patterns enhances to reduce the clustering rate. Bejing family the predominant (34%) were subdivided by pncA sequence into three sensitive DST pncA wild type, three MDR-DST isolates showing cys14Arg mutation in pncA, two sensitive DST isolates with pncA Gly97Asp mutation, and three sensitive DST pncAVal128Gly mutation. The next most common CASI_DELHI family (16%) were subdivided by pncA sequencing into CASI_DELHI (st 381, MDR) including two pncA silent mutation ser65ser (tcc > tct) and CASI_DELHI (st26, sensitive) which included six pncA (wild-type) results, and Latin-American-Mediterranean 6 family (6%) all had PncA Gly97Asp mutation. We concluded that spoNC provides good snap shot for MDR surveillance and its country origin and performs early identification of outbreaks in Egypt.

Keywords: Egypt, multidrug resistant, Mycobacterium, spoligotyping, spoligotyping with pncA sequencing


How to cite this article:
Ismail S, Al Amry K, Aggor G, El Naggar H, Selim S. Spoligotyping with pncA sequencing strategy conferring the transmission of multidrug-resistant tuberculosis in Egypt. Int J Mycobacteriol 2019;8:211-7

How to cite this URL:
Ismail S, Al Amry K, Aggor G, El Naggar H, Selim S. Spoligotyping with pncA sequencing strategy conferring the transmission of multidrug-resistant tuberculosis in Egypt. Int J Mycobacteriol [serial online] 2019 [cited 2019 Nov 19];8:211-7. Available from: http://www.ijmyco.org/text.asp?2019/8/3/211/266488




  Introduction Top


The total population of Egypt in 2016 was 96 million, with statistics of tuberculosis (TB) cases as follows: notified were 8283, total new and relapse cases were 7974 (57% pulmonary) and TB cases with multidrug-resistant (MDR) or rifampicin-resistant-TB was 14% (12–16) for new cases and 20% (17–24) for previously treated cases. In 2015, thoug the cohort of 7860 new and relapse patients registered, the treatment success rate was 85%.[1] Increasing proportion of MDR strains and increased migratory movements, MTBC genotyping together with classic epidemiological investigations is today widely used to monitor the dynamics of TB transmission in populations.[2] TB importance as a major public health problem dramatically linked with the emergence of the MDR-TB, which defined as a combined resistance to rifambicine and isonizid.[3] In Egypt, previous studies carried out that identified 22 different spoligotypes (STs) among the 44 MTB isolates, of which 13 were unique and the remaining 31 isolates were grouped into nine clusters belong to strains that share identical STs.[4] Mycobacterium tuberculosis genotype of the Egyptian TB belongs to the ancestral Manu lineage which considered the predominant that could be a missing link in the split between ancestral and modern tubercle bacilli during the evolution of M. tuberculosis.[5] Spoligotyping technique is based on the reverse blot detection of 43 variable spacer sequences in the direct repeat (DR) region that is present in all M. tuberculosis complex (MTBC) isolates when we compare spoligotyping with IS6110-RFLP and MIRU-VNTR methods it could be the perfect method that yield rapid results in the form of an internationally standardized binary pattern, making it ideal for high-throughput analysis, above the simplest method to be performed.[6] In previous studies carried out in Iraq to indicate the genetic diversity of Mycobacterium isolates that explored that Iraq having its own most predominant ST lineage (SIT1144/T1) which not found among neighboring countries also CAS lineage,[7] another study carried out in Iraq (Duhuk) indicate that T, CAS and Haarlm were the most prevalent STs.[8] Discrimination of clinical M. tuberculosis isolates in Syria demonstrates that the high prevalence of the TUR lineage and low prevalence of Beijing lineage.[9] The Latin-American-Mediterranean (LAM) was the predominant lineage in Brazil.[10] The World Health Organization does not include pyrazinamide (PZA) in the group of antimycobacterial drugs to be routinely tested for resistance although PZA has been continuously used to treat TB, because PZA drug susceptibility testing (DST) has a difficult and often inaccurate.[11] In M. tuberculosis, the anti-TB drug (PZA) modified to an active state by amidase that encodes by pncA gene, mutations in pncA lead to the majority of resistance to PZA.[12] In the pncA locus, a region of ~ 600 nt covering the entire pncA gene and putative promotor region have mutations that are highly divers.[13] In previous studies in most articles that had at least the pnc A coding region sequenced, isolates encompassing 42 different countries and four continents with very diverse geographic locations, there were variable numbers of phenotypically PZA resistant (PZAr) and phenotypically PZA sensitive (PZAs).[14] Furthermore, there were 608 unique polymorphisms in 397 positions throughout the pncA locus, with a pooled estimated prevalence of 16.2% among all TB cases and 60.5% among MDR cases a identified through recently published meta-analysis of global PZA resistance.[15] Although spoligotyping alone had the least discriminatory power, this was combined with pncA sequencing using the spoNC approach; this power was improved and provided good discrimination for MDR-TB surveillance and early identification of outbreaks.[16] For improve the resolution of spoligotyping clusters pncA sequencing used as a secondary genetic marker that gives a novel strategy providing high volume and faster approach for genotyping in endemic settings besides resistance information for epidemiological programmatic purpose in Egypt.

In this study, we get first insight study to investigate the circulating MTBC lineage in Egypt which is considered a main cause of TB outbreaks and endemicity using a novel strategy that combined both spoligotyping and pncA sequencing (spoNC) techniques.


  Methods Top


Sampling

A total of 50 M. tuberculosis isolates, 21 MDR and 29 susceptible to isoniazid and rifampicin (RIF) by routine phenotypic DST and MIGIT960, were selected from 400 positive culture isolates, from patients from different governorates in Egypt referred to the Central Labs Ministry of Health in 2016 for daily inspection .

All the results obtained by DST were confirmed by using line probe assay in Institute of Tropical Medicine, MycobacteriumUnit, Belgium.

Study setting and population

This study was carried out in a prospective cohort study of new and previous cases of sputum smear-positive pulmonary TB cases. For this study, isolates were obtained from sputum samples that were collected from previously untreated and treated pulmonary TB patients during normal routine testing [Table 1]
Table 1:

Click here to view
.

Culture and drug susceptibility testing

Sputum samples were collected and handed to TB Unit at Central Labs as soon as collected. Quality control assessments were performed for all sputum samples, to determine the presence of blood, mucus, or saliva. Smear microscopy was performed by following the Ziehl–Neelsen method, and samples were cultured on two slopes of (LJ) medium after decontamination with the N-acetyl-L-cysteine-sodium hydroxide method. Proportion method was carried out with the following drug concentrations: 0.2/ml and 1/ml isoniazid, 1/ml rifampin, 2/ml streptomycin, and 6/ml ethambutol indicating the resistant and sensitive isolates [Table 2]. Quality assurance activities included regular checking of the reagents used in test procedures (including expiration dates) and regular maintenance and calibration of equipment.
Table 2:

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Data collection methods

Clinical, bacteriological (smear microscopy results during treatment, DST results, and treatment outcomes), and sociodemographic (name, sex, and age) data were taken from patients files and treatment charts.

DNA isolation

To obtain genomic DNA for spoligotyping, mycobacterial colonies freshly grown on LJ medium were re-suspended in 200 μl of tris-ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris–HCL, 1 mM EDTA [PH 8.0]), followed by heat inactivation at 100°C for 5 min and centrifugation at 10,000 g for 15 s to pellet cell debris. The supernatant, containing DNA, was stored at −20C and used in polymerase chain reactions (PCRs.)

Spoligotyping

Spoligotyping was performed using primers DRa and DRb, corresponding to DR region of the MTBC genome, according to the procedures described by Kamerbeek et al., 2001.[6] Amplification and hybridization were performed using a membrane prepared in-house. Detection of hybridized DNA was achieved using enhanced chemiluminescence (ECL) detection liquid (Amersham Biosciences) followed by exposure to X-ray film (hyperfilm ECL; Amersham Biosciences), in accordance with the instructions of the manufacturer Results were read manually to obtain a complete pattern of the presence or absence of the respective spacers.

pncA sequence

DNA was extracted as described elsewhere.[17],[18] The pnc A gene (Rv2043c, NCBI gene identifier [ID] 888260), including the proximal promoter region, was amplified. On a subset of samples, the distal promoter region (100 bp upstream of the start codon) was also included in the amplified region according to the protocol described by the authors.[19] Amplicons were sequenced with an automated DNA sequencer. The pncA gene sequence of isolates from Samara, Russian Federation, was determined from whole-genome sequencing data as described by the author.[20] Mutations in the pncA gene were identified by comparison with the wild-type (WT) M. tuberculosis H37Rv pncA gene sequence.


  Results Top


DNA finger print analysis

DST, spoligotyping, and pncA results were entered into spoligotyping 4 data base. For molecular data (spoligotyping and pncA and drug susceptibility), systematic quality control was performed through double data entry.

Spoligotyping patterns in a binary format in combination with pncA results were entered in an Excel spreadsheet and compared with the spoligotyping database SpolDB4 using MIRU-VNTR plus.

Spoligotyping patterns compared to the international Spol DB4.0 database using MIRU-VNTR plus, a freely available web-based program (20 m) allowing assignment of shared international ST numbers to know profiles. STs that were not present in the Spol DB4.0 were referred to as “orphan” types. Identical spoligotyping patterns were considered to be in a cluster. Dendograms were generated using the dice coefficient and the unweighted pair group method with arithmetic averages (UPGMA). The clustering rate was defined as (nc − c)/n, where “nc” is the total number of clustered cases, “c” is the number of clusters, and “n” is the total number of cases in the sample. A cluster was defined as two or more patterns with identical DNA genotypes.

spoNC analysis

A total of 49 STs with different pncA sequences were obtained from the 50 isolates analyzed. All 49 spoligotyped collected from newly and previous treated cases also from male and females. Patterns from 45 isolates belonged to seven families in the spol DB4.0, whereas four isolates couldn't be matched to any spoligolineage (orphan) but pncA results characterized these orphan patterns as three WTs for pncA sequencing, only one with silent mutation, one isolate of 49 that have no ST pattern which may be due to failing in PCR amplification.

pncA results showing that only four of 21 MDR isolates had mutations (Cys14Arg) and 4 of 21 MDR isolates had silent mutations (Ser65Ser) which will not altered the genotype sequence of these MDR isolates, rest of 21 MDR isolates had WT pncA results. 29 sensitive isolates showed that 11 isolate had mutations (Gly97Asp, Val128Gly), 2 isolates had silent mutations and rest of 29 sensitive DST isolates had WT pncA results.

The largest spoligotyping family was Bejing lineage that accounted for 34% (17 isolates) of total isolates, 5 Bejing family cluster (st 255) which was subdivided by pncA sequence into 4 sensitive DST isolates; 2 of them with silent mutation ser65ser (tcc > tct) and 2 with failed pncA sequence. So, Bejing family (st 1) isolates with cluster size 12 were subdivided by pncA sequence into 3 sensitive DST pncA WT, 3 MDR-DST isolates showing cys14Arg mutation in pncA, 2 sensitive DST isolates with pncA Gly97Asp mutation, and 3 sensitive DST pncAVal128Gly mutation. The next most common family was the CASI_DELHI family with 8 isolates (16%) which were subdivided by pncA sequencing into CASI_DELHI (st 381, MDR) including 2 pncA silent mutation ser65ser (tcc > tct) and CASI_DELHI (st26, sensitive) which included 6 pncA (WT) results, 6 isolates with sensitive DST results belonging to LAM6 family and representing 12% (st64) of all PncA Gly97Asp mutation. Furthermore, the study showed that 5 isolates (10%, sensitive) belonged to the H1 families which showed pncA WT results and the same percent (10%, sensitive) were shown for U family which were subdivided into 2 sensitive DST isolates (st1462) showing pncA WT and 3 MDR isolates (st602) with WT pncA. Furthermore, there were 3 isolates which belonged to EAI family (6%, st 1391, MDR) with pncA WT, and only one isolate belonged to MANU2 (sensitive) showing pncA WT.

ATC6AAC, ATC at codon 7 changed to AAC; WT_GTC7GGC, double-pattern WT_GTC at codon 7 changed to GGC [Table 3] and [Figure 1].
Table 3:

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Figure 1: Spoligotyping data

Click here to view



  Discussion Top


To understand the genetic variations of MTBC, many phylogenetic studies that investigate evolution and spread of MTBC.[21],[22] Six major global MTBC lineages have been defined (1) Indo-Oceanic, (2) East-Asian including Beijing, (3) East-African-Indian, (4) Euro-American, (5) West Africa or Mycobacterium africanum I, (6) West Africa or M. africanum II; these identifications mainly based on RD classification system using large sequence polymorphisms.[23] Beijing strains of particular (sub)-lineages spread takes place in certain world regions and it seems to be more virulent and associated with enhanced resistance levels.[23] To indicate the transmission in population, there is an urgent need to the establishment of an early warning surveillance system to detect genotypic clusters among clinical isolates of M. tuberculosis. Constrained resources and the prevalence of endemic strains wide-scale genotyping in countries with high TB burden which complicate and hampered the molecular discrimination of epidemiologically linked clusters.[24] The first encyclopedia of pnc A sequence variations linked to either a PZAr or PZAs phenotype had been carried out through large-scale study which linking pnc A sequence diversity with phenotypic, structural biology and population biology data. These genetic variants were classified and identified as (i) very high confidence resistance mutations that were found only in PZAr strains (Category A), (ii) high-confidence resistance mutations found in >70% of PZAr strains (Category E), (iii) mutations with an unclear role found in <70% in PZAr strains (Category D), and (iv) genetic variants (including the WT) not involved in phenotypic resistance (Category B) which likely pave the way for application of new genome-based sequencing technologies to predict PZAr.[25] By analyzing the data obtained in our study using a new approach (spoNC) performed in Egyptian isolates, it was found that TB burden in Egypt has strong correlation with foreign phylogenetic lineage (Beijing, U, Haarlem, X, LAM6, EAI and CASI-Delhi) coming from different countries such as China, Europe, and Latin America, as well as Uganda. Unfortunately, most of these foreign lineages belong to Beijing lineage that is considered an aggressive type of phylogenetic TB lineage that never been reported before in surrounding countries such as Syria, Iraq, Saudi Arabia, and Jordan.[9],[10] This lineage belongs to China and has different mutations with pncA results that were previously reported.[24] It is considered a high confidence category (A) mostly distributed in: Suez, Luxor, Behira, Giza, Minya, and Cairo. Furthermore, LAM6 phylogenetic lineage – the second predominant lineage circulating in Egypt – belongs to (LAM) lineage that prevails in Southern Europe, Africa,[26] and South America.[27] In Saudi Arabia and Turkey, 7.2% and 5.3% strains belong to LAM lineages, respectively;[28],[29] hence, it is not surprise to found this lineage distributed in Egypt. This lineage is distributed in several governorates as shown in Sharquia, Cairo, Giza, Alex, Monufia, with pncA mutations and is considered as high confidence category (A) mutations that indicate this lineage distributed all over Egypt. All new cases of TB patient with susceptible DST results for RIF and isoniazid have resistant pncA mutations, suggesting that pncA mutations are transmissible and usually linked to Beijing and LAM6 phylogenetic spoligotyping lineage, and these two lineages have high confidence mutations which is suggested to be main reason for high TB burden in Egypt. The family of M. tuberculosis was dominant in Iraq also, reported in Iran [30] and Turkey.[31] CAS lineage founded to be endemic in India, Sudan, Pakistan, and Afghanistan.[30],[32] Haarlem lineage reported in Iraq,[8] which was first isolated in the Netherlands [33] and prevalent in I Central Africa during European colonization.[34] U and MANU lineage also have been reported in Iraq. LAM lineage (MDR strain) and Haarlem lineage (sensitive strain) have been reported before in Syria.[9] Finally, although Egypt has noticeable migratory movement with Turkey, TUR lineage could not be demonstrated in this study in contrast to other neighboring countries as Iran, Iraq, and Syria.


  Conclusion Top


In Egypt, geographical situation, touristic importance, unstable political situation in neighboring countries such as Syria and Iraq, capability of M. tuberculosis for borderline transmission play a main role in rising rates of sensitive and MDR TB. Thus, there is an urgent need to applicable early wide-scale epidemiological systems that help us to detect the expected transmissible phylogenetic clusters responsible for high burden setting and outbreaks in Egypt. Hence, in this study, we get the first insight about the circulating phylogenetic lineage of MBTC that could be found using a novel molecular strategy by combining spoNC.

We recommend a large-scale study to be carried out in Egypt to screen and mapping TB movement in combination with MIRU-VNTR technique that high discrimination with WT pncA isolates to give us a reliable data about TB situation and main phylogenetic lineage of Mycobacterium causing endemicity of TB and to give expectations of outbreaks in Egypt. An additional aspect that is highlighted by our study is the great advantage in sharing large data sets generated by several groups by comparing data which had been obtained from Egypt and other data found in SPOLDATAbase which have different phylogenetic lineages belonging to different countries. In addition, this study gives us what to date about situation of TB in Egypt even the study had been carried out in small basis datasets.

Acknowledgement

The authors wish to thanks Dr. Leen Rigouts, Prof. Mycobacteriology Unit, institute of tropical medicine, ITM, Antwerp, Belgium for all her efforts for training and analyzing the data.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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