Mycobacteria Interspersed Repetitive Units-Variable Number of Tandem Repeat , Spoligotyping and Drug Resistance of Isolates from Pulmonary Tuberculosois Patients in Kenya

Background: Molecular typing allows a rapid and precise species differentiation and is essential in investigating the spread of specific genotypes and any relationship with drug resistance. Methodology: To compare the discrimination power of 24-loci Mycobacteria interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) to spoligotyping in determining the circulating genotypes of Mycobacterium tuberculosis in isolates from pulmonary tuberculosis patients in Kenya, a total of 204 isolates were typed. Results: Spoligotyping identified 22 spoligo lineages; while 36(17.6%) isolates were not determined. MIRU-VNTR typing identified 12 genotypes; Kenya H37_Rv_ like, S-like that had never been reported before and which were not identified by spoligotyping were identified. Others were Uganda I and II, LAM, Beijing, TUR, EAI, Delhi/C, S and Haarlem. Only 8 (3.9%) were not defined by MIRUVNTR. Delhi/CAS, EAI, S, S-like, LAM and Beijing had strains that showed resistance to all the five drugs tested. Two strains of EAI and 2 of S genotypes were resistant to all the five drugs tested. Beijing genotype commonly associated with drug resistance was found to be third in drug resistance (14.7%) after Delhi/CAS (28.9%) and LAM (17.6%) with the highest resistance towards isoniazid and pyrazinamide (3.9% each). MIRU-VNTR typing was more discriminative than spoligotyping; identifying 10 unique H37_Rv-like isolates designated KeniaH37_Rv_like genotype and 14 S-like genotype. Conclusion: MIRU-VNTR typing has not been reported in any other study in Kenya and How to cite this paper: Ndungu, P.W., Kariuki, S., Revathi, G., Ng’ang’a, Z. and Niemann, S. (2017) Mycobacteria Interspersed Repetitive Units-Variable Number of Tandem Repeat, Spoligotyping and Drug Resistance of Isolates from Pulmonary Tuberculosois Patients in Kenya. Advances in Microbiology, 7, 205-216. https://doi.org/10.4236/aim.2017.73017 Received: February 7, 2017 Accepted: March 21, 2017 Published: March 24, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Background
For a long time differentiation of clinical Mycobacterium tuberculosis (MTB) isolates was based on morphological and phenotypic characteristics such as growth morphology on Loweinstein Jensen medium (eunogic or dysnogic) or biochemical properties such as niacin test and nitrate reduction.Since these tests need sufficient bacterial growth, are time consuming, do not allow unambiguous species identification and may not be performed by laboratories routinely, there was need for molecular methods that could allow a more rapid and precise species differentiation.Molecular typing of MTB is essential for epidemiological purposes such as investigating the spread of specific genotypes [1].
The first methods that allowed molecular fine typing of clinical isolates were IS6110 DNA fingerprinting and spoligotyping [2] [3].These methods are used as intensive markers in epidemiological studies of MTB isolates and have revealed first insights in the population structure of clinical isolates in different geographical settings.There are several major spoligotypes of Mycobacterium tuberculosis namely Beijing, East African Indian (EAI) with at least five subtypes EAI1, EAI2, EAI3, EAI4, EAI5; Haarlem (H) with subtypes H1, H2, H3; Central and Middle Eastern Asia (CAS) with two subtypes CAS1 and CAS 2; European clade of IS6110 low banders X family with three subtypes X1, X2, X3; the poorly defined T family with subtypesT1, T2, T3, T4; Latino-American and Mediterranean family (LAM) with subtypes LAM1-LAM10 [4].
Spoligotyping analyzes the direct repeat (DR) locus in the genome of MTB which is composed of a cluster of 36 base pairs (bp) repeat references interspersed with unique spacers of 35 to 41 bp.By using a reverse southern blotting technique the variability in the spacer sequences can be interrogated and recorded in digital code [5].Although this method provides a digital typing data it only measures variability in a single locus and does not generally provide sufficient discrimination for outbreak investigation [6].
The most currently used typing method is based on variable number tandem repeats (VNTR) of Mycobacteria interspersed repetitive units (MIRUS).The repeated units are 52 to 77 nucleotides in length and the number of repeated units can be determined by the size of the entire locus [7].This polymerase chain reaction (PCR) based typing method was originally based on 12 human minisatellite-like regions of the MTB genome [8] [9].A population based study in the USA indicated that the use of the 12 locus-based MIRU-VNTR typing as a first line method, together with spoligotyping, provides adequate discrimination in most cases for large scale, prospective genotyping of MTB in the USA.IS6110 fingerprinting can subsequently be used as a secondary typing method to type the clustered isolates, when additional discrimination is needed [10].
MIRU-VNTR has the same discriminatory power in comparison to IS6110 RFLP [11].It has also been useful in studying the population structure of MTB [12] [13] and is suitable for global epidemiological surveillance of TB due to its high resolution, simplicity, sensitivity, high reproducibility and easy inter-laboratory comparison [14] [15] [16].MIRU-VNTR typing has become an important method as it allows high throughput, discriminatory and reproducible analysis of clinical isolates [17] [18].First evaluations have shown that it can provide unique high-resolution insights into the population structure of the MTB and provides clear criteria for the identification of the different MTB lineages and sub-lineages [18] [19].

Study Area
A total of six TB clinics were selected for this study.These were Kangemi, Riruta and Mbagathi TB clinics, Aga Khan in Nairobi as well as Kiambu and Nazareth Hospitals in Kiambu county.

Study Design
This was a hospital based prospective study.

Study Population
The study population comprised 204 adult patients with pulmonary TB attending five clinics in Nairobi.

Sputa Collection and Processing
An early morning and a spot sample were collected from each patient.The sputa were processed according to the protocol by Murray [20].They were cultured in MGIT liquid medium using the Bactec 960 system and Lowentein Jensen(LJ) solid media.All the cultures were subjected to the Capilia TB assay (TAUNS Laboratories, Inc.) to rule out Mycobacteria other than tuberculosis (MOTTS).Drug sensitivity tests were done using five first line-drugs isoniazid (INH) (0.10 μg/ml), rifampicin (RIF) (1 μg/ml), streptomycin (SM) (1 μg/ml), ethambutol (EMB) (5 μg/ml) and pyrazinamide(PZA) (100 μg/ml) using BACTEC MGIT 960 STR 4.0 and PZA kits.The procedure was performed as described by Becton, Dickinson and company 7 loveton circle Sparks, Maryland 21152 USA.H37Rv ATCC 25618 was used for susceptibility control and was included in all test sets.

Deoxyribonucleic Acid (DNA) Extraction
The isolates were grown on Löwenstein-Jensen (LJ) medium at 37˚C until growth became clearly visible.An appropriate number of bacterial cells were transfered into a microcentrifuge tube containing 400 μl Tris-EDTA (TE) buffer.They were incubated for 20 min at 80˚C in a water bath to kill the bacteria and 50 μl of 10 mg/ml lysozyme was added and vortexed then incubated for at least 1 hour at 37˚C.Then 75 μl of 10% Sodium dodecyl sulphate (SDS)/proteinaseK mix (5 μl Proteinase K (10 mg/ml) + 70 μl 10% SDS) was added, vortexed shortly and incubated for 10 min at 65˚C.One hundred μl of 5 M Sodium chloride (NaCl) was added followed by 100 μl -N-cetyl-N, N, N,-trimethyl ammonium bromide/NaCl (CTAB/NaCl) mix (prewarmed at 65˚C) and vortexed until the liquid content became white and was incubated for 10 min at 65˚C.Approximately 750 μl chloroform/isoamyl alcohol mix (24:1) was added and vortexed for 10 seconds then centrifuged for 10 min at 12,000 g at room temperature.The aqueous supernatant was transferred in a new tube and 0.6 volume (450 μl) isopropanol added, mixed carefully and incubated for 30 min at −20˚C.This was then centrifuged for 15 minutes at 12,000 g.Most of supernatant was removed and 500 μl of cold 70% ethanol was added and spinned for 5 min at 12,000 g.The supernatant was discarded and the tube spinned for 5 minutes at 12,000 g.The last μl of the supernatant was discarded cautiously and the pellet dried for about 10 min at room temperature.Eighty μl TE buffer was added and the DNA dissolved for 10 minutes at 60˚C.Quality and concentration of the DNA preparation was controlled by agarose gel electrophoresis.

Spoligotyping and MIRU-VNTR Typing
All the samples were spoligotyped by the method described by Kremer [21].
Briefly PCR master mix was added to 5 μl of DNA sample.This was loaded run in the PCR and run as follows: 5 minutes at 95˚C for, 1 minute at 94˚C, 1 minute at 58˚C, 30 seconds at 72˚C, 5 minutes at 72˚C and α at 4˚C.Some 190 μl 2XSSPE + 0.1%SDS was added to the DNA sample.Each sample was loaded on the blotter with a membrane laid on it and incubated at 65˚C for 1 hour.The membrane was removed from the blotter washed in SSPE buffer on a shaker at 60˚C for 15 minutes.The membrane was hybridized at 42˚C for 10 minutes in the hybridization oven after which it was removed and placed on the film chamber.A film was placed on top of the membrane and detection reagent added and allowed to stand for one minute.The film was then developed in the dark room.
MIRU typing was done using the 24-loci based MIRU-VNTR typing kit (User manual www.genoscreen.com).Briefly, a 96-well plate, using a PCR spreadsheet was prepared.In a PCR free area, 8 μl of triplex mixes per well were loaded in the 96-well plate according to the PCR spreadsheet.Two μl of DNA suspension were added, including positive and negative controls.The 96-well plate was tightly sealed with an adhesive PCR film and centrifuged at 13,000xg.This was then loaded in the PCR machine and the PCR cycle run as follows: 15 min at 95˚C, 1 min at 94˚C, 30 s at 59˚C, 1 min 30 s at 72˚C (these were repeated for 40 cycles), 10 min at 72˚C, and α at 4˚C.For fragment analysis 9.5 μl of HiDi for-mamide and 0.5 μl Genescan was prepared per plate.A 96-well sequencer plate was loaded with 10 μl of the mixture.The 96-well plate containing the PCR products was centrifuged and the adhesive PCR film removed.Two μl of PCR products per well were added to the 96-well sequencer plate according to the spreadsheet.The plate was sealed with an adhesive film and centrifuged to remove bubbles.The DNA was denatured at 95˚C for 5 min using PCR machine and kept on ice before loading.The plate was then loaded to the sequencer.
Fragment analysis was done using ABI DNA analyzer.

Data Sequence Analysis
Spoligotyping data was computed using the bionumerics software (Applied maths, Sint Marten-Latems, Belgium).
A web server MIRU-VNTR plus (http://www.miru-vntrplus.org)created using 186 strains representing the major MTB lineage was used in interpreting the MIRU data.For each strain, species, lineage and epidemiologic information is stored together with copy numbers of 24 loci, spoligotyping patterns, regions of difference (RD) profiles and susceptibility data.Using this MIRU-VNTR service, the strains were compared with the reference strains and assigned the lineages and genotypes.The comparisons were based on MIRU-, spoligo-and susceptibility data.

Ethical Considerations
The study was approved by both Scientific Steering Committee (SCC) and Ethical Review Committee (ERC) (ERC NO. 1622).Permission was also granted by the administration of the respective hospitals and the Nairobi City Council Health Committee.The patients also gave informed consent.
Thirty six isolates (17.6%) could not be identified by spoligotyping.Based on the 24-loci system, ten genotypes were identified by MIRU-VNTR typing; Kenia H37_Rv_like and S-like, which had not been determined before and which were not determined by spoligotyping were determined by MIRU-VNTR method.
Others were EAI, Delhi/CAS, S, Uganda I and II, LAM, Beijing, TUR, and Haarlem.Only eight isolates could not be identified because they were not among those that were available in the database therefore could be new (Table 2).Delhi/CAS was the most prevalent with 59 (28.9%) followed by LAM and Beijing 36 (17.6%) and 30 (14.7%) respectively.TUR, EAI, S and KeniaH37-RV-Like were 10 (4.9%) each; the rest were less than 10.
Two strains of EAI and 2 of S genotypes were resistant to all the five drugs tested.Beijing which is commonly associated with drug resistance showed highest resistance to INH and PZA (3.9% each) and least to Rif and EMB (1.9%).
The large number of patterns shows a large diversity of spoligotypes in our study.This diversity could be attributed to human travel and migration of asylum seekers such as refugees especially because of the political instability in the neighboring Somalia and Sudan which has resulted in influx of refugees from these countries, as well as international travel.
Other studies in other regions also showed wide diversity in various countries.
In Turkey by [25], 95 drug resistant isolates collected from three different cen- In India, spoligotyping resulted in 49 patterns.The largest cluster was composed of spoligotype international T types (SITS) 26, Central Asian (CAS)1-Delhi lineage, followed by SIT 11(Eastern-African-Indian) EAI 3-Indian lineage.
A large number of isolates (75%) belonged to genotypic lineages such as CAS, EAI, and Manu with a high specificity for the Indian subcontinent [26].A total of 273 clinical isolates showed 120 different spoligotyping patterns majority of which belonged to major clades of MTB, Haarlem 22.6%, LAM 23.3% and T 32.6% [27].
A study by [28]  In our study sensitivity testing showed Delhi/ CAS as having the highest resistance followed by Beijing.Beijing has been associated with resistance in different parts of the world.In 49 studies in 35 countries, it was found to be associated with drug resistance in several of those countries [33] while in China drug resistance for Beijing was 75.4% [34].Our study also showed that Beijing had highest resistance for Isoniazid and pyrazinamide.Resistance to isoniazid is important because it has been shown to result in MDR-TB if not taken care of.In Vietnam a study showed Beijing having greatest resistance towards isoniazid and streptomycin than any other drug [35].

Conclusion
MIRU-VNTR typing identified presence of Kenia H37Rv-like and S-like genotypes that were unique as well as Euro-American lineages that could not be identified by spoligotyping.Earlier studies in Kenya have only reported on spoligotyping; no other study has reported on MIRU-VNTR typing.This means that unique or new genotypes may have been missed by earlier studies and therefore MIRU-VNTR is of importance in determining emerging genotypes in the country.Resistance of Beijing genotype to isoniazid should be of concern since it could translate to MDR-TB.

Table 1 .
Spoligotyping lineage subtypes of the sequenced samples from patients sampled from five clinics in Nairobi.

Table 2 .
Distribution of the MIRU genotypes of the isolates taken from patients sampled from five clinics in Nairobi.
VNTR typing as opposed to spoligotyping which is the only typing method reported in Kenya, where 36 isolates could not be identified.Although there was dominance of the EAI, Beijing and East-African-Indian global lineages, MIRU-VNTR typing revealed presence of Euro-American lineage represented by Haarlem, Uganda I and II, S,TUR types all of which are dominant in the European region.This means that there has been cross transmission of TB strains from other continents.