Genomic and geospatial epidemiology of Mycobacterium tuberculosis in Oman: first national insight using whole genome sequencing.

OBJECTIVES: Mycobacterium tuberculosis (MTB) is a global public health issue. Although Oman reduced the burden of tuberculosis (TB) by 85% in under 25 years, the annual incidence rate remains stagnant. Whole genome sequencing (WGS) is used to investigate the transmission dynamics of MTB complex. This study aimed to resolve traditional genotype clusters and exploring the geospatial distribution to understand the epidemiology of TB in Oman. METHODS: Confirmed cases with spoligotyping clusters were randomly selected. WGS of 70 isolates were selected for final analysis. Correlation of epidemiological and geospatial data was conducted. RESULTS: A total of 233 cases were registered in 2021; 169 had confirmed growth, with an incidence rate of 5.2/100,000 population for 2021. A total of 70 genomes were analyzed, and five large clusters and three medium clusters were identified. The lineages L1, L2, L3, and L4 and several sublineages belonging to the Indo-Oceanic family and East African Indian family were identified as predominant in Oman. There were no multidrug-resistant cases identified. CONCLUSION: There is a considerable genetic variation among the strains in Oman. This predominance could be linked with the high percentage of non-national population, which represents different countries and frequent traveling to high TB burden countries. WGS combined with geospatial investigations of MTB are required to better understand the disease transmission in Oman, which will support TB elimination efforts.

Dynamics of Mycobacterium tuberculosis Lineages in Oman, 2009 to 2018.

Study aim. Effective Tuberculosis (TB) control measures in Oman have reduced the annual incidence of tuberculosis cases by 92% between 1981 and 2016. However, the current incidence remains above the program control target of <1 TB case per 100,000 population. This has been partly attributed to a high influx of migrants from countries with high TB burdens. The present study aimed to elucidate Mycobacterium tuberculosis infection dynamics among nationals and foreigners over a period of 10 years. Methods. The study examined TB cases reported between 2009 and 2018 and examined the spatial heterogeneity of TB cases and the distribution of M. tuberculosis genotypes defined by spoligotypes and MIRU-VNTR among Omanis and foreigners. Results. A total of 484 spoligoprofiles were detected among the examined isolates (n = 1295). These include 943 (72.8%) clustered and 352 (27.2%) unique isolates. Diverse M. tuberculosis lineages exist in all provinces in Oman, with most lineages shared between Omanis and foreigners. The most frequent spoligotypes were found to belong to EAI (318, 30.9%), CAS (310, 30.1%), T (154, 14.9%), and Beijing (88, 8.5%) lineages. However, the frequencies of these lineages differed between Omanis and foreigners. Of the clustered strains, 192 MTB isolates were further analysed via MIRU-VNTR. Each isolate exhibited a unique MIRU-VNTR profile, indicative of absence of ongoing transmission. Conclusions. TB incidence exhibits spatial heterogeneity across Oman, with high levels of diversity of M. tuberculosis lineages among Omanis and foreigners and sub-lineages shared between the two groups. However, MIRU-VNTR analysis ruled out ongoing transmission.

First case report of Mycobacterium canariasense native mitral valve endocarditis.

Mycobacterium canariasense is a relatively newly discovered, rapidly growing nontuberculous Mycobacterium first described in 17 patients with fever in the Canary Islands, Spain, in 2004. To date, there have been very few case reports in literature, and to our knowledge, infective endocarditis due to M. canariasense has not been reported. In this case report, we present a 33-year-old man who was an intravenous drug user with native mitral valve infective endocarditis caused by M. canariasense after presenting with septic emboli to the toes and kidneys. The rapidly growing mycobacterium isolated from blood culture and valve tissue was identified by 16S rRNA sequencing as M. cosmeticum but was finally identified as M. canariasense by rpoB gene sequencing. The patient underwent mitral valve replacement surgery and received combined antibiotic therapy of intravenous ciprofloxacin, intravenous amikacin, and oral clarithromycin with a successful outcome. This case highlights the importance of molecular identification of nontuberculous Mycobacterium to guide antimicrobial therapy in such serious infections.

Mycobacterium farcinogenes osteomyelitis of the proximal tibia: A case report.

Non-tuberculous mycobacteria (NTM) are an unusual cause of osteomyelitis. Mycobacterium farcinogenes is an uncommon cause of human disease. We describe here the first case of M. farcinogenes osteomyelitis in a 49-year-old man who underwent left knee anterior cruciate ligament and medial meniscal repair which was complicated by recurrent septic arthritis and surgical site infection. As a consequence, he underwent multiple surgical debridements. Ultimately, left proximal tibial osteomyelitis was diagnosed and M. farcinogenes was recovered from the tissue biopsy culture. Clinical improvement was achieved following surgical removal of the prosthesis along with prolonged combination antimicrobial therapy.

Use of nanotechnology for infectious disease diagnostics: application in drug resistant tuberculosis.

BACKGROUND: The increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb). METHODS: Lateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5' end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines. RESULTS: The three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively. CONCLUSION: The AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.

Significance of Coexisting Mutations on Determination of the Degree of Isoniazid Resistance in Mycobacterium tuberculosis Strains.

The emergence and spread of drug-resistant tuberculosis (TB) pose a threat to TB control in Sri Lanka. Isoniazid (INH) is a key element of the first-line anti-TB treatment regimen. Resistance to INH is mainly associated with point mutations in katG, inhA, and ahpC genes. The objective of this study was to determine mutations of these three genes in INH-resistant Mycobacterium tuberculosis (MTb) strains in Sri Lanka. Complete nucleotide sequence of the three genes was amplified by polymerase chain reaction and subjected to DNA sequencing. Point mutations in the katG gene were identified in 93% isolates, of which the majority (78.6%) were at codon 315. Mutations at codons 212 and 293 of the katG gene have not been reported previously. Novel mutations were recognized in the promoter region of the inhA gene (C deletion at -34), fabG1 gene (codon 27), and ahpC gene (codon 39). Single S315T mutation in the katG gene led to a high level of resistance, while a low level of resistance with high frequency (41%) was observed when katG codon 315 coexisted with the mutation at codon 463. Since most of the observed mutations of all three genes coexisted with the katG315 mutation, screening of katG315 mutations will be a useful marker for molecular detection of INH resistance of MTb in Sri Lanka.

RFLP clusters of rifampicin resistant and susceptible Mycobacterium tuberculosis strains in Western province of Sri Lanka.

INTRODUCTION: Continuous studies on genetic diversity of Mycobacterium tuberculosis could enhance the awareness on transmission, control and prevention of tuberculosis (TB). In this study, we investigated current genetic diversity of TB and rifampicin resistant TB by, Restriction Fragment Length Polymorphism (RFLP) based on fingerprinting of the IS6110 insertion sequence, in the Western province of Sri Lanka, the famous touristic destination with the highest TB burden in the country. METHODOLOGY: Genomic DNA extracted from susceptible and rifampicin resistant TB strains (confirmed for rpoB gene point mutations) were digested with PvuII restriction enzyme, electrophoresed and subjected to Southern transfer. The blots were hybridised with IS6110 probe and visualized using a chemiluminescence detection. RESULTS: The number of copies of IS6110 per isolate varied from 1 to 14. The dendrogram revealed a total of 68 distinct strains among 77 TB isolates and they belonged to nine clusters. Both rifampicin resistant and susceptible strains were distributed in all clusters. This evaluation revealed the absence of genetically identical or strong relatedness between susceptible and resistant isolates. However, clonal expansion was detected in transmission of both TB and rifampicin resistant TB. In addition, the resistant isolates having the novel mutation had no clonal relatedness. CONCLUSION: This is the first observational study regarding clonal expansion of TB in Sri Lanka. Thus, further investigation on genotypes, clonal expansion and transmission of drug resistance using additional markers would be useful for controlling TB.

DNA probe based colorimetric method for detection of rifampicin resistance of Mycobacterium tuberculosis.

Rifampicin resistance of Mycobacterium tuberculosis is due to the occurrence of point mutations of the rpoB gene and the site of mutations vary geographically. Commercialized molecular based methods are not able to comprehensively detect rifampicin resistance as they target a limited number of gene mutations which are thought to be common. The aim of the study was to establish a low cost DNA probe based colorimetric method that can be customized for detection of rifampicin resistance of M. tuberculosis. Thus, enzyme-linked oligosorbent assay (ELOSA) was developed for the detection of polymerase chain reaction (PCR) amplified fragments of rpoB gene of M. tuberculosis DNA on microtiter plates. Forty two M. tuberculosis isolates (rifampicin resistant and susceptible isolates identified by agar proportion method) were used for developing and validating the assay. The point mutations of resistant isolates had been previously determined by DNA sequencing. Two fragments of rpoB gene were labeled with digoxigenin by PCR. The amplified products were hybridized with selected allele specific probes for three mutations and its wild types (six probes) which were captured onto streptavidin coated microtiter plates and detected by color development. Both sensitivity and specificity of all probes were ≥96% and there was excellent discrimination (area under the curve (AUC)>0.9) between rifampicin susceptible cases and resistant cases. The probe-based colorimetric assay (PCR-ELOSA) developed in this study showed good agreement with reference mutations that were confirmed by DNA sequencing. In conclusion, PCR-ELOSA is a reliable and economical assay that can be customized for detection of rifampicin resistance.

The manual mycobacteria growth indicator tube and the nitrate reductase assay for the rapid detection of rifampicin resistance of M. Tuberculosis in low resource settings.

BACKGROUND: Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis. METHODOLOGY: Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 µg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics. RESULTS: Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively. CONCLUSION: The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.

Geographical profile of rpoB gene mutations in rifampicin resistant Mycobacterium tuberculosis isolates in Sri Lanka.

The nature and frequency of mutations in the rpoB gene of rifampicin (RIF) resistant Mycobacterium tuberculosis clinical isolates varies considerably between different geographical regions. The objective of the present study was the identification of rpoB gene mutations responsible for RIF resistance in M. tuberculosis isolates in Sri Lanka. Three regions of the rpoB gene of M. tuberculosis, one corresponding to a 437-bp region, including the rifampicin resistance-determining region (RRDR) and two other regions (1395 bp and 872 bp) spanning the RRDR, were polymerase chain reaction amplified, and were subjected to DNA sequencing. The two mutations found within the RRDR in the 31 RIF resistant strains isolated in this study were at codon 526 (n=15, 48.4%) CAC (His)→TAC (Tyr) and codon 531 (n=3, 9.7%) TCG (Ser)→TTG (Leu). A significant proportion (n=15, 48.3%) showed mutations spanning the RRDR, including two novel mutations at codon 626 (n=13, 41.9%) GAC (Asp)→GAG (Glu) and 184 (n=2, 6.4%) GAC (Asp)→GAT (Asp), a silent mutation. Two isolates revealed double mutations (codons 626+526 and 626+184). The presence of a high frequency of new mutations, and the different frequencies of the universally prevailing mutations, as reported here, emphasizes the need for expanding the geographical database of mutations for effective application of an rpoB-based diagnosis of multidrug resistant tuberculosis.