Sub-Lineage Specific Phenolic Glycolipid Patterns in the Mycobacterium tuberculosis Complex Lineage 1.

"Ancestral" Mycobacterium tuberculosis complex (MTBC) strains of Lineage 1 (L1, East African Indian) are a prominent tuberculosis (TB) cause in countries around the Indian Ocean. However, the pathobiology of L1 strains is insufficiently characterized. Here, we used whole genome sequencing (WGS) of 312 L1 strains from 43 countries to perform a characterization of the global L1 population structure and correlate this to the analysis of the synthesis of phenolic glycolipids (PGL) - known MTBC polyketide-derived virulence factors. Our results reveal the presence of eight major L1 sub-lineages, whose members have specific mutation signatures in PGL biosynthesis genes, e.g., pks15/1 or glycosyltransferases Rv2962c and/or Rv2958c. Sub-lineage specific PGL production was studied by NMR-based lipid profiling and strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis showed in average a more prominent growth in human macrophages. In conclusion, our results show a diverse population structure of L1 strains that is associated with the presence of specific PGL types. This includes the occurrence of mycoside B in one sub-lineage, representing the first description of a PGL in an M. tuberculosis lineage other than L2. Such differences may be important for the evolution of L1 strains, e.g., allowing adaption to different human populations.

Family cluster of multi-drug resistant tuberculosis in Kingdom of Saudi Arabia.

We describe the clinical and genetic characteristics of multi-drug resistant tuberculosis (MDR-TB) in a family cluster in the western region of Kingdom of Saudi Arabia diagnosed between 2012 and 2016. All cases had risk factors for tuberculosis acquisition and they were not household contacts of the index case. Genetic analysis detected both MDR-TB and pre-extensively drug-resistant tuberculosis (pre-XDR TB) strains in the index case and confirmed tuberculosis transmission between two cases. Lack of early diagnosis of MDR-TB by molecular testing and lack of extended contact tracing contributed to the transmission of MDR-TB among this family cluster over four years.

First case report of chronic pulmonary lung disease caused by Mycobacterium abscessus in two immunocompetent patients in Saudi Arabia.

Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium that is a common water contaminant, and an exclusive opportunistic pathogen in immunosuppressed patients; however, it occasionally causes lung infections in immunocompetent persons as well. We report two cases of chronic pulmonary infection caused by M abscessus in two young migrant workers; they were nonreactive to HIV antigen, without any history of pulmonary disorders and no evidence of any immunocompromized status. Both were initially suspected as having pulmonary tuberculosis, and antituberculosis drugs were administered. The preliminary culture found the isolate as a rapidly growing nontuberculous mycobacteria and later by advanced molecular genotyping of the isolates revealed it as M abscessus. The patients were treated with multiple drugs including clarithromycin. The symptoms resolved slowly, the smears and culture became negative, and they recovered completely. This is the first case of its type to be reported from Saudi Arabia.

Microevolution of Mycobacterium tuberculosis in a tuberculosis patient.

Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab profiles, revealing patterns of microevolution in a clonal infection.

Molecular strain typing of Mycobacterium tuberculosis isolates to detect cross-contamination events. Proposed modifications to prevent its recurrence.

OBJECTIVE: To investigate possible cross-contamination events of Mycobacterium tuberculosis cultures, and also to shed light on cross-contamination problems in our laboratories. METHODS: At the TB Research Unit in the Department of Comparative Medicine Research Centre of King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia, we received 22 TB isolates sub-cultured on Lowenstein-Jensen media from a local laboratory in Riyadh on 1st July 2005. We finger printed all 22 isolates in question using a polymerase chain reaction-based spoligotype molecular technique. The epidemiological and clinical data were reviewed. RESULTS: All 22 cases had been proven to be cross-contaminated as a result of processing all specimens using a contaminated buffer. All of these patients had no clinical course consistent with tuberculosis. The discordant clinical pictures, and a deoxyribonucleic acid fingerprint that matches those of other culture-positive specimens processed concurrently, in addition to a lack of an epidemiological link between the patients suggest cross-contamination events. CONCLUSION: Using molecular techniques has become an absolute necessity to detect cross-contamination events in our laboratory, to prevent the deleterious consequences of cross-contamination in patients.

Mycobacterium riyadhense sp. nov., a non-tuberculous species identified as Mycobacterium tuberculosis complex by a commercial line-probe assay.

A non-chromogenic, slowly growing Mycobacterium strain was isolated from a maxillary sinus lavage from a symptomatic patient in Riyadh, Saudi Arabia. It was initially identified as a member of the Mycobacterium tuberculosis complex by a commercial line-probe assay. Its 16S rRNA, hsp65 and rpoB gene and 16S-23S internal transcribed spacer sequences were unique; phylogenetic analysis based on the 16S rRNA gene sequence groups this organism close to Mycobacterium szulgai and Mycobacterium malmoense. Its unique biochemical properties and mycolic acid profile support separate species status. We propose the name Mycobacterium riyadhense sp. nov. to accommodate this strain. The type strain is NLA000201958(T) (=CIP 109808(T) =DSM 45176(T)).

First insight into the population structure of Mycobacterium tuberculosis in Saudi Arabia.

This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of M. tuberculosis, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia.

Usefulness of molecular techniques to identify ongoing tuberculosis transmission in Saudi Arabia.

This study represents the first time that molecular tracing techniques have been used to identify patterns of tuberculosis TB infection in Saudi Arabia. The 2 strains were isolated from a socio-economically advantage family who share a number of common facilities including a car and a driver. There are several factors that may play vital roles in on-going transmission of TB in Saudi Arabia including a high number of expatriates, the Hajj pilgrimage, and the social habits of Saudi citizens. Our sibling case series is believed to be a frequent pattern of disease transmission in this country. Control measures such as health education, active case finding, and prompt and supervised medical treatment are needed. More studies using molecular techniques are recommended to find the incidence of cross infection in Saudi Arabia. In addition, molecular techniques have to be established in all reference laboratories to help the detection of ongoing active transmission, molecular epidemiology and detect sources of infection.

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Role of tuberculosis laboratories in Saudi Arabia. A call to implement standardized procedures.

There is no doubt that the laboratory is the backbone for the diagnosis of tuberculosis (TB). Only through testing in the laboratory can the physician confirm suspicion of TB despite any previous clinical and x-ray findings. Recent visits to several laboratories in the Kingdom of Saudi Arabia showed that some need considerable improvement. Unless there are standardized procedures to diagnose TB, and safety measures are implemented in all laboratories, it will be impossible to diagnose accurately and control TB. The laboratories should be redesigned to conform to international TB Diagnostic Centers, with well trained staff and proper safety procedures.