Aina Sievers

Development and Application of Two Multiplex Real-Time PCR Assays for the Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

ABSTRACT Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.

Susceptibility of Mycobacterium bovis BCG vaccine strains to antituberculous antibiotics.

ABSTRACT Mycobacterium bovis BCG is one of the most commonly administered vaccines. Complications, including disseminated BCG disease, are rare but increasingly reported in immunodeficient children. There is growing recognition of the importance of differences between BCG vaccine strains. We determined the susceptibilities of five genetically distinct BCG vaccine strains to 12 antituberculous drugs.

Mycobacterium tuberculosis and Sulfamethoxazole Susceptibility

We noted with interest the paper by Forgacs et al. ([2][1]) reporting on a case of tuberculosis with an apparent response to treatment with trimethoprim-sulfamethoxazole (TMP-SMX) and the subsequent in vitro studies indicating that the majority of Mycobacterium tuberculosis strains may be

Tuberculosis in australia: An analysis of cases identified in reference laboratories in 1986-88

&NA; The Special Interest Group in Mycobacteria within the Australian Society for Microbiology has carried out a collaborative study of cases of tuberculosis diagnosed in Australian reference laboratories in the years 1986, 1987 and 1988. Annual totals of 574, 584 and 613 respectively, suggest that the incidence of bacteriologically‐positive tuberculosis is continuing at 3–4 cases per 100,000 population. The highest rates were detected in males over 50 and females over 65 years of age. Three‐quarters of the total cases relate to pulmonary disease. Resistance to at least 1 anti‐tuberculosis drug was detected in 78 (12.7%) of isolates tested in 1988. The negligible decline in incidence of tuberculosis in Australia, the high prevalence in S.E. Asian countries, and the fact that HIV‐infection is an important risk factor, make it imperative that Australias diagnostic and management programmes be maintained.

Epidemiology and control of tuberculosis in Victoria, a low-burden state in south-eastern Australia, 2005-2010.

SETTING Victoria, Australia. OBJECTIVE To describe the epidemiology and control of tuberculosis (TB) in Victoria, 2005-2010. DESIGN Retrospective review of laboratory-confirmed TB in Victoria, 2005-2010. State TB reference laboratory records were matched with Department of Health notification records to obtain laboratory, demographic, clinical and treatment data. RESULTS The incidence of TB fell in the Australian-born population but increased overall, reflecting an increase in the proportion of overseas-born cases from 88.9% to 95.8% between 2005 and 2010 (P = 0.03). Patients from India and Viet Nam accounted for over one third of all cases. For overseas-born cases, the median time between arrival and diagnosis was 4 years. Half of all diagnoses were pulmonary disease, of which 45.4% were Ziehl-Neelsen smear-positive. Treatment was most commonly self-administered (76.9%), and very few patients defaulted or failed treatment (1.1%). Only 4.1% of cases were linked to another laboratory-confirmed case. Multidrug-resistant TB remained uncommon (1.7% of cases). CONCLUSIONS TB in Victoria remains low by global standards and continues to overwhelmingly affect the overseas-born population. Current TB control strategies in Victoria are effective, but strengthened control in high-burden countries will also improve TB control locally.

THE DIAGNOSTIC USE OF THE POLYMERASE CHAIN REACTION FOR THE DETECTION OF MYCOBACTERIUM TUBERCULOSIS

&NA; Detection of Mycobacterium tuberculosis by microscopy is difficult in specimens containing fewer than 104 bacteria/mL and growth in culture can take up to 6 wks. In this study the Polymerase Chain Reaction (PCR) was investigated as a rapid diagnostic technique for the detection of M. tuberculosis. The presence of DNA polymerase inhibitors in sputum specimens poses a potentially serious problem as false negative results can occur. In this study polymerase inhibitors were detected by inclusion of an internal plasmid control in each test. DNA from specimens in which the internal control failed to amplify was purified with a DNA binding matrix before retesting by PCR. A total of 169 sputum specimens were examined and 4 were found to have inhibitors. The correlation between detection of M. tuberculosis by PCR with a combination of culture, Ziehl‐Neelsen (ZN) staining and patient history was 97.6%. This study confirms that PCR offers a more sensitive and rapid alternative for the detection of M. tuberculosis to ZN staining and culture, with results being available within 24 hrs of a specimen being received in the laboratory.

Genetic diversity of Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis in Beira city, Mozambique

BACKGROUND Tuberculosis (TB) represents a serious public health problem in Mozambique, with an estimated incidence rate of 548 cases per 100,000 population in 2011. Information on the molecular epidemiology of Mycobacterium tuberculosis (MTB) strains circulating in Mozambique is limited. This study provides the first description of the genetic diversity of MTB strains circulating in Beira city, the second largest town in Mozambique. METHODS A total of 67 MTB isolates were tested to determine genetic lineages and diversity. The genetic lineages were determined using real-time PCR while genetic diversity was assessed by obtaining Mycobacterial Interspersed Repetitive Unit-Variable Numbers of Tandem Repeat profiles. RESULTS Only three of the six major lineages were represented, with 41 (61%) strains belonging to lineage 1, 25 (37%) belonging to lineage 4 and the remaining isolate belonging to lineage 3. No lineage 2 strains (containing the Beijing family) were identified. A high degree of diversity amongst the strains from both lineages 1 and 4 were observed. Comparison of the profiles of representative strains with those of reference strains in the MIRU-VNTRplus database revealed that all lineage 1 isolates clustered with the Eastern African Indian (EAI) 5 sub-family. The lineage 4 strains clustered with a variety of different sub-family strains, including the Latin-American-Mediterranean (LAM) 1 sub-family, the Haarlem, Uganda 1 and Cameroon sub-families and the T2-S sub-family. CONCLUSIONS The TB epidemic in Beira city is caused by a diverse group of MTB strains predominantly belonging to lineages 1 and 4.