OMNIgene.SPUTUM suppresses contaminants whilst maintaining Mycobacterium tuberculosis viability and obviates cold-chain transport

The study was funded by DNA Genotek Inc. Canada, the manufacturer of OMNIgene.SPUTUM (grant number SMD0-Z0B014).Tuberculosis (TB) diagnostics are centralised, requiring long-distance transportation of specimens in most resource-limited settings. We evaluated the ability of OMNIgene.SPUTUM (OM-S) to obviate cold-chain transport of TB specimens. A two-arm (same-day and after 5 days sample processing) study was conducted to assess contamination rates and Mycobacterium tuberculosis viability in OM-S-treated samples against the standard decontamination procedure (SDP) in Mozambique, using Lowenstein Jensen (LJ) and mycobacterial growth indicator tube (MGIT) culture and molecular bacterial load assay. 270 specimens were processed using OM-S and SDP in same-day and 5-day arms. Contamination was lower in OM-S-treated than SDP-treated cultures: 12% versus 15% and 2% versus 27% in the same-day and 5-day arms, respectively. M. tuberculosis recovery in OM-S-treated LJ cultures was 10% and 56% higher in the same-day and 5-day arms, respectively, than SDP-treated cultures, but lower in MGIT (52% and 28% lower in the same-day and 5-day arms, respectively). M. tuberculosis viable count was 1log estimated CFU·mL−1 lower in 5-day OM-S-treated sputa. OM-S was more effective at liquefying sputum with a shorter sample processing time: 22 min for culture. OM-S is simple to use and has demonstrated a high potency to suppress contaminants, maintenance of viability at ambient temperatures and higher M. tuberculosis recovery, particularly in the solid LJ cultures. Optimisation of OM-S to achieve higher MGIT culture positivity and shorter time to result will increase its application and utility in the clinical management of TB.Publisher PDFPeer reviewe

Heat-inactivation renders sputum safe and preserves Mycobacterium tuberculosis RNA for downstream molecular tests

The study was made possible by funding from the European and Developing Countries Clinical Trials Partnership (EDCTP) – Pan African Biomarker Expansion program (PanBIOME) grant SP.2011.41304.008. Support was also obtained the University of St Andrews School of Medicine research grant.The World Health Organization End tuberculosis (TB) strategy has called for development of- and increased access to- effective tools for diagnosis and treatment of TB disease. Mycobacterium tuberculosis (Mtb), the causative agent of TB is categorized as highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category three laboratory. We have evaluated the use of heat-inactivation to render TB samples safe to work with whilst preserving RNA for downstream molecular tests. Using Mycobacterium bovis Bacillus Calmette Guérin (BCG) cultures and TB positive sputa we show that boiling for 20 min at 80-, 85-, and 95- ºC inactivates all Mtb bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85 and the untreated sputa flagged positive at 3 days of incubation in Mycobacterium Growth Indicator Tube. Quantification of reference genes, 16S rRNA, tmRNA, pre-16S rRNA and rpoB by Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA-species dependent, <1log10, 1.1log10, 1.3log10 and 2.4log10 estimated CFU/ml for 16S rRNA, tmRNA, pre-16S and rpoB respectively. The RNA loss was independent of inactivation temperature. These findings show that heat-inactivation could obviate the need for category three laboratory to perform RNA-based testing of TB samples.PostprintPeer reviewe

A tuberculosis molecular bacterial load assay (TB-MBLA)

Funding: European and Developing Countries Clinical Trials Partnership (EDCTP) – Pan African Biomarker Expansion program (PanBIOME) grant SP.2011.41304.008. Support was also obtained the University of St Andrews School of Medicine research grant.Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers’ safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.Publisher PDFPeer reviewe

Reduction of blood C-reactive protein concentration complements the resolution of sputum bacillary load in patients on anti-tuberculosis therapy

Funding: This study was conducted under the PanACEA Biomarkers Expansion (PanBIOME) programme and the establishment of Maputo Tuberculosis Trial Unit (MaTuTU Project) which was funded in parts through the European and Developing Countries Clinical Trials Partnership (EDCTP), PZA-study and Federal Ministry of Education and Research (BMBF), Germany.Background: Tuberculosis (TB) is a difficult-to-treat disease requiring the combination of four antibiotics for a minimum of 6 months. Rapid and quantitative biomarkers to monitor treatment response are urgently needed for individual patient management and clinical trials. C-reactive protein (CRP) is often used clinically as a rapid marker of inflammation caused by infection. We assessed the relationship of TB bacillary load and CRP as biomarkers of treatment response. Methods: Xpert MTB/RIF-confirmed pulmonary TB cases were enrolled for treatment response assessment in Mozambique. Treatment response was measured using the Tuberculosis Molecular Bacterial Load Assay (TB-MBLA) in comparison with standard-of-care Mycobacterium Growth Indicator Tube (MGIT) culture at baseline and at weeks 1, 2, 4, 8, 12, 17, and 26 of treatment. Blood CRP concentration was measured at baseline, week 8, and week 26. Treatment response was defined as increase in MGIT culture time to positivity (TTP), and reduction in TB-MBLA-measured bacillary load and blood CRP concentration. Results: Out of the 81 screened presumptive TB cases, 69 were enrolled for 6-month treatment follow-up resulting in 94% treatment completion rate. Four participants did not complete TB treatment and 22 participants had missing CRP or TB-MBLA results and were excluded from TB-MBLA-CRP analysis. The remaining 43 participants—median age, 31 years old [interquartile range (IQR): 18–56]; 70% (30/43) male; and 70% (30/43) infected with HIV—were considered for analysis. Culture TTP and bacillary load were inversely correlated, Spearman’s r = −0.67, p < 0.0001. Resolution of sputum bacillary load concurred with reduction of blood CRP, r = 0.70, p < 0.0001. At baseline, bacillary load had a median (IQR) of 6.4 (5.5–7.2), which reduced to 2.4 (0.0–2.9) and 0.0 (0.0–0.0) log10 CFU/ml at months 2 and 6 of treatment, respectively. Correspondingly, blood CRP reduced from 1.9 (1.6–2.1) at baseline to 1.3 (0.9–1.7) and 0.4 (0.1–0.8) log10 mg/dl at months 2 and 6 of treatment, respectively. CRP reduction trialed bacteriological resolution at a rate of −0.06 log10 mg/dl compared to a bacillary load of 0.23 log10 CFU/ml per week. Consequently, 14 (33%) and 37 (88%) patients had reduced CRP to normal concentration and bacillary load to zero by the end of treatment, respectively. Pre-treatment CRP concentration and bacillary load, and resolution during treatment were slightly lower in HIV co-infected patients but not significantly different from HIV-uninfected TB patients. Conclusion: TB-MBLA-measured bacillary load and blood CRP complement each other in response to anti-TB therapy. Slow CRP reduction probably reflects residual TB bacilli in the lung not expectorated in sputum. Combining both measures can improve the accuracy of these biomarkers for monitoring TB treatment response and shorten turnaround time since the results of both assays could be available in 24 h.Publisher PDFPeer reviewe

Optimising molecular diagnostic capacity for effective control of tuberculosis in high-burden settings

The World Health Organization's 2035 vision is to reduce tuberculosis (TB) associated mortality by 95%. While low-burden, well-equipped industrialised economies can expect to see this goal achieved, it is challenging in the low- and middle-income countries that bear the highest burden of TB. Inadequate diagnosis leads to inappropriate treatment and poor clinical outcomes. The roll-out of the Xpert® MTB/RIF assay has demonstrated that molecular diagnostics can produce rapid diagnosis and treatment initiation. Strong molecular services are still limited to regional or national centres. The delay in implementation is due partly to resources, and partly to the suggestion that such techniques are too challenging for widespread implementation. We have successfully implemented a molecular tool for rapid monitoring of patient treatment response to anti-tuberculosis treatment in three high TB burden countries in Africa. We discuss here the challenges facing TB diagnosis and treatment monitoring, and draw from our experience in establishing molecular treatment monitoring platforms to provide practical insights into successful optimisation of molecular diagnostic capacity in resource-constrained, high TB burden settings. We recommend a holistic health system-wide approach for molecular diagnostic capacity development, addressing human resource training, institutional capacity development, streamlined procurement systems, and engagement with the public, policy makers and implementers of TB control programmes.PostprintPeer reviewe

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