Tuberculosis bacteria detection and counting in fluorescence microscopy images using a multi-stage deep learning pipeline

The manual observation of sputum smears by fluorescence microscopy for the diagnosis and treatment monitoring of patients with tuberculosis (TB) is a laborious and subjective task. In this work, we introduce an automatic pipeline which employs a novel deep learning-based approach to rapidly detect Mycobacterium tuberculosis (Mtb) organisms in sputum samples and thus quantify the burden of the disease. Fluorescence microscopy images are used as input in a series of networks, which ultimately produces a final count of present bacteria more quickly and consistently than manual analysis by healthcare workers. The pipeline consists of four stages: annotation by cycle-consistent generative adversarial networks (GANs), extraction of salient image patches, classification of the extracted patches, and finally, regression to yield the final bacteria count. We empirically evaluate the individual stages of the pipeline as well as perform a unified evaluation on previously unseen data that were given ground-truth labels by an experienced microscopist. We show that with no human intervention, the pipeline can provide the bacterial count for a sample of images with an error of less than 5%.Publisher PDFPeer reviewe

Systematic assessment of clinical and bacteriological markers for tuberculosis reveals discordance and inaccuracy of symptom-based diagnosis for treatment response monitoring

This work was supported by Commonwealth PhD studentship award to Dr Bariki Mtafya (Award number: TZS-2016-718) at University of St Andrews and European and Developing Countries Clinical Trials Partnership through TWENDE project (grant number; TWENDE-EDCTP-CSA-2014-283) and PanACEA II (grant number; 97118-PanACEA-TRIA.2015.1102) awarded to Professor Stephen Gillespie and Dr Wilber Sabiiti at the University of St Andrews, UK.Background : Clinical symptoms are the benchmark of tuberculosis (TB) diagnosis and monitoring of treatment response but is not clear how they relate to TB bacteriology, particularly the novel tuberculosis Molecular Bacterial Load Assay (TB-MBLA). Methods : Presumptive cases were bacteriologically confirmed for TB and assessed for symptom and bacteriological resolution using smear microscopy (SM), culture and TB-MBLA over 6-month treatment course. Kaplan Meier and Kappa statistics were used to test relationship between symptom- and bacteriological-positivity. Results : A cohort of 46 bacteriologically confirmed TB cases were analysed for treatment response over a six-month treatment course. Pre-treatment symptom and bacteriological positivity concurred in over 70% of the cases. This agreement was lost in over 50% of cases whose chest pain, night sweat, and loss of appetite had resolved by week 2 of treatment. Cough resolved at a 3.2% rate weekly and was 0.3% slower than the combined bacteriological (average of MGIT and TB-MBLA positivity) resolution rate, 3.5% per week. Drop in TB-MBLA positivity reflected fall in bacillary load, 5.7±1.3- at baseline to 0.30±1.0- log10 eCFU/mL at month 6, and closer to cough resolution than other bacteriological measures, accounting for the only one bacteriologically positive case out of seven still coughing at month 6. Low baseline bacillary load patients were more likely to be bacteriologically negative, HR 5.6, p=0.003 and, HR 3.2, p=0.014 by month-2 and 6 of treatment respectively. Conclusion : The probability of clinical symptoms reflecting bacteriological positivity weakens as patient progresses on anti-TB therapy, making symptom-based diagnosis a less reliable marker of treatment response.Publisher PDFPeer reviewe

Molecular bacterial load assay (MBLA) concurs with culture on the NaOH-induced Mycobacterium tuberculosis loss of viability

This work was supported by the commonwealth studentship award for Bariki Mtafya at University of St Andrews in UK and European and Developing Countries Clinical Trials Partnership (EDCTP) through TWENDE and PanACEA II grants.Effective methods to detect viable Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB) are urgently needed. To date, cultivation of Mtb is the gold standard which depends on initial sample processing with N-Acetyl-L-Cysteine/Sodium hydroxide (NALC/NaOH), chemicals that compromise Mtb viability and, consequently the performance of downstream tests. We applied culture and the novel Molecular bacterial load assay (MBLA) to measure the loss of Mtb viability following NALC/NaOH treatment of Mtb H37Rv pure culture and clinical sputa from pulmonary TB patients. Compared to untreated controls, NALC/NaOH treatment of Mtb, reduced MBLA detectable bacillary load (estimated colony forming units/milliliter (eCFU/mL) by 0.66±0.21log10- at 23°C (P=0.018) and 0.72±0.08log10- at 30°C (P=0.013). Likewise, NALC/NaOH treatment reduced viable count on solid culture by 0.84±0.02log10- at 23°C (P<0.001) and 0.85±0.01log10- CFU/mL at 30°C (P<0.001) respectively. The reduction in viable count was reflected by a corresponding increase in time to positivity of MGIT liquid culture, 1.2 days at 23°C (P<0.001), and 1.1 days at 30°C (P<0.001). This NaOH-induced Mtb viability loss was replicated in clinical sputum samples, with bacterial load dropping by 0.65±0.17log10 from 5.36±0.24log10- to 4.71±0.16log10- eCFU/mL for untreated and treated sputa respectively. Applying the Bowness et al model, revealed that the treated MGIT time to culture positivity of 142hrs was equivalent to 4.86±0.28log10CFU, consistent with MBLA-measured bacterial load. Our study confirms the contribution of NALC/NaOH treatment to loss of viable bacterial count. Tests that obviate the need of decontamination may offer alternative option for accurate detection of viable Mtb and treatment response monitoring.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

Increased and Expedited Case Detection by Xpert MTB/RIF Assay in Childhood Tuberculosis: A Prospective Cohort Study

The Xpert MTB/RIF assay is a quick and accurate tuberculosis diagnostic tool in children. Compared with microscopy, 3-fold more tuberculosis cases were detected with a similar turnaround time, resulting in a potentially shortened time to tuberculosis diagnosi

Performance of Tuberculosis Molecular Bacterial Load Assay Compared to Alere TB-LAM in Urine of Pulmonary Tuberculosis Patients with HIV Co-Infections

This research article was published by MDPI in 2023Alternative tools are needed to improve the detection of M. tuberculosis (M. tb) in HIV co-infections. We evaluated the utility of Tuberculosis Molecular Bacterial Load Assay (TB-MBLA) compared to lipoarabinomannan (LAM) to detect M. tb in urine. Sputum Xpert MTB/RIF-positive patients were consented to provide urine at baseline, weeks 2, 8, 16, and 24 of treatment for TB-MBLA, culture, and LAM. Results were compared with sputum cultures and microscopy. Initial M. tb. H37Rv spiking experiments were performed to validate the tests. A total of 63 urine samples from 47 patients were analyzed. The median age (IQR) was 38 (30–41) years; 25 (53.2%) were male, 3 (6.5%) had urine for all visits, 45 (95.7%) were HIV positive, of whom 18 (40%) had CD4 cell counts below 200 cells/µL, and 33 (73.3%) were on ART at enrollment. Overall urine LAM positivity was 14.3% compared to 4.8% with TB-MBLA. Culture and microscopy of their sputum counterparts were positive in 20.6% and 12.7% of patients, respectively. Of the three patients with urine and sputum at baseline, one (33.33%) had urine TB-MBLA and LAM positive compared to 100% with sputum MGIT culture positive. Spearman’s rank correction coefficient (r) between TB-MBLA and MGIT was −0.85 and 0.89 with a solid culture, p > 0.05. TB-MBLA has the promising potential to improve M. tb detection in urine of HIV-co-infected patients and complement current TB diagnostics

Effect of seven anti-tuberculosis treatment regimens on sputum microbiome : a retrospective analysis of the HIGHRIF study 2 and PanACEA MAMS-TB clinical trials

Funding: European and Developing Countries Clinical Trials Partnership and German Ministry of Education and Research.Background Respiratory tract microbiota has been described as the gatekeeper for respiratory health. We aimed to assess the impact of standard-of-care and experimental anti-tuberculosis treatment regimens on the respiratory microbiome and implications for treatment outcomes. Methods In this retrospective study, we analysed the sputum microbiome of participants with tuberculosis treated with six experimental regimens versus standard-of-care who were part of the HIGHRIF study 2 (NCT00760149 ) and PanACEA MAMS-TB (NCT01785186 ) clinical trials across a 3-month treatment follow-up period. Samples were from participants in Mbeya, Kilimanjaro, Bagamoyo, and Dar es Salaam, Tanzania. Experimental regimens were composed of different combinations of rifampicin (R), isoniazid (H), pyrazinamide (Z), ethambutol (E), moxifloxacin (M), and a new drug, SQ109 (Q). Reverse transcription was used to create complementary DNA for each participant's total sputum RNA and the V3-V4 region of the 16S rRNA gene was sequenced using the Illumina metagenomic technique. Qiime was used to analyse the amplicon sequence variants and estimate alpha diversity. Descriptive statistics were applied to assess differences in alpha diversity pre-treatment and post-treatment initiation and the effect of each treatment regimen. Findings Sequence data were obtained from 397 pre-treatment and post-treatment samples taken between Sept 26, 2008, and June 30, 2015, across seven treatment regimens. Pre-treatment microbiome (206 genera) was dominated by Firmicutes (2860 [44%] of 6500 amplicon sequence variants [ASVs]) at the phylum level and Streptococcus (2340 [36%] ASVs) at the genus level. Two regimens had a significant depressing effect on the microbiome after 2 weeks of treatment, HR20mg/kgZM (Shannon diversity index p=0·0041) and HR35mg/kgZE (p=0·027). Gram-negative bacteria were the most sensitive to bactericidal activity of treatment with the highest number of species suppressed being under the moxifloxacin regimen. By week 12 after treatment initiation, microbiomes had recovered to pre-treatment level except for the HR35mg/kgZE regimen and for genus Mycobacterium, which did not show recovery across all regimens. Tuberculosis culture conversion to negative by week 8 of treatment was associated with clearance of genus Neisseria, with a 98% reduction of the pre-treatment level. Interpretation HR20mg/kgZM was effective against tuberculosis without limiting microbiome recovery, which implies a shorter efficacious anti-tuberculosis regimen with improved treatment outcomes might be achieved without harming the commensal microbiota.Publisher PDFPeer reviewe

INNOVATIONS AND FRAGMENTS OF TRADITION IN SLOVENIAN VILLAGES

In this paper the author reports on some results of an investigation of innovations in ten different types of village in Slovenia. The investigation was designed to establish (1) the rate of development of rural communities in Slovenia (measured according to the diffusion of innovations), and (2) the influence of tradition (from the degree of the distribution of »isoetes«, i.e. local customs). , Two groups of innovations were investigated: innovations in land cultivation methods on peasant farms, and innovations in the equipment of rural households. The data were collected using the method of interviews, which were held with both pure farmers and peasants-workers in 1968 and again in 1973. The obtained results regarding the spread of farm innovations are shown in Diagram 1; the results regarding the spread of innovations in household equipment are presented graphically in Diagram 2; while Diagram 3 indicates the »standing« of each village as regards the degree of innovation it has achieved with all the 25 controled innovations. As was expected, the highest degree of innovation is shown by villages with the largest proportion of inhabitants in temporary employment abroad and villages vhich contain a comparatively numerous category of peasants-workers

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

Rapid and Accurate Detection of Mycobacterium tuberculosis in Sputum Samples by Cepheid Xpert MTB/RIF Assay-A Clinical Validation Study

Background: A crucial impediment to global tuberculosis control is the lack of an accurate, rapid diagnostic test for detection of patients with active TB. A new, rapid diagnostic method, (Cepheid) Xpert MTB/RIF Assay, is an automated sample preparation and real-time PCR instrument, which was shown to have good potential as an alternative to current reference standard sputum microscopy and culture.Methods: We performed a clinical validation study on diagnostic accuracy of the Xpert MTB/RIF Assay in a TB and HIV endemic setting. Sputum samples from 292 consecutively enrolled adults from Mbeya, Tanzania, with suspected TB were subject to analysis by the Xpert MTB/RIF Assay. The diagnostic performance of Xpert MTB/RIF Assay was compared to standard sputum smear microscopy and culture. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis.Results: Xpert MTB/RIF Assay achieved 88.4% (95% CI = 78.4% to 94.9%) sensitivity among patients with a positive culture and 99% (95% CI = 94.7% to 100.0%) specificity in patients who had no TB. HIV status did not affect test performance in 172 HIV-infected patients (58.9% of all participants). Seven additional cases (9.1% of 77) were detected by Xpert MTB/RIF Assay among the group of patients with clinical TB who were culture negative. Within 45 sputum samples which grew non-tuberculous mycobacteria the assay's specificity was 97.8% (95% CI = 88.2% to 99.9%).Conclusions: The Xpert MTB/RIF Assay is a highly sensitive, specific and rapid method for diagnosing TB which has potential to complement the current reference standard of TB diagnostics and increase its overall sensitivity. Its usefulness in detecting sputum smear and culture negative patients needs further study. Further evaluation in high burden TB and HIV areas under programmatic health care settings to ascertain applicability, cost-effectiveness, robustness and local acceptance are required