Clinical evaluation of new diagnostic tests and development of testing strategies for tuberculosis diagnosis in Africa

Tuberculosis (TB) continues to kill more than 1.5 Mio people every year and causes a significant morbidity burden in the about 9 Mio patients who survived this infectious disease. Rapid and accurate TB diagnosis is considered one cornerstone of the global fight against TB. The “End TB Strategy” of the World Health Organization (WHO) is enforcing the need to develop new TB diagnostic tests, which are addressing the shortcomings of standard diagnostic tests that are currently used in TB epidemic and resource constrained settings like sub-Saharan Africa. In addition, to improved diagnostic tests, innovate testing strategies are needed to detect TB and control TB transmissions within specific risk groups such as prisoners, children and also TB contacts. In the frame of the presented habilitation project, three new diagnostic tests, namely the new Xpert MTB/RIF assay, which was endorsed by WHO in 2010, and two urine- based LAM tests, were evaluated in various clinical diagnostic studies in Tanzania. Further, a cross-sectional TB prevalence study was conducted in 13 Ethiopian prisons to study risk factors for TB in inmates and how successful currently implemented diagnostics algorithms are to detect TB in detention facilities. Finally, the isolated TB strains from these research studies were further analyzed using genotyping techniques in order to analyse mechanisms of TB transmission, spread of drug resistance and pathogenicity of TB strains in different high TB risk populations in Africa. For the Xpert MTB/RIF evaluation, the studies included adult and pediatric cohorts of patients with suspicion of TB. Further, the Xpert MTB/RIF assay was evaluated in a study with household contacts of smear-positive TB patients. Finally, the assay´s capacity to monitor TB treatment was assessed in TB patients who were enrolled in a therapeutic drug trial. The results of these studies contributed relevant information on the diagnostic accuracy of the assay and are also reflected by the current Xpert MTB/RIF policy recommendations from WHO. The most relevant findings were that Xpert MTB/RIF had a significant higher sensitivity compared to smear microscopy and detected up to 60% of smear negative, culture positive adult and paediatric TB cases. Xpert MTB/RIF performs equally well in HIV-positive and HIV-negative TB suspects and only one test in adults is sufficient to reach almost maximal sensitivity. Due to easy handling, rapid availability of test results and good performance in field conditions, the Xpert MTB/RIF test should be considered as the preferred test in contact tracing scenarios in Tanzania. However, due to sustained positive Xpert MTB/RIF results until the end of antimicrobial therapy in up to 27% of smear-positive TB patients, this assay is not useful to monitor microbiological response to TB treatment. The diagnostic capacity of the two LAM-assays was evaluated in a cohort of children with presumed TB. This study showed that LAM-sensitivity was highest, with maximal 70%, in HIV positive TB cases but had poor sensitivity, 28%, in HIV-negative TB suspects. Importantly, those groups of children suffering most from (co-) morbidities and high mortality were more likely to be LAM-positive. Therefore, specifically HIV-positive children with presumed active TB infection and advanced morbidity might benefit most from urine LAM-testing. This is in line with current WHO recommendations on the use of urine-based LAM tests. However, further scientific evidence is needed for a final evaluation of the use of LAM tests for the diagnosis of TB in clinical routine in Africa. The prison studies revealed that TB prevalence with about 450 TB cases among 100.000 convicts was twice as large as in the standard Ethiopian population. About 30% of existing TB cases were not detected by the prison health staff, whereby half on these were smear negative. Risk factors for TB in prisons were related to subject characteristics and behavior (e.g. alcohol drinking and TB contact at home) as well as to prison capacities (e.g. windows in prison cells). Genotypic analyses revealed that TB strains from prisoners were forming joint clusters with TB strains isolated from the Ethiopian standard population. These findings support the concept of interrelated TB epidemics among populations inside and outside prisons. Both sides need to be addressed in TB control programmes, and e.g. systematic and comprehensive TB screenings among new prisoners at entry as well as long-term prisoners might be an important strategy in order to end TB in high risk populations in Africa

Clinical evaluation of new diagnostic tests and development of testing strategies for tuberculosis diagnosis in Africa

Tuberculosis (TB) continues to kill more than 1.5 Mio people every year and causes a significant morbidity burden in the about 9 Mio patients who survived this infectious disease. Rapid and accurate TB diagnosis is considered one cornerstone of the global fight against TB. The “End TB Strategy” of the World Health Organization (WHO) is enforcing the need to develop new TB diagnostic tests, which are addressing the shortcomings of standard diagnostic tests that are currently used in TB epidemic and resource constrained settings like sub-Saharan Africa. In addition, to improved diagnostic tests, innovate testing strategies are needed to detect TB and control TB transmissions within specific risk groups such as prisoners, children and also TB contacts. In the frame of the presented habilitation project, three new diagnostic tests, namely the new Xpert MTB/RIF assay, which was endorsed by WHO in 2010, and two urine- based LAM tests, were evaluated in various clinical diagnostic studies in Tanzania. Further, a cross-sectional TB prevalence study was conducted in 13 Ethiopian prisons to study risk factors for TB in inmates and how successful currently implemented diagnostics algorithms are to detect TB in detention facilities. Finally, the isolated TB strains from these research studies were further analyzed using genotyping techniques in order to analyse mechanisms of TB transmission, spread of drug resistance and pathogenicity of TB strains in different high TB risk populations in Africa. For the Xpert MTB/RIF evaluation, the studies included adult and pediatric cohorts of patients with suspicion of TB. Further, the Xpert MTB/RIF assay was evaluated in a study with household contacts of smear-positive TB patients. Finally, the assay´s capacity to monitor TB treatment was assessed in TB patients who were enrolled in a therapeutic drug trial. The results of these studies contributed relevant information on the diagnostic accuracy of the assay and are also reflected by the current Xpert MTB/RIF policy recommendations from WHO. The most relevant findings were that Xpert MTB/RIF had a significant higher sensitivity compared to smear microscopy and detected up to 60% of smear negative, culture positive adult and paediatric TB cases. Xpert MTB/RIF performs equally well in HIV-positive and HIV-negative TB suspects and only one test in adults is sufficient to reach almost maximal sensitivity. Due to easy handling, rapid availability of test results and good performance in field conditions, the Xpert MTB/RIF test should be considered as the preferred test in contact tracing scenarios in Tanzania. However, due to sustained positive Xpert MTB/RIF results until the end of antimicrobial therapy in up to 27% of smear-positive TB patients, this assay is not useful to monitor microbiological response to TB treatment. The diagnostic capacity of the two LAM-assays was evaluated in a cohort of children with presumed TB. This study showed that LAM-sensitivity was highest, with maximal 70%, in HIV positive TB cases but had poor sensitivity, 28%, in HIV-negative TB suspects. Importantly, those groups of children suffering most from (co-) morbidities and high mortality were more likely to be LAM-positive. Therefore, specifically HIV-positive children with presumed active TB infection and advanced morbidity might benefit most from urine LAM-testing. This is in line with current WHO recommendations on the use of urine-based LAM tests. However, further scientific evidence is needed for a final evaluation of the use of LAM tests for the diagnosis of TB in clinical routine in Africa. The prison studies revealed that TB prevalence with about 450 TB cases among 100.000 convicts was twice as large as in the standard Ethiopian population. About 30% of existing TB cases were not detected by the prison health staff, whereby half on these were smear negative. Risk factors for TB in prisons were related to subject characteristics and behavior (e.g. alcohol drinking and TB contact at home) as well as to prison capacities (e.g. windows in prison cells). Genotypic analyses revealed that TB strains from prisoners were forming joint clusters with TB strains isolated from the Ethiopian standard population. These findings support the concept of interrelated TB epidemics among populations inside and outside prisons. Both sides need to be addressed in TB control programmes, and e.g. systematic and comprehensive TB screenings among new prisoners at entry as well as long-term prisoners might be an important strategy in order to end TB in high risk populations in Africa

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo.

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27(-) phenotype was associated with active TB in HIV(-) (p = 0.0003) and HIV(+) (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV(-) subjects, MTB-specific CD4 T cell populations from HIV(+) TB-asymptomatic subjects were often dominated by CD27(-) cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression

Prevalence of Pulmonary Tuberculosis among Prison Inmates in Ethiopia, a Cross-Sectional Study

Setting Tuberculosis (TB) is one of the major health problems in prisons. Objective This study was done to assess the prevalence and determinants of active tuberculosis in Ethiopian prisons. Design A cross-sectional study was conducted from January 2013 to December 2013 in 13 zonal prisons. All incarcerated inmates underwent TB symptom screening according to WHO criteria. From identified TB-suspects two sputum samples were analyzed using smear microscopy and solid culture. A standardized questionnaire assessing TB risk factors was completed for each TB suspect. Results 765 (4.9%) TB suspects were identified among 15, 495 inmates. 51 suspects were already on anti-TB treatment (6.67%) and 20 (2.8%) new culture-confirmed TB cases were identified in the study, resulting in an overall TB prevalence of 458.1/100, 000 (95% CI: 350-560/100, 000). Risk factors for active TB were alcohol consumption, contact with a TB case before incarceration and no window in prison cell. HIV prevalence was not different between TB suspects and active TB cases. Further, the TB burden in prisons increased with advancing distance from the capital Addis Ababa. Conclusions The overall TB prevalence in Ethiopian prisons was high and extremely variable among different prisons. TB risk factors related to conditions of prison facilities and the impact of implemented TB control measures need to be further studied in order to improve TB control among inmates

Tuberculosis and Sexual and Reproductive Health of Women in Four African Countries

Tuberculosis (TB) is a major reason of maternal mortality in low-income countries, and it increases the probability of adverse sexual and reproductive health (SRH) outcomes, including ectopic pregnancy and perinatal mortality. The data presented here is from the TB Sequel observational cohort conducted in four African countries. For this sub-study, we selected only female participants, who were diagnosed with drug susceptible TB and followed-up until the end of anti-TB treatment. The data collection included questionnaires, clinical examination and laboratory tests at TB diagnosis, day 14, month 2, 4 and 6. A total of 486 women, with 88.3% being 18-49 years old, were included in the analysis. Around 54.7% were HIV positive. Most of the participants (416/486;85.6%) in our cohort were considered cured at month 6. Only 40.4% of non-pregnant women of reproductive age used contraception at TB diagnosis. A total of 31 out of 486 women experienced pregnancy during TB treatment. Pregnancy outcomes varied between live birth (16/31;51.6%), induced abortion (6/31;19.4%), miscarriage (4/31;12.9%) and stillbirth (3/31;9.6%). Integration and linking of SRH services with TB programmes are vital to increase contraception use and protect women from obstetric risks associated with pregnancy during TB treatment

Evaluation of the burden of unsuspected pulmonary tuberculosis and co-morbidity with non-communicable diseases in sputum producing adult inpatients

A high burden of tuberculosis (TB) occurs in sub-Saharan African countries and many cases of active TB and drug-resistant TB remain undiagnosed. Tertiary care hospitals provide an opportunity to study TB co-morbidity with non-communicable and other communicable diseases (NCDs/CDs). We evaluated the burden of undiagnosed pulmonary TB and multi-drug resistant TB in adult inpatients, regardless of their primary admission diagnosis, in a tertiary referral centre. In this prospective study, newly admitted adult inpatients able to produce sputum at the University Teaching Hospital, Lusaka, Zambia, were screened for pulmonary TB using fluorescent smear microscopy and automated liquid culture. The burden of pulmonary TB, unsuspected TB, TB co-morbidity with NCDs and CDs was determined. Sputum was analysed from 900 inpatients (70.6% HIV infected) 277 (30.8%) non-TB suspects, 286 (31.8%) TB suspects and 337 (37.4%) were already receiving TB treatment. 202/900 (22.4%) of patients had culture confirmed TB. TB co-morbidity was detected in 20/275 (7.3%) NCD patients, significantly associated with diabetes (P = 0.006, OR 6.571, 95%CI: 1.706-25.3). 27/202 (13.4%) TB cases were unsuspected. There were 18 confirmed cases of MDR-TB, 5 of which were unsuspected. A large burden of unsuspected pulmonary TB co-morbidity exists in inpatients with NCDs and other CDs. Pro-active sputum screening of all inpatients in tertiary referral centres in high TB endemic countries is recommended. The scale of the problem of undiagnosed MDR-TB in inpatients requires further study

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

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

Health-related quality of life and psychological distress among adults in Tanzania: a cross-sectional study

Little data is available on health-related quality of life (HRQoL) and mental health of the general population in Tanzania. We aimed to describe HRQoL and level of psychological distress among adults in Mbeya and Songwe Regions of Tanzania. Methods We conducted a cross-sectional study between April and October 2019 in Mbeya and Songwe Regions. Data were collected using the Medical Outcomes Short Form-36 (SF-36) questionnaire and the Page Kessler Psychological Distress Scale (K10). We described demographic characteristics of participants and used log-binomial regression to identify participant characteristics associated with psychological distress (K10 score ≥ 20). Results A total of 393 adults were enrolled. The participants had a median age of 29 years (IQR 23–40) and 54.2% were male. Participants reported a physical component summary score (PCS) with a mean of 54.7 (SD7.1) and a mental component summary score (MCS) with a mean of 55.5 (SD5.1). Older participants (≥ 40 year) and those that were divorced/widowed reported lower physical functioning, energy/vitality and emotional well-being compared to their counterparts ( p < 0.05). In terms of psychological distress, majority of participants (78.4%; 305/389) reported that they were likely to be well (K10 score < 20), while 13.4% (52/389) reported to have mild (K10 score 20–24), 5.7% (22/389) moderate (K10 score 25–29), and 2.6% (10/389) severe (K10 score ≥ 30) psychological distress. Conclusions Physical function and mental well-being in this adult population from Tanzania were lower than that reported in other similar research in Tanzania and other African countries. This study provides valuable references for other research initiatives and clinical services in this region

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