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

Emerging viral infectious disease threat: Why Tanzania is not in a safe zone

Emerging diseases are global threat towards human existence. Every country is exposed to potentially emergence of infectious diseases. Several factor such as changes in ecology, climate and human demographics play different roles in a complex mechanism contributing to the occurrence of infectious diseases. Important aspects towards control in case of outbreaks are surveillance, preparedness and early response. Tanzania should therefore take opportunity of the calm situation currently present, to prepare. Except for HIV/AIDS, Tanzania has not experienced a major public health threat. However, the question is, is the country safe from emerging and re-emerging infectious diseases? In this article we try to explore the danger of emerging infectious disease (EID) epidemics in Tanzania and the risks attached if an outbreak is to occur. The aim is to formulate recommendations to the government, responsible authorities and general population of what can be done to improve the level of EID preparedness in the country. In conclusion, it is important to strengthen the capacity of community and healthcare staffs on how to respond to potential infectious disease outbreaks. Community-based surveillance systems should be incorporated into the national systems for early detection of public health events. It is also critical to enhance one health approach to increase cross-sectoral information sharing, surveillance and interventional strategies as regards to preparedness and response to disease outbreaks

Model-based relationship between the molecular bacterial load assay and time-to-positivity in liquid culture

The molecular bacterial load (MBL) assay is a new tuberculosis biomarker which provides results in ∼4 hours. The relationship between MBL and time-to-positivity (TTP) has not been thoroughly studied and predictive models do not exist. We aimed to develop a model for MBL and identify the MBL-TTP relationship in patients. The model was developed on data from 105 tuberculosis patients from Malawi, Mozambique and Tanzania with joint MBL and TTP observations quantified from patient sputum collected for 12 weeks. MBL was quantified using polymerase chain reaction (PCR) of mycobacterial RNA and TTP using the Mycobacterial Growth Indicator Tube (MGIT) 960 system. Treatment consisted of isoniazid, pyrazinamide and ethambutol in standard doses together with rifampicin 10 or 35 mg/kg. The developed MBL-TTP model included several linked sub-models; a component describing decline of bacterial load in sputum, another component describing growth in liquid culture and a hazard model translating bacterial growth into a TTP signal. Additional components for contaminated and negative TTP samples were included. Visual predictive checks performed using the developed model gave good description of the observed data. The model predicted greater total sample loss for TTP than MBL due to contamination and negative samples. The model detected an increase in bacterial killing for 35 versus 10 mg/kg rifampicin (p=0.002). In conclusion, a combined model for MBL and TTP was developed that described the MBL-TTP relationship. The full MBL-TTP model or each sub-model used separately. Secondly, the model can be used to predict biomarker response for MBL given TTP data or vice versa in historical or future trials.PostprintPeer 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

Wuchereria bancrofti infection is linked to systemic activation of CD4 and CD8 T cells

Background Susceptibility to HIV has been linked to systemic CD4+ T cell activation in cohorts of seronegative individuals with high HIV-exposure risk. We recently described an increased risk of HIV transmission in individuals infected with Wuchereria bancrofti, the causative agent for lymphatic filariasis, in a prospective cohort study. However, the reason for this phenomenon needs further investigation. Methodology/Principal findings Two-hundred and thirty-five HIV negative adults were tested using Trop Bio ELISA for detection of W. bancrofti infection and Kato Katz urine filtration and stool based RT-PCR for detection of soil transmitted helminths and schistosomiasis. FACS analysis of the fresh peripheral whole blood was used to measure T cell activation markers (HLA-DR, CD38), differentiation markers (CD45, CD27), markers for regulatory T cells (FoxP3, CD25) and the HIV entry receptor CCR5. Frequencies of activated HLA-DRpos CD4 T cells were significantly increased in subjects with W. bancrofti infection (n = 33 median: 10.71%) compared to subjects without any helminth infection (n = 42, median 6.97%, p = 0.011) or those with other helminths (Schistosoma haematobium, S. mansoni, Trichuris trichiura, Ascaris lumbricoides, hookworm) (n = 151, median 7.38%, p = 0.009). Similarly, a significant increase in HLA-DR(pos)CD38(pos) CD4 T cells and effector memory cells CD4 T cells (CD45RO(pos)CD27(neg)) was observed in filarial infected participants. Multivariable analyses further confirmed a link between W. bancrofti infection and systemic activation of CD4 T cells independent of age, fever, gender or other helminth infections. Conclusions/Significance W. bancrofti infection is linked to systemic CD4 T cell activation, which may contribute to the increased susceptibility of W. bancrofti infected individuals to HIV infection

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

Dispensing antibiotics without prescription at the community pharmacies and accredited drug dispensing outlets in Tanzania : a cross-sectional study

This study was part of the larger 3-country Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) project funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care, Award (MR/S004785/1).Worldwide, antimicrobial resistance is increasing rapidly and is highly associated with misuse of antimicrobials. The HATUA study (a broader 3-country study) investigated the antibiotic dispensing practices of pharmaceutical providers to clients, particularly the propensity to dispense without prescription. A cross-sectional study using a ‘mystery client’ method was conducted in 1,148 community pharmacies and accredited drugs dispensing outlets (ADDO) in Mwanza (n = 612), Mbeya (n = 304) and Kilimanjaro (n = 232) in Tanzania. Mystery clients asked directly for amoxicillin, had no prescription to present, did not discuss symptoms unless asked [when asked reported UTI-like symptoms] and attempted to buy a half course. Dispensing of amoxicillin without prescription was common [88.2, 95%CI 86.3%–89.9%], across all three regions. Further-more, a majority of outlets sold a half course of amoxicillin without prescription: Mwanza (98%), Mbeya (99%) and Kilimanjaro (98%). Generally, most providers in all three regions dispensed amoxicillin on demand, without asking the client any questions with [Chi2 = 11.8851 and p-value = 0.003]. In Mbeya and Kilimanjaro, providers in ADDOs were more likely to do this than those in pharmacies but no difference was observed in Mwanza. While the Tanzanian government has laws, regulations and guidelines that prohibit antibiotic dispensing without prescription, our study suggests non-compliance by drug providers. Enforcement, surveillance, and the provision of continuing education on dispensing practices is recommended, particularly for ADDO providers.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

Xpert MTB/RIF Ultra assay for the diagnosis of pulmonary tuberculosis in children: a multicentre comparative accuracy study

We evaluated the diagnostic performance of the novel next-generation Xpert MTB/RIF Ultra (Xpert Ultra) in comparison to Xpert MTB/RIF (Xpert) assay for the detection of paediatric pulmonary tuberculosis in high burden settings.; From May 2011 to September 2012, children with suspected pulmonary tuberculosis were enrolled at two Tanzanian sites and sputum samples were examined using sputum smear, Xpert and culture. Xpert Ultra was tested between January and June 2017 using sputum pellets, which had been stored at -80°C. The diagnostic accuracy of Ultra versus Xpert was determined using well-defined case definitions as reference standard.; In total, 215 children were included. The median age was 5.4 years, the HIV prevalence was 52% and 13% had culture-confirmed pulmonary tuberculosis. When only the first available sample of each patient was analysed, the sensitivity of Xpert Ultra was 64.3 % (95% CI: 44.1 to 81.4) while that of Xpert was 53.6% (95%CI: 33.9 to 72.5). The specificity of Xpert Ultra based on analysis of all available samples was 98.1% (95%CI: 93.4 to 99.7), that of Xpert was 100%.; Xpert Ultra was found to have a higher sensitivity, but slightly reduced specificity compared to Xpert in detecting pulmonary tuberculosis in children

Non-prescribed antibiotic dispensing practices for symptoms of urinary tract infection in community pharmacies and accredited drug dispensing outlets in Tanzania : a simulated clients approach

This study was funded by HATUA project. The Holistic Approach to Unravel Antibacterial Resistance in East Africa is a 3-year Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care. The award is also part of the EDCTP2 programme supported by the European Union.Background Antibiotic dispensing without prescription is a major determinant of the emergence of Antimicrobial Resistance (AMR) which has impact on population health and cost of healthcare delivery. This study used simulated clients describing UTI like symptoms to explore compliance with regulation, variations in dispensing practices and drug recommendation, and quality of seller-client interaction on the basis of the gender of the client and the type of drug outlets in three regions in Tanzania. Method A total of 672 Accredited Drug Dispensing Outlets (ADDOs) and community pharmacies were visited by mystery clients (MCs). The study was conducted in three regions of Tanzania namely Kilimanjaro (180, 26.79%), Mbeya (169, 25.15%) and Mwanza (323, 48.07%) in March–May 2020. During data collection, information was captured using epicollect5 software before being analyzed using Stata version 13. Results Overall, 89.43% (CI: 86.87–91.55%) of drug sellers recommended antibiotics to clients who described UTI like symptoms but held no prescription and 58.93% were willing to sell less than the minimum recommended course. Female clients were more likely than male to be asked if they were taking other medications (27.2% vs 9.8%), or had seen a doctor (27.8% vs 14.7%), and more likely to be advised to consult a doctor (21.6% vs 9.0%); pharmacies addressed these issues more often than ADDOs (17.7% vs 13.2, 23.9% vs 16.6%, 17.7 vs 10.9% respectively). Sellers recommended 32 different drugs to treat the same set of symptoms, only 7 appear in the Tanzanian Standard Treatment Guidelines as recommended for UTI and 30% were 2nd and 3rd line drugs. ADDO sellers recommended 31 drug types (including 2nd and 3rd line) but had permission to stock only 3 (1st line) drugs. The most commonly suggested antibiotics were Azithromycin (35.4%) and ciprofloxacin (20.5%). Azithromycin was suggested more often in pharmacies (40.8%) than in ADDOs (34.4%) and more often to male clients (36.0%) than female (33.1%). Conclusion These findings support the need for urgent action to ensure existing regulations are adhered to and to promote the continuing professional development of drug sellers at all outlet levels to ensure compliance with regulation, high quality service and better antibiotic stewardship.Publisher PDFPeer reviewe