Proteomic analysis of peripheral blood mononuclear cells isolated from patients with pulmonary tuberculosis: A pilot study from Zanzibar, Tanzania

This study aimed at exploring the proteomic profile of PBMCs to predict treatment response in pulmonary tuberculosis (PTB). This was a pilot study conducted among 8 adult patients from Zanzibar, Tanzania with confirmed PTB. Blood samples were collected at baseline, at 2 months of treatment, and at the end of treatment at 6 months. Proteins were extracted from PBMCs and analyzed using LC-MS/MS based label free quantitative proteomics. Overall, 3,530 proteins were quantified across the samples, and 12 differentially expressed proteins were identified at both 2 months of treatment and at treatment completion, which were involved in cellular and metabolic processes, as well as binding and catalytic activity. Seven were downregulated proteins (HSPA1B/HSPA1A, HSPH1, HSP90AA1, lipopolysaccharide-binding protein, complement component 9, calcyclin-binding protein, and protein transport protein Sec31A), and 5 proteins were upregulated (SEC14 domain and spectrin repeat-containing protein 1, leucine-rich repeat-containing 8 VRAC subunit D, homogentisate 1,2-dioxygenase, NEDD8-activating enzyme E1 regulatory subunit, and N-acetylserotonin O-methyltransferase-like protein). The results showed that proteome analysis of PBMCs can be used as a novel technique to identify protein abundance change with anti-tuberculosis treatment. The novel proteins elucidated in this work may provide new insights for understanding PTB pathogenesis, treatment, and prognosis.publishedVersio

Bedtime Salivary Cortisol as a Screening Test for Cushing Syndrome in Children

Background Diagnosing Cushing syndrome (CS) can be challenging. The 24-hour urine free cortisol (UFC) measurement is considered gold standard. This is a laborious test, dependent on correct urine collection. Late-night salivary cortisol is easier and is used as a screening test for CS in adults, but has not been validated for use in children. Objective To define liquid chromatography tandem mass spectrometry (LC-MS/MS)-based cutoff values for bedtime and morning salivary cortisol and cortisone in children, and validate the results in children with and without CS. Methods Bedtime and morning salivary samples were collected from 320 healthy children aged 4 to 16 years. Fifty-four patients from the children’s outpatient obesity clinic and 3 children with pituitary CS were used for validation. Steroid hormones were assayed by LC-MS/MS. Cutoff levels for bedtime salivary cortisol and cortisone were defined by the 97.5% percentile in healthy subjects. Results Bedtime cutoff levels for cortisol and cortisone were 2.4 and 12.0 nmol/L, respectively. Applying these cutoff levels on the verification cohort, 1 child from the obesity clinic had bedtime salivary cortisol exceeding the defined cutoff level, but normal salivary cortisone. All 3 children with pituitary CS had salivary cortisol and cortisone far above the defined bedtime cutoff levels. Healthy subjects showed a significant decrease in salivary cortisol from early morning to bedtime. Conclusions We propose that bedtime salivary cortisol measured by LC-MS/MS with a diagnostic threshold above 2.4 nmol/L can be applied as a screening test for CS in children. Age- and gender-specific cutoff levels are not needed.publishedVersio

Diagnosis and management of extrapulmonary tuberculosis in low-resource settings, a study from Zanzibar, Tanzania

Tuberculosis (TB) is still a major global public health concern. Of the 7 million incident TB cases recognized by the World Health Organization in 2018, 15% were extrapulmonary tuberculosis (EPTB) cases. Diagnosing EPTB remains a challenge despite continuing efforts and progress in the development of new TB diagnostic tools. Signs and symptoms of EPTB are often non-specific and similar in a range of other conditions. Laboratory confirmation of extrapulmonary disease is constrained by the difficulty of obtaining appropriate biological specimens, conventional diagnostic methods with low sensitivity and lack of an accurate, rapid, low-cost diagnostic test for EPTB. In addition, laboratory facilities are costly and often limited in low-resource settings. This may lead to diagnostic delay in initiating antituberculosis (anti-TB) treatment, on the other hand, starting empirical treatment, without laboratory confirmation, emphasises the need of close monitoring of treatment response. The overall aim of this thesis was to improve the diagnosis and management of EPTB cases in a low-resource setting. Keeping the primary objective in mind, the secondary aims were to implement and assess the performance of a new diagnostic test based on immunochemical detection of the Mycobacterium tuberculosis complex specific antigen MTP64 (MPT64 test), for diagnosing EPTB in the routine diagnostics at Mnazi Mmoja Hospital (MMH) in Zanzibar. Further, to evaluate the health care seeking pathways and the diagnostic delays experienced by presumptive EPTB patients, identify factors associated with longer diagnostic delay, and to assess the impact of anti-TB treatment on self-rated health among EPTB cases. Next, to describe the clinical presentation of EPTB, and follow the study participants during anti-TB treatment to assess clinical parameters, independent of laboratory investigations, which could aid in the monitoring of treatment response among EPTB cases. Presumptive EPTB patients of all ages were prospectively enrolled at MMH for thirteen months from august 2014. At inclusion, data were collected in a face-to face interview using the semi-structured study questionnaire, the results from a full physical examination were recorded and the adult patients initiating anti-TB treatment answered the EQ-5D-3L to evaluate the study participant`s self-rated health before treatment. Further, a biological specimen was collected from the site of assumptive EPTB infection and subjected to the MPT64 test, GeneXpert® MTB/RIF assay and routine laboratory diagnostics. The included patients initiating anti-TB treatment were assessed after the intensive phase of treatment and at treatment completion. The adult study participants again reported their self-rated health using the EQ-5D-3L after completing anti-TB treatment. Utilizing a predefined composite reference standard, the patients were classified as TB (confirmed TB, probable TB or possible TB) or non-TB cases to assess the diagnostic tests performances and other variables. We included 132 patients (median age 27 years, interquartile range 8-41 years), who were defined in accordance with the composite reference standard as TB cases (n=64 in paper I and III; n=69 in paper II) and non-TB cases (n=62 in paper I and III, n=63 in paper II). Six patients were classified as uncategorized cases in paper I and III. A higher proportion of positive test results was found for the MPT64 test in TB cases (45/69, 65%) as compared to ZN staining (8/69, 12%), culture (8/60, 13%) and the GeneXpert® MTB/RIF assay (6/38, 16%). The MPT64 test showed an overall sensitivity and specificity of 69% and 95%, respectively, with the best test performance demonstrated among TB lymphadenitis cases and in paediatric TB. Many EPTB cases experienced a delay exceeding two months from symptom onset until treatment was initiated, with health system delay as the main contributor to overall delay. The majority of adult TB cases described reduced work capacity with a median of 60 days due to the ongoing illness and using the EQ-5D instrument, a significantly improved self-perceived health status was noted after as compared to before anti-TB treatment. We further evaluated three clinical parameters, weight gain, clinical improvement and regression of objective findings during anti-TB treatment and found that most of the TB cases fulfilled ≥2 parameters after the intensive phase of anti-TB treatment. The MPT64 test is implementable in the routine diagnostic laboratory in this low-resource setting and has the potential to improve the diagnosis of EPTB, especially for lymph node TB and paediatric TB in this and similar settings. With many EPTB patients experiencing long delays in the initiation of treatment together with the reported reduced work capacity among the adult TB patients and improvement of self-reported health status after treatment, reducing the diagnostic delay and timely initiation of appropriate treatment can have crucial impact on the economic loss and morbidity of the affected patients. We propose that a combination of only clinical parameters can be incorporated in a simple assessment tool to aid health care workers in low-resource settings to monitor treatment response among EPTB patients. The findings from this study can be used to improve EPTB patient management in the current setting, but larger and more studies in various routine diagnostic settings are needed to expand the knowledge base regarding the MPT64 test and further evaluate and validate various simple clinical parameters to be incorporated as the suggested easy treatment response assessment tool

Diagnostic delay in extrapulmonary tuberculosis and impact on patient morbidity: A study from Zanzibar.

BACKGROUND:Early and proper treatment of tuberculosis could have an important impact on the morbidity, mortality and the economic situation of patients. There is insufficient knowledge on the extent of diagnostic delay and the associated factors in extrapulmonary tuberculosis (EPTB). The aims of this study were to assess the health care seeking behaviour, EPTB knowledge and diagnostic delay in presumptive EPTB patients at the main referral hospital in Zanzibar, factors associated with longer delay, and the impact of untreated EPTB on self-rated health. MATERIALS AND METHODS:Prospective data collection using a semi-structured questionnaire in patients presenting with symptoms suggestive of EPTB. The time between the onset of symptoms and first visit to a health care provider (patient delay), and then to the initiation of treatment (health system delay) and total delay were analysed according to sociodemographic and clinical factors and health care seeking trajectories. The EQ-5D-3L was used among the adult EPTB patients to assess the impact of treatment on self-rated health. RESULTS:Of the 132 patients with median age of 27 years (interquartile range 8-41), 69 were categorized as TB cases and 63 as non-TB cases. The median patient, health system and total delays were 14, 34 and 62 days respectively, among the EPTB patients. A longer health system delay with repeated visits to the same health care level was reported. Significantly better self-rated health status was described after treatment. The knowledge regarding extrapulmonary disease was low. CONCLUSION:Many EPTB patients, presenting to the main referral hospital in Zanzibar, experience a long delay in the initiation of treatment, specially patients with TB lymphadenitis. The health system delay is the major contributor to the total delay. The improvement of self-rated health after treatment implies that timely treatment has the potential to reduce morbidity and the economic loss for the patient

MPT64 antigen detection test improves routine diagnosis of extrapulmonary tuberculosis in a low-resource setting: A study from the tertiary care hospital in Zanzibar

Background: Extrapulmonary tuberculosis (EPTB) is a diagnostic challenge. An immunochemistry-based MPT64 antigen detection test (MPT64 test) has reported higher sensitivity in the diagnosis of EPTB compared with conventional methods. The objective of this study was to implement and evaluate the MPT64 test in routine diagnostics in a low-resource setting. Methods: Patients with presumptive EPTB were prospectively enrolled at Mnazi Mmoja Hospital, Zanzibar, and followed to the end of treatment. Specimens collected were subjected to routine diagnostics, GeneXpert® MTB/RIF assay and the MPT64 test. The performance of the MPT64 test was assessed using a composite reference standard, defining the patients as tuberculosis (TB) cases or non-TB cases. Results: Patients (n = 132) were classified as confirmed TB (n = 12), probable TB (n = 34), possible TB (n = 18), non-TB (n = 62) and uncategorized (n = 6) cases. Overall, in comparison to the composite reference standard for diagnosis, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the MPT64 test was 69%, 95%, 94%, 75% and 82%, respectively. The MPT64 test performance was best in TB lymphadenitis cases (n = 67, sensitivity 79%, specificity 97%) and in paediatric TB (n = 41, sensitivity 100%, specificity 96%). Conclusions: We show that the MPT64 test can be implemented in routine diagnostics in a low-resource setting and improves the diagnosis of EPTB, especially in TB lymphadenitis and in children

Evaluation of treatment response in extrapulmonary tuberculosis in a low-resource setting

Background Diagnosing extrapulmonary tuberculosis (EPTB) is challenging and many patients are initiated on empirical anti-TB treatment without a laboratory confirmed diagnosis. Monitoring treatment response is thus important to ensure correct diagnosis and proper disease management. The definition of satisfactory response to treatment in EPTB remains unclear. The objectives of this study were to describe the clinical presentation of EPTB and the effect of treatment on clinical parameters. Further, to assess if simple clinical parameters, without laboratory data, could evaluate treatment response. Methods Prospective cohort study of presumptive EPTB patients at Mnazi Mmoja Hospital, Zanzibar. By using a composite reference standard, patients were categorized as TB or non-TB cases. The TB patients were followed during anti-TB treatment. Results There were 64 TB and 62 non-TB cases. The frequency of symptoms at baseline were comparable in TB and non-TB patients, with lymphadenitis and pleuritis as the most common manifestations. Among TB cases, there was a trend towards regression of lymphadenopathy after 2 months, and at treatment completion 24/28 (86%) cases showed full regression. Weight gain ≥5% was reported in 36/49 (73%) of the TB patients at 2 months and in 38/46 (83%) at treatment completion. After 2 months of treatment, a combination of clinical parameters; improvement of symptoms (50/50), ≥5% weight gain (36/49) and regression of physical signs (45/49) correlated with the treatment response. Conclusions An algorithm including only simple clinical parameters could be used as an easy tool to assess treatment responses in low-resource settings. However, this needs to be tested on a larger sample size

IP-10 dried blood spots assay monitoring treatment efficacy in extrapulmonary tuberculosis in a low-resource setting

Treatment efficacy is difficult to evaluate in extrapulmonary tuberculosis (EPTB) patients. Interferon-γ inducible protein (IP-)10 has been suggested as a biomarker for response to treatment. We have investigated if IP-10 from dried plasma spots (DPS) or dried blood spots (DBS) can be used in treatment monitoring of EPTB patients in a low-resource setting of Zanzibar. IP-10 levels in plasma, DPS and DBS samples collected before, during (2 months) and after TB treatment of 36 EPTB patients (6 culture and/or Xpert MTB/RIF positive and 30 clinically diagnosed) and 8 pulmonary tuberculosis (PTB) patients, were quantified by an enzyme-linked immunosorbent assay. There was a high positive correlation between IP-10 measured in plasma and DPS and DBS, respectively. We found a significant decline in IP-10 levels from baseline to end of treatment in plasma, DPS and DBS, both in EPTB and PTB patients. The declines were observed already after 2 months in HIV negative patients. In conclusion, the DPS/DBS IP-10 assay allows for easy and manageable monitoring in low-resource settings and our findings suggest that IP-10 may serve as a biomarker for treatment efficacy in EPTB patients, albeit further studies in cohorts of patients with treatment failure and relapse are needed

IP-10 dried blood spots assay monitoring treatment efficacy in extrapulmonary tuberculosis in a low-resource setting

Treatment efficacy is difficult to evaluate in extrapulmonary tuberculosis (EPTB) patients. Interferon-γ inducible protein (IP-)10 has been suggested as a biomarker for response to treatment. We have investigated if IP-10 from dried plasma spots (DPS) or dried blood spots (DBS) can be used in treatment monitoring of EPTB patients in a low-resource setting of Zanzibar. IP-10 levels in plasma, DPS and DBS samples collected before, during (2 months) and after TB treatment of 36 EPTB patients (6 culture and/or Xpert MTB/RIF positive and 30 clinically diagnosed) and 8 pulmonary tuberculosis (PTB) patients, were quantified by an enzyme-linked immunosorbent assay. There was a high positive correlation between IP-10 measured in plasma and DPS and DBS, respectively. We found a significant decline in IP-10 levels from baseline to end of treatment in plasma, DPS and DBS, both in EPTB and PTB patients. The declines were observed already after 2 months in HIV negative patients. In conclusion, the DPS/DBS IP-10 assay allows for easy and manageable monitoring in low-resource settings and our findings suggest that IP-10 may serve as a biomarker for treatment efficacy in EPTB patients, albeit further studies in cohorts of patients with treatment failure and relapse are needed

MPT64 antigen detection test improves routine diagnosis of extrapulmonary tuberculosis in a low-resource setting: A study from the tertiary care hospital in Zanzibar

<div><p>Background</p><p>Extrapulmonary tuberculosis (EPTB) is a diagnostic challenge. An immunochemistry-based MPT64 antigen detection test (MPT64 test) has reported higher sensitivity in the diagnosis of EPTB compared with conventional methods. The objective of this study was to implement and evaluate the MPT64 test in routine diagnostics in a low-resource setting.</p><p>Methods</p><p>Patients with presumptive EPTB were prospectively enrolled at Mnazi Mmoja Hospital, Zanzibar, and followed to the end of treatment. Specimens collected were subjected to routine diagnostics, GeneXpert® MTB/RIF assay and the MPT64 test. The performance of the MPT64 test was assessed using a composite reference standard, defining the patients as tuberculosis (TB) cases or non-TB cases.</p><p>Results</p><p>Patients (n = 132) were classified as confirmed TB (n = 12), probable TB (n = 34), possible TB (n = 18), non-TB (n = 62) and uncategorized (n = 6) cases. Overall, in comparison to the composite reference standard for diagnosis, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the MPT64 test was 69%, 95%, 94%, 75% and 82%, respectively. The MPT64 test performance was best in TB lymphadenitis cases (n = 67, sensitivity 79%, specificity 97%) and in paediatric TB (n = 41, sensitivity 100%, specificity 96%).</p><p>Conclusions</p><p>We show that the MPT64 test can be implemented in routine diagnostics in a low-resource setting and improves the diagnosis of EPTB, especially in TB lymphadenitis and in children.</p></div

Demographic and baseline characteristics of the 126 categorized study participants, n(%).

<p>Demographic and baseline characteristics of the 126 categorized study participants, n(%).</p