Host biomarkers for monitoring therapeutic response in extrapulmonary tuberculosis.

PURPOSE: The aim of this study was to explore the utility of inflammatory biomarkers in the peripheral blood to predict response to treatment in extrapulmonary tuberculosis (EPTB). METHODS: A Luminex xMAP-based multiplex immunoassay was used to measure 40 inflammatory biomarkers in un-stimulated plasma of 91 EPTB patients (48 lymphadenitis, and 43 pleuritis) before and at 2 and 6 months of treatment. RESULTS: Overall a significant change was observed in 28 inflammatory biomarkers with treatment in EPTB patients. However, MIG/CXCL9, IP-10/CXCL10, and CCL23 decreased in all patients' groups with successful treatment at both time points. At 2 months, 29/64 (45%) patients responded partially while 35/64 (55%) showed complete regress. Among good responders, a higher number of biomarkers (16/40) reduced significantly as compared to partial responders (1/40). Almost half (14/29) of partial responders required longer treatment than 6 months to achieve satisfactory response. The levels of MIG, IP-10, MIF, CCL22 and CCL23 reduced significantly among 80, 74, 60, 71, 51% good responders, as compared to 52, 52, 52, 59, 52% partial responders, respectively. A biosignature, defined by a significant decrease in any one of these five biomarkers, corresponded with satisfactory response to treatment in 97% patients at 2 month and 99% patients at 6 months of treatment. CONCLUSION: Change in inflammatory biomarkers correlates with treatment success. A five biomarker biosignature (MIG, IP-10, MIF, CCL22 and CCL23) could be used as an indicator of treatment success.

Isolation of non-tuberculous mycobacteria among tuberculosis patients, a study from a tertiary care hospital in Lahore, Pakistan.

BACKGROUND: There is scarce knowledge on the prevalence of diseases caused by non-tuberculous mycobacteria (NTM) in Pakistan. In the absence of culture and identification, acid-fast bacilli (AFB) causing NTM disease are liable to be misinterpreted as tuberculosis (TB). Introduction of nucleic acid amplification testing for Mycobacterium tuberculosis complex (MTBC) offers improved diagnostic accuracy, compared with smear microscopy, and also assists in differentiating MTBC from other mycobacteria. This study aimed to investigate the prevalence of NTM among patients investigated for TB and describe NTM disease and treatment outcomes at a tertiary care hospital in Pakistan. METHODS: This is a retrospective study, data on NTM isolates among culture-positive clinical samples over 4 years (2016-19) was retrieved from laboratory records. Information on clinical specimens processed, AFB smear results, and for the AFB positive isolates, results of species identification for MTBC, and for NTM isolates, results of species characterization and drug susceptibility testing was collected. Additional clinical data including patient characteristics, treatment regimens, and outcomes were collected for patients with NTM disease treated at Gulab Devi Hospital, Lahore. RESULTS: During the study period, 12,561 clinical specimens were processed for mycobacterial culture and 3673 (29%) were reported positive for AFB. Among these 3482 (95%) were identified as MTBC and 191 (5%) as NTM. Among NTM, 169 (88%) were isolated from pulmonary and 22 (12%) from extrapulmonary specimens. Results of NTM speciation were available for 60 isolates and included 55% (n = 33) M. avium complex and 25% (n = 15) M. abscesses. Among these patients, complete clinical records were retrieved for 12 patients with pulmonary disease including nine infected with M. avium complex and three with M. abscessus. All 12 patients had a history of poor response to standard first-line anti-TB treatment. Ten patients were cured after 18 months of treatment, whereas, one with M. abscessus infection died and another was lost to follow up. CONCLUSION: In TB endemic areas, NTM can be misdiagnosed as pulmonary TB leading to repeated failed anti-TB treatment and increased morbidity, emphasizing the need for improved diagnosis.

Viable Mycobacterium tuberculosis in sputum after pulmonary tuberculosis cure.

BACKGROUND: Pulmonary tuberculosis (TB) with detectable Mycobacterium tuberculosis in the sputum is a major source of transmission. In resource limited TB endemic settings, cure is declared through sputum smear examination for acid fast bacilli without performing culture. This may lead to erroneous treatment outcomes as viable bacteria may be missed due to the low sensitivity of direct smear method. The aim of this study was to investigate if sterilizing cure is achieved among the new pulmonary TB cases declared cured by sputum smear conversion and to evaluate the impact of addition of ethambutol in the continuation phase in achieving it. METHODS: New sputum smear-positive pulmonary TB patients registered at a tertiary care hospital in Pakistan from November 2013 to March 2014 were followed under standard Directly Observed Treatment Short Course strategy for 6 months. Half of these patients received ethambutol in addition to isoniazid and rifampicin in the continuation phase. Sputum specimens were examined on microscopy at 2 months and at the end of treatment. Sputa of patients with negative direct smear examination at the end of treatment were cultured. RESULTS: Among 5746 TB suspects, 1595 were new sputum smear positive pulmonary TB cases, and 533 were registered at our hospital. Among these, 504 converted sputum negative at 2 months and 348 converted at the end of 6 months of treatment and were declared cured. Sputa of 204/348 patients were cultured, and 12/204 (6%) were culture-positive. Culture positivity at 6 months was not associated with bacterial load, smoking, diabetes, presence of cavities, history of contact with TB patients, age, sex, socioeconomic status, or addition of ethambutol in the continuation phase. CONCLUSION: Viable cultivable bacilli were detected in 6% of cured patients, which would have significant impact on the control of TB. This highlights the need for an inexpensive and accurate surrogate marker for culture as it is not feasible to perform culture in routine for monitoring treatment response in the low-resource settings. The treatment outcome did not improve by addition of ethambutol emphasizing the need to find the optimal duration of treatment for individual or carefully selected groups of patients.

High prevalence of diabetes and anthropometric heterogeneity among tuberculosis patients in Pakistan.

BACKGROUND: In Pakistan, the prevalence of diabetes (DM) among adults is 6.9% and expected to double by 2040. DM may facilitate transmission and halter the elimination of tuberculosis (TB). We aimed to determine the prevalence of DM among patients with TB in Pakistan, and to investigate anthropometric biochemical and haemodynamic associations between TB patients with and without DM. METHODS: We conducted a cross-sectional study at Gulab Devi Chest Hospital in Lahore, Punjab. A total of 3027 newly diagnosed smear-positive TB patients ≥25 years of age were screened for DM by HbA1c regardless of previous DM history. RESULTS: The prevalence of screen-detected DM and known DM among the TB participants was 13.5% and 26.1%, respectively, resulting in a combined DM prevalence of 39.6%. Most participants were male (64.4%). Using bivariate analyses, participants with DM were significantly older (49.8 vs. 40.6 years) with higher haemoglobin (men, 12.1 vs. 11.8 g/dl, women 11.5 vs. 10.7 g/dl), body mass index (21.0 vs. 17.6 kg/m2 ) and waist-hip ratio (men, 0.87 vs. 0.81, women, 0.87 vs. 0.79) (all P < 0.05) than participants without DM. Stratifying by screen-detected and known DM, these differences remained significant when using multivariate analysis. CONCLUSION: We report a high prevalence of DM among patients with TB who may be anthropometrically and biochemically distinct from TB patients without DM, and this heterogeneity further transcends the different DM groups.

Persistently high plasma procalcitonin levels despite successful treatment of tuberculous pleuritis and tuberculous lymphadenitis patients.

In a prospective cohort study, we evaluated plasma PCT levels in 48 TB lymphadenitis (TBLN) and 41 TB pleuritis (TBPE) patients. Measurements of PCT were done in unstimulated plasma of microbiologically and clinically confirmed TBLN and TBPE patients registered for anti-TB treatment at a tertiary care hospital in Lahore, Pakistan. Plasma levels of PCT were found to be raised in 89% of the patients at baseline with a median of 1.5 ng/ml. Levels were higher (p = 0.001) in TBLN as compared to TBPE (2.69, 0.96 ng/ml). PCT levels were not related to the bacterial burden depicted by culture positivity in these patients. PCT showed a negative correlation with the severity of constitutional symptoms (rho = - 0.238, p = 0.034), and inflammatory biomarkers; ferritin (rho = - 0.43, p < 0.001), INF-γ (rho = - 0.314, p = 0.003), TNF-α (rho = - 0.220, p = 0.039), IL-6 (rho = - 0.224, p = 0.035), and several chemokines of CCL and CCXL group. Raised plasma levels of PCT did not decrease with anti-TB treatment, indicating it is not a good biomarker to monitor treatment response in TBLN and TBPE patients. More studies with a larger number of confirmed EPTB cases are needed to define the role of PCT and its interaction with other biomarkers in EPTB.

Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Identification of host biomarkers from dried blood spots for monitoring treatment response in extrapulmonary tuberculosis.

There is a lack of objective tools for monitoring treatment response in extrapulmonary tuberculosis (EPTB). This study aimed to explore the utility of inflammatory biomarkers from the dry blood spots (DBS) as a tool for monitoring treatment response in EPTB. In a prospective cohort study, 40 inflammatory biomarkers were investigated in DBS samples from 105 EPTB cases using a Luminex platform. The samples were taken before, and, at the end of the 2nd and 6th months of treatment. A total of 11 inflammatory host biomarkers changed significantly with treatment in all EPTB patients. CXCL9/MIG, CCL20, CCL23, CXCL10/IP-10, CXCL1, CXCL2, and CXCL8 significantly declined in our cohort of EPTB (48 TB pleuritis and 57 TB lymphadenitis) patients at both time points. A biosignature consisting of MIG, CCL23, and CXCL2, corresponded with the treatment response in 81% of patients in the 2nd month and 79% of patients at the end of treatment. MIG, CCL23, IP-10, and CXCL2 changed significantly with treatment in all patients including those showing partial clinical response at the 2nd month of treatment. The changes in the levels of inflammatory biomarkers in the DBS correspond with the treatment success and can be developed as a routine test in low-resource settings.

The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis.

Extrapulmonary tuberculosis often poses a diagnostic challenge. This study aimed to assess the value of histological examination in diagnosing tuberculous lymphadenitis (LNTB) when performed simultaneously with rapid molecular assay (Xpert MTB/RIF) testing. People presumed to have LNTB were prospectively enrolled in a tertiary care hospital. Excision biopsy was performed and tested by histology, Xpert, and culture. Of 390 lymph nodes, 11 (2.8%) were positive by AFB microscopy, 124 (31.8%) by Xpert, 137 (35.1%) by culture, and histopathology was consistent with TB in 208 (53.3%). Altogether, LNTB was diagnosed in 228 and bacteriologically confirmed TB in 178 cases. Against culture, histopathology versus Xpert had higher sensitivity (93 vs. 62%) but lower specificity (68 vs. 83%). In patients with short clinical history, a significantly higher number of Xpert-positive specimens were culture-positive. Among patients with histology suggestive of TB, no difference was seen in response to treatment between bacteriology positive and negative, but a significant slow response was noted in bacteriology confirmed TB with nonspecific histology. In a country like Pakistan, with high TB and low HIV prevalence, diagnosis is possible for more than 95% of LNTB when Xpert and histopathology examination is used in combination, compared to less than 60% by Xpert alone.

Enhanced serodiagnostic potential of a fusion molecule consisting of Rv1793, Rv2628 and a truncated Rv2608 of Mycobacterium tuberculosis.

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.

Predictors of slow clinical response and extended treatment in patients with extra-pulmonary tuberculosis in Pakistan, A hospital-based prospective study.

The optimal duration of treatment in different forms of extrapulmonary tuberculosis (EPTB) is not clearly defined. This study aimed to identify predictors of slow clinical response and extended anti-TB treatment in EPTB patients. Socio-demographic, clinical, and microbiological characteristics of EPTB patients registered for anti-TB treatment at a tertiary care hospital, were analysed for identification of predictors of extended treatment. A total of 251 patients (137 lymphadenitis, and 114 pleuritis) were included in the analysis. Treatment was extended to more than 6 months in 58/251 (23%) patients. In the multivariate regression analysis, culture-positive EPTB (p = 0.007) [OR (95% CI) = 3.81 (1.43, 10.11)], history of diabetes (p = 0.014) [OR (95% CI) = 25.18 (1.94, 325.83)], smokeless tobacco use (p = 0.002) [OR (95% CI) = 17.69 (2.80, 111.72)], and slow regression of local signs and symptoms after 2 months of treatment (p < 0.001) [OR (95% CI) = 17.09 [(5.79, 50.39)] were seen to be significantly associated with treatment extension. Identification of predictors of extended treatment can help clinical decisions regarding optimal duration of treatment. Further studies are needed to identify subgroups of EPTB patients who can benefit from a shorter or longer treatment regimen.

Gut microbiome dysbiosis and correlation with blood biomarkers in active-tuberculosis in endemic setting.

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment.

Detection of active mycobacterium tuberculosis at 6th month exit among declared successfully treated cases in Pakistan.

OBJECTIVES: Tuberculosis (TB) is a significant public health concern, and the basis of successful anti-tuberculosis treatment (ATT) rests on the complete eradication of live bacilli from a patient. This study was conducted to detect the live TB bacilli in Lowenstein Jensen culture media among exit cases of TB who were declared successfully treated, either cured or treatment completed. METHODS: This cross-sectional study was conducted across Pakistan. Fifty-eight active TB DOTS centers were selected. The sample size of 3355 TB cases were equally distributed in all DOTS facilities. A detailed questionnaire was developed to record the information from TB DOTS and patients. After successful treatment, the sputum was taken from TB cases and examined to detect live bacilli on L-J culture. RESULTS: A total of 3355 TB cases were enrolled in the study. The male to female proportion was 1704(50.9%) and 1651(49.2%). Initially, 1993(59.4%) cases were cured, and 1362(40.6%) were declared as treatment completed cases. At exit, 324(9.65%) cases were again ZN smear-positive, and 328(9.77%) were positive on L-J culture, after being declared successfully treated for TB. CONCLUSIONS: To eradicate live TB bacilli, all TB cases should be subjected to L-J culture at the end of ATT.

Extrapulmonary tuberculosis in Pakistan- A nation-wide multicenter retrospective study.

BACKGROUND: Pakistan is fifth among high burden countries for tuberculosis. A steady increase is seen in extrapulmonary tuberculosis (EPTB), which now accounts for 20% of all notified TB cases. There is very limited information on the epidemiology of EPTB. This study was performed with the aim to describe the demographic characteristics, clinical manifestations and treatment outcomes of EPTB patients in Pakistan. METHOD: We performed descriptive analysis on routinely collected data for cohorts of TB patients registered nationwide in 2016 at health facilities selected using stratified convenient sampling. FINDINGS: Altogether 54092 TB including 15790 (29.2%) EPTB cases were registered in 2016 at 50 study sites. The median age was 24 years for EPTB and 30 years for PTB patients. The crude prevalence of EPTB in females was 30.5% (95%CI; 29.9-31.0) compared to 27.9% (95%CI; 27.3-28.4) in males. The likelihood of having EPTB (OR), was 1.1 times greater for females, 2.0 times for children, and 3.3 times for residents of provinces in the North-West. The most common forms of EPTB were pleural (29.6%), lymphatic (22.7%) and abdominal TB (21.0%). Pleural TB was the most common clinical manifestation in adults (34.2%) and abdominal TB in children (38.4%). An increase in the prevalence of pleural and osteoarticular and decline in lymphatic and abdominal TB was observed with advancing age. Diversity in demography and clinical manifestations were noted between provinces. The treatment success rate for all type EPTB was significantly high compared to bacteriology confirmed PTB with the exception of EPTB affecting CNS with a high mortality rate. CONCLUSIONS: The study provides an insight into demography, clinical manifestations and treatment outcomes of EPTB. Further studies are needed to explain significant diversities observed between provinces, specific risk factors and challenges concerning EPTB management.

Tuberculosis-Related Diabetes: Is It Reversible after Complete Treatment?

Individuals with newly diagnosed tuberculosis (TB) were screened for diabetes (DM) with fasting plasma glucose (FPG) in Pakistan. A significant decrease in FPG was observed when TB was treated. Of those with newly diagnosed DM, 46% and 62% no longer had hyperglycemia after 3 and 6 months, respectively. Individuals with known DM also showed a significant decrease in fasting plasma levels when treated for TB, but after 3 months none had normoglycemia, and after 6 months 9.2% were normoglycemic. Thus, TB-related DM may abate when the stress terminates, as is the case in gestational DM. However, because stress hyperglycemia may be associated with subsequent risk of developing DM, follow-up is recommended.

Field evaluation of a blood based test for active tuberculosis in endemic settings.

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30-70%, and 93-99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.