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.

Trends in Non-Tuberculous Mycobacterial Lung Disease and Treatment Outcomes in a Low-Tuberculosis Prevalence Setting: A Retrospective Analysis.

BACKGROUND: Information on the management of non-tuberculous mycobacterial (NTM) lung infection and disease is scarce. The aim of this study was to investigate the trends in NTM lung infections, and the factors associated with the initiation of treatment and treatment outcomes. METHODS: A retrospective analysis was carried out on patient medical records from Haukeland University Hospital, Bergen, Norway, from 2000 to 2021. RESULTS: Among 154 patients with NTM lung infection, the majority (70%) were older than 65 years, and 49% had an underlying pulmonary comorbidity. The most frequently observed mycobacterial species was M. avium complex (MAC), followed by M. malmoense and M. abscessus. In total, 72 (47%) patients received antibiotic treatment. Patients with high symptom scores, aged below 65, and with MAC infection had more than three times the odds of receiving antibiotic treatment. A favourable response and culture conversion was observed in 53 of 72 (74%) patients. However, 17 (32%) of them had a relapse. Out of 82 patients who did not receive treatment, 45 (55%) had spontaneous culture conversion, and 8 (18%) of them had a relapse. No factor was identified to be significantly associated with a favourable treatment response. CONCLUSION: A favourable response to treatment was seen in 74% of patients with a high relapse rate.

Novel Insights into the Pathogenesis of Human Post-Primary Tuberculosis from Archival Material of the Pre-Antibiotic Era, 1931-1947.

OBJECTIVES: Primary and post-primary tuberculosis (TB) are distinct entities. The aim of this study was to study the histopathology of primary and post-primary TB by using the unique human autopsy material from the pre-antibiotic era, 1931-1947. MATERIAL AND METHODS: Autopsy data were collected from the autopsy journals, and the human tissue was collected from the pathology archives at the Department of Pathology, the Gades Institute. RESULTS: Histological presentations of TB lesions showed great diversity within a single lung. Post-primary TB starts as a pneumonia forming early lesions, characterized by the infiltration of foamy macrophages containing mycobacterial antigens within alveoli, and progressing to necrotic pneumonias with an increasing density of mycobacterial antigens in the lesions. These necrotic pneumonic lesions appeared to either resolve as fibrocaseous lesions or lead to cavitation. The typical granulomatous inflammation, the hallmark of TB lesions, appeared later in the post-primary TB and surrounded the pneumonic lesions. These post-primary granulomas contained lesser mycobacterial antigens as compared to necrotic pneumonia. CONCLUSIONS: Immunopathogenesis of post-primary TB is different from primary TB and starts as pneumonia. The early lesions of post-primary TB may progress or regress, holding the key to understanding how a host can develop the disease despite an effective TB immunity.

Maternal and paternal tuberculosis is associated with increased asthma and respiratory symptoms in their offspring: a study from Northern Europe.

Background: Given the profound impact of tuberculosis (TB) on immunity and given murine studies suggesting that infections may influence immunity across generations, we hypothesize that parental TB might impact health and disease in future offspring. Objective: This study investigated the impact of maternal and paternal TB on offspring asthma and respiratory symptoms. Methods: We included data from the third follow-up of the Respiratory Health in Northern Europe study (RHINE). Information on own asthma status, asthma-like symptoms and other respiratory symptoms, as well as information about parental TB and asthma, were collected using standardized questionnaires. The associations between parental TB and RHINE participants' asthma and respiratory symptoms were analyzed using multiple logistic regression, with adjustment for parental education, smoking habits and asthma. Results: Of 8,323 study participants, 227 (2.7%) reported only paternal TB, 282 (3.4%) only maternal TB, and 33 (0.4%) reported that both parents had TB. We found a higher risk of asthma (aOR: 1.29, 95% CI: 1.05-1.57) in offspring with a history of parental TB as compared to offspring without parental TB., Parental TB was significantly associated with allergic asthma in offspring (aOR: 1.58, 95% CI: 1.29-2.05), while no significant association between parental TB and asthma without allergy (aOR: 1.00, 95% CI: 0.76-1.32) in offspring was observed. Conclusion: Results from this study indicate that parental TB might be a risk factor for offspring's asthma and respiratory symptoms. We raise the hypothesis that the immunological impact of infections might be transmitted to influence offspring phenotype in humans.

Previous tuberculosis infection associated with increased frequency of asthma and respiratory symptoms in a Nordic-Baltic multicentre population study.

Background: Tuberculosis (TB) infection induces profound local and systemic, immunological and inflammatory changes that could influence the development of other respiratory diseases; however, the association between TB and asthma is only partly understood. Our objective was to study the association of TB with asthma and respiratory symptoms in a Nordic-Baltic population-based study. Methods: We included data from the Respiratory Health in Northern Europe (RHINE) study, in which information on general characteristics, TB infection, asthma and asthma-like symptoms were collected using standardised postal questionnaires. Asthma was defined based on asthma medication usage and/or asthma attacks 12 months prior to the study, and/or by a report of ≥three out of five respiratory symptoms in the last 12 months. Allergic/nonallergic asthma were defined as asthma with/without nasal allergy. The associations of TB with asthma outcomes were analysed using logistic regressions with adjustments for age, sex, smoking, body mass index and parental education. Results: We included 8379 study participants aged 50-75 years, 61 of whom reported having had TB. In adjusted analyses, participants with a history of TB had higher odds of asthma (OR 1.99, 95% CI 1.13-3.47). The associations were consistent for nonallergic asthma (OR 2.17, 95% CI 1.16-4.07), but not for allergic asthma (OR 1.20, 95% CI 0.53-2.71). Conclusion: We found that in a large Northern European population-based cohort, persons with a history of TB infection more frequently had asthma and asthma symptoms. We speculate that this may reflect long-term effects of TB, including direct damage to the airways and lungs, as well as inflammatory responses.

Significance of non-granulomatous cytomorphology on fine needle aspirate in lymphadenitis cases classified as tuberculous by using a composite reference standard.

BACKGROUND: Fine needle aspiration cytology (FNAC) is established as a first line investigation for tuberculous lymphadenitis (TBLA). We aimed to describe the various cytomorphologic features of tuberculosis (TB) on FNAC and their contribution in the diagnostic decision-making in suspected TBLA cases. METHODS: Patients with presumptive TBLA were prospectively enrolled (n = 266) and subjected to routine diagnostic work-up for TB, including FNAC samples, and followed until the end of treatment. Patients were categorized as TB or non-TB cases based on a composite reference standard of which the various cytomorphologic patterns were compared. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy was calculated using cross-tabulation. RESULTS: Fifty-six patients were categorized as bacteriologically confirmed TB, 102 as clinically confirmed TB and 108 as non-TB. The most common cytomorphologic pattern among TB cases (59%) was granulomatous inflammation with necrosis, however, about one-third of tuberculous lymphadenitis patients presented with non-granulomatous inflammation, with 21% showing only necrosis and 13% presenting with a reactive pattern. The overall sensitivity and specificity of FNAC was 85% and 66%, respectively. CONCLUSIONS: We found that about one-third of TBLA patients presented without granulomas on FNA, highlighting the importance of considering TB in a wide spectrum of cytomorphology in a high TB burden setting. Our study supports the use of FNAC as a first-line investigation tool for diagnosing TBLA in a low-resource setting due to its relative simplicity and good sensitivity. However, the low specificity of FNAC, emphasizes the need for a second-tier confirmatory test with improved specificity.

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.

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.

Improving diagnosis of tuberculous lymphadenitis by combination of cytomorphology and MPT64 immunostaining on cell blocks from the fine needle aspirates.

BACKGROUND: Extra pulmonary tuberculosis (EPTB) constitutes 18% of all tuberculosis (TB) cases and tuberculous lymphadenitis (TBL) constitutes 20-40% of EPTB. Diagnosis of TBL is challenging because of the paucibacillary nature of the disease. OBJECTIVE: To investigate the diagnostic potential of a new antigen detection test based on the detection of M. tuberculosis complex specific antigen MPT64 from fine needle aspirate (FNA) cytology smears and biopsies obtained from patients with clinically suspected TBL using immunohistochemistry (IHC). MATERIALS AND METHODS: This study was conducted at Khyber Teaching Hospital and Rehman Medical Institute, Peshawar, Pakistan, from January 2018 to April 2019. Samples, including FNA (n = 100) and biopsies (n = 8), were collected from 100 patients with presumptive TBL. Direct smears and cell blocks were prepared from the FNA samples. All samples were subjected to hematoxylin-eosin (H&E) staining, Ziehl-Neelsen (ZN) staining, and immunostaining with polyclonal anti-MPT64 antibody. The culture was performed only for biopsy specimens. All patients were followed until the completion of anti-TB treatment. The response to treatment was included in the composite reference standard (CRS) and used as the gold standard to validate the diagnostic tests. RESULTS: The sensitivity, specificity, positive and negative predictive values for ZN staining were 4.4%,100%,100%,56%, for culture were 66%,100%,100%,50%, for cytomorphology were 100%,90.91%,90%,100%, and for immunostaining with anti-MPT64 were all 100%,respectively. The morphology and performance of immunohistochemistry were better with cell blocks than with smears. CONCLUSION: MPT64 antigen detection test performed better than ZN and cytomorphology in diagnosing TBL. This test applied to cell blocks from FNA is robust, simple, and relatively rapid, and improves the diagnosis of TBL.

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.

Improved diagnosis of extrapulmonary tuberculosis in adults with and without HIV in Mbeya, Tanzania using the MPT64 antigen detection test.

Extrapulmonary tuberculosis (EPTB) in People Living with HIV (PLWHIV) is a diagnostic challenge. Our immunochemistry based MPT64 antigen detection test has shown improved sensitivity compared to current laboratory tests in the resource limited diagnostic setting. The aim of this study was to validate the implementability and diagnostic performance of the test in PLWHIV and HIV negative adults in a HIV endemic Tanzanian setting. Adult (>18 y) presumptive EPTB patients were prospectively enrolled at Mbeya Zonal Referral Hospital and followed to the end of treatment or until an alternative diagnosis was reached. Suspected sites of infection were sampled and were subject to routine diagnostics, GeneXpert MTB/RIF assay and the MPT64 test. The performance of the diagnostics tests was assessed using a composite reference standard that included clinical suspicion, mycobacterial culture, response to anti-tuberculosis (TB) therapy, cytological and radiological findings. Patients (N = 168) were categorized as 21 confirmed TB, 23 probable TB and 44 possible TB cases, 69 patients were categorized as non-TB cases and 11 were uncategorized. In the TB group, the three most common infections were adenitis (41%), peritonitis (19%) and pleuritis (14%). The TB and non-TB groups did not differ in HIV seropositivity (46% vs 42%) Among HIV negative and PLWHIV, the MPT64 test had a sensitivity of (91% vs 78%), specificity (75% vs 86%), positive predictive value (80% vs 88%), negative predictive value (89% vs 74%), and accuracy (84% vs 81%), respectively. Performance was not significantly reduced in PLWHIV, and sensitivity was higher than in the currently used tests, including the GeneXpert MTB/RIF assay. The MPT64 test improved the diagnosis of EPTB, irrespective of HIV status. The test performed better than currently used diagnostic test. The test was implementable in a tertiary level hospital with basic pathology services in a HIV endemic Tanzanian setting.

A Phase I/II randomized trial of H56:IC31 vaccination and adjunctive cyclooxygenase-2-inhibitor treatment in tuberculosis patients.

Host-directed-therapy strategies are warranted to fight tuberculosis. Here we assess the safety and immunogenicity of adjunctive vaccination with the H56:IC31 candidate and cyclooxygenase-2-inhibitor treatment (etoricoxib) in pulmonary and extra-pulmonary tuberculosis patients in a randomized open-label phase I/II clinical trial (TBCOX2, NCT02503839). A total of 222 patients were screened, 51 enrolled and randomized; 13 in the etoricoxib-group, 14 in the H56:IC31-group, 12 in the etoricoxib+H56:IC31-group and 12 controls. Three Serious Adverse Events were reported in the etoricoxib-groups; two urticarial rash and one possible disease progression, no Serious Adverse Events were vaccine related. H56:IC31 induces robust expansion of antigen-specific T-cells analyzed by fluorospot and flow cytometry, and higher proportion of seroconversions. Etoricoxib reduced H56:IC31-induced T-cell responses. Here, we show the first clinical data that H56:IC31 vaccination is safe and immunogenic in tuberculosis patients, supporting further studies of H56:IC31 as a host-directed-therapy strategy. Although etoricoxib appears safe, our data do not support therapy with adjunctive cyclooxygenase-2-inhibitors.

Successful Outpatient Management of Children at a Secondary Care Hospital in Pakistan in a Dengue Fever Epidemic and Their Clinical Outcomes.

BACKGROUND: There is limited published literature on the feasibility of WHO 2009 guidelines for the management of dengue fever (DF) in Pakistani children. This study aimed to assess the outcome of children with DF who received outpatient treatment according to these guidelines during a DF epidemic. METHOD: This was a prospective cohort study conducted at Federal General Hospital, a secondary care hospital, Islamabad, Pakistan, from 1st August to 31st October 2019. Using WHO DF 2009 guidelines, children ≤13 years, diagnosed as confirmed DF (NS1 Ag +), were classified into the outpatient (DF) or the inpatient group (DF with warning signs or severe dengue (SD)). The inpatient group was admitted to the Pakistan Institute of Medical Sciences, a tertiary care hospital, and discharged on recovery. These children were followed for the primary outcome, i.e., recovery or hospitalization by day 14 of enrollment. Additionally, clinical and laboratory features (Hb, HCT, TLC, PLT, and ALT) of the patients in the outpatient who remained stable with those who progressed to inpatient care during follow-up were compared; also, time of recovery of blood counts was assessed. RESULTS: Of 93 children with DF, 87 (93.5%) received outpatient care at enrollment. Of these, 6 (7.8%) deteriorated by day 7 and were admitted to inpatient care. SD was present in 6/93 (6.4%) patients at presentation and were admitted. All children showed signs of recovery until day 14. Male gender (p=0.049), lower normal mean platelet (p=0.02), and high mean hematocrit (p=0.001) were associated with disease progression. CONCLUSION: The majority of children with confirmed DF who received outpatient treatment according to WHO 2009 guidelines were successfully managed. Additionally, confirmed DF with warning signs or SD were admitted and recovered. Regular follow-ups according to the guidelines are pertinent. Thrombocytopenia and high HCT were associated with disease progression.

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.

MPT64 antigen detection test improves diagnosis of pediatric extrapulmonary tuberculosis in Mbeya, Tanzania.

Pediatric extrapulmonary tuberculosis (EPTB) is a diagnostic challenge. A new immunochemistry based MPT64 antigen detection test has shown improved sensitivity compared to current laboratory tests. The aim of this study was to implement and validate the test performance in a resource limited African setting. Presumptive pediatric (0-18 y) EPTB patients were prospectively enrolled at Mbeya Zonal Referral Hospital, and followed to the end of treatment or until a final diagnosis was reached. Specimens from suspected sites of infection were subject to routine diagnostics, GeneXpert MTB/RIF assay and the MPT64 test. The performance of the tests was assessed using mycobacterial culture as well as a composite reference standard. 30 patients were categorized as TB cases, 31 as non-TB cases and 2 were uncategorized. In the TB group, the three most common infections were adenitis (30%), peritonitis (30%) and meningitis (20%). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the MPT64 test was 92%, 88%, 87%, 92% and 90%, respectively. Mortality was equally high among TB/non-TB cases (23% vs 21%), and malnutrition was the main comorbidity among TB cases. The MPT64 test was implementable in the routine diagnostics in a low-resource setting and improved the diagnosis of pediatric EPTB.

Role of a Digital Clinical Decision-Support System in General Practitioners' Management of COPD in Norway.

Background: The study investigated if a web-based clinical decision-support system (CDSS) tool would improve general practitioners' (GPs) accuracy of diagnosis and classification of patients with chronic obstructive pulmonary disease (COPD), and whether nonpharmacological and pharmacological treatment would be better aligned with the COPD guidelines. Methods: GPs were randomized to either a single use of the CDSS or continuing standard of care. The clinical recommendations of the CDSS were based on the GOLD guidelines and provided suggestions for treatment and management of COPD. Data were collected digitally from GPs and patients in both groups using a tablet computer. A follow-up questionnaire was sent to the GPs 1 year after the conclusion of the study. Results: A total of 25 GPs (31% women, mean age 41 years) participated, 12 randomized to using the CDSS tool and 13 followed standard of care when assessing their next five to ten COPD patients. In sum, 149 patients with presumed COPD were included (88 CDSS group, 61 standard-of-care group). In the CDSS group, no COPD misdiagnoses occurred, 98% received vaccine recommendations, and all smokers (n=39) received smoking-cessation advice. The standard-of-care group had 23% misdiagnosis (P<0.001), only 67% received vaccine recommendations (P<0.001), and 87% smoking-cessation advice (P=0.022. All told, 31% of patients did not receive medication as recommended according to guidelines, with no significant differences between the groups. GPs rated the CDSS as very useful. Mean usage time was 3 minutes, 26 seconds. A majority (13 of 19, 68%) of the GPs continued using the CDSS after the conclusion of the study. CAT score identified twice as many patients as having more symptoms than the mMRC, indicating the added value of the multi-item questionnaire. Conclusion: Use of the CDSS was associated with preventing misdiagnosis of COPD and improved adherence to recommended nonpharmacological measures, but a single use did not improve pharmacological treatment considerations.

Immunochemistry-Based Diagnosis of Extrapulmonary Tuberculosis: A Strategy for Large-Scale Production of MPT64-Antibodies for Use in the MPT64 Antigen Detection Test.

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done.

Paediatric Contacts of Adult COVID-19 Patients: Clinical Parameters, Risk Factors, and Outcome.

BACKGROUND: There is insufficient data in Pakistan and in South Asia regarding paediatric COVID-19 demographics and related parameters. The main aim of this study was to assess the paediatric population exposed to SARS-CoV-2 infection, their clinical parameters, risk factors, and outcome. METHODS: This was a descriptive retrospective study conducted at the Pakistan Institute of Medical Sciences and Federal General Hospital Islamabad from 23rd July 2020 to 22nd August 2020. All paediatric contacts (≤13 years) of one hundred adult COVID-19 patients were included. Data of the index cases was taken from the medical records. Paediatric data was collected on the phone using a predesigned proforma. RESULTS: There were 137 paediatric contacts of 100 adult COVID-19 index cases. The index cases were predominantly males (67%) and belonged to the middle socioeconomic class (89%), and 14% succumbed to the disease. Females had more paediatric contacts. The mean age of contacts was 6.6 years, and the majority (80%) developed no symptoms. Among the symptomatic contacts, fever and cough were the most common symptoms. None of the contacts developed dyspnoea or required hospitalization. Majority of the contacts had been vaccinated with the BCG vaccine. Testing for COVID-19 was done in only 77 (56%) contacts, 25 (32%) by the government team, and 52 (67%) privately. A higher number of symptomatic contacts were positive (15/17 (88%)) as compared to that of the asymptomatic contacts (6/60 (10%)) (p = 0.002). Development of symptoms in the contacts was associated with the history of respiratory illnesses, recurrent infections, use of hematinics, a positive COVID-test result, and health professionals being index cases (p ≤ 0.01). Parents with higher education and in the health profession and the families of symptomatic contacts reported better compliance with quarantine regulations. CONCLUSION: A significant number of children were exposed to adult COVID-19 patients. Most paediatric contacts remained asymptomatic. Children with preexisting medical conditions and with parents in health profession were susceptible to infection.

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.

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.