the diagnosis of extrapulmonary tuberculosis (eptB) is often challenging due to paucibacillary nature of the disease. Xpert MtB/Rif Ultra (Ultra) has been developed to improve detection of Mycobacterium tuberculosis complex (Mtc) in paucibacillary specimens. the objective of the study was to assess the performance of Ultra for the diagnosis of eptB in a high-income low tB prevalence country. extrapulmonary samples received for tB diagnostics at two hospitals in norway between January 2015 and January 2016 were prospectively and consecutively included. Defrosted samples were subjected to Ultra. Culture and routine PCR tests were used as reference standard. A total of 82 samples, 10 culture and/or routine PCR positive (confirmed TB) samples and 72 culture and routine PCR negative samples were included in analysis. The overall sensitivity and specificity of Ultra were 90% (9/10, 95% CI 56-100) and 99% (71/72, 95% CI 93-100), respectively. Ultra was positive in 6/7 smear negative confirmed TB samples. To conclude, Ultra showed a high sensitivity and specificity in extrapulmonary specimens and may contribute to a rapid diagnosis of eptB in a low tB prevalence setting.

Background: Extrapulmonary tuberculosis (EPTB) poses diagnostic challenges due to the paucibacillary nature of the disease. The immunochemistry-based MPT64 antigen detection test (MPT64 test) has shown promising results for diagnosing EPTB in previous studies performed in low-resource settings, with higher sensitivity than microscopy and culture. The aim of this study was to investigate the performance of the MPT64 test in a routine clinical setting in a high-income low TB prevalence country. Methods: Extrapulmonary samples sent for TB diagnostics to microbiology and pathology laboratories at three regional tertiary care hospitals in Norway in a one-year period were included and subjected to the MPT64 test in parallel to the routine TB diagnostic tests. Results: Samples from 288 patients were included and categorised as confirmed TB cases (n = 26), clinically diagnosed TB cases (n = 5), non-TB cases (n = 243) and uncategorised (n = 14), using a composite reference standard (CRS). In formalin-fixed biopsies, the sensitivity (95% CI) of the MPT64 test, microscopy, PCR-based tests pooled, and culture was 37% (16-62), 20% (4-48), 37% (16-62) and 50% (23-77), respectively, against the CRS. The MPT64 test showed a good positive predictive value (88%) and an excellent specificity (99, 95% CI 92-100) in formalin-fixed biopsies. In fine-needle aspirates, pus and fluid samples, the test performance was lower. Conclusions: The MPT64 test was implementable in pathology laboratories as part of routine diagnostics, and although the sensitivity of the MPT64 test was not better than culture in this setting, the test supplements other rapid diagnostic methods, including microscopy and PCR-based tests, and can contribute to strengthen the diagnosis of EPTB in formalin-fixed biopsies in the absence of culture confirmation.

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.

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.

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