An estimated 1.5 million deaths were attributable to TB in 2018.1World Health OrganizationGlobal Tuberculosis Report 2019. License: CC BY-NC-SA 3.0 IGO., Geneva, Switzerland2019Google Scholar However, some uncertainty exists as to the exact global figures, given that approximately 30% of incident cases are not diagnosed, and because of the difficulties of ascertaining TB as cause of death (CoD).2García-Basteiro A.L. Brew J. Williams B. Borgdorff M. Cobelens F. What is the true tuberculosis mortality burden? Differences in estimates by the World Health Organization and the Global Burden of Disease study.Int J Epidemiol. 2018; 47: 1-12Crossref PubMed Scopus (16) Google Scholar Undoubtedly, complete diagnostic autopsies (CDAs) constitute the gold standard for establishing a diagnosis of TB at death. However, CDAs are seldom performed in high-TB-burden countries because of the scarcity of trained pathologists, the time-consuming nature of the procedure, and the meager acceptability of the practice by relatives.3Bassat Q. Ordi J. Vila J. et al.Development of a post-mortem procedure to reduce the uncertainty regarding causes of death in developing countries.Lancet Glob Health. 2013; 1: e125-e126Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar In recent years, an alternative minimally invasive autopsy (MIA), a procedure well accepted by the next of kin, has been developed.4Maixenchs M. Anselmo R. Zielinski-Gutiérrez E. et al.Willingness to know the cause of death and hypothetical acceptability of the minimally invasive autopsy in six diverse African and Asian settings: a mixed methods socio-behavioural study.PLoS Med. 2016; 13e1002172Crossref PubMed Scopus (44) Google Scholar,5Castillo P. Martínez M.J. Ussene E. et al.Validity of a minimally invasive autopsy for cause of death determination in adults in Mozambique: an observational study.PLoS Med. 2016; 13e1002171Crossref PubMed Scopus (56) Google Scholar MIA can be conducted relatively rapidly with the use of biopsy needles for sampling key organs, which leave barely visible marks, which is thus more acceptable to relatives. This method has shown good sensitivity for diagnosing TB as CoD.6Garcia-Basteiro A. Hurtado J.C. Castillo P. et al.Unmasking the hidden tuberculosis mortality burden in a large post mortem study in Maputo Central Hospital.Eur Respir J. 2019; 54: 1900312Crossref PubMed Scopus (9) Google Scholar Nonetheless, MIA has thus far used standard histological and microbiological approaches for TB diagnosis (identification of granulomatous lesions, acid-fast bacilli smears, in-house polymerase chain reaction methods),7Cox J.A. Lukande R.L. Lucas S. Nelson A.M. Van Marck E. Colebunders R. Autopsy causes of death in HIV-positive individuals in sub-Saharan Africa and correlation with clinical diagnoses.AIDS Rev. 2010; 12: 183-194PubMed Google Scholar which remain time consuming, require specific expertise, and have limited sensitivity. Thus, we evaluated the diagnostic accuracy of the molecular Xpert MTB/RIF Ultra (hereafter referred to as Xpert Ultra) assay in samples obtained by MIA to detect CoD by TB. This was an ancillary study to a large observational postmortem evaluation (CADMIA study) aimed at validating MIA against CDA for any CoD determination in different age groups in Maputo, Mozambique.3Bassat Q. Ordi J. Vila J. et al.Development of a post-mortem procedure to reduce the uncertainty regarding causes of death in developing countries.Lancet Glob Health. 2013; 1: e125-e126Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,5Castillo P. Martínez M.J. Ussene E. et al.Validity of a minimally invasive autopsy for cause of death determination in adults in Mozambique: an observational study.PLoS Med. 2016; 13e1002171Crossref PubMed Scopus (56) Google Scholar Both the CDA and MIA pathological and microbiological methods have been comprehensively described elsewhere.8Castillo P. Ussene E. Ismail M.R. et al.Pathological methods applied to the investigation of causes of death in developing countries: minimally invasive autopsy approach.PLoS One. 2015; 10e01Crossref Scopus (56) Google Scholar,9Martínez M.J. Massora S. Mandomando I. et al.Infectious cause of death determination using minimally invasive autopsies in developing countries.Diagn Microbiol Infect Dis. 2016; 84: 80-86Crossref PubMed Scopus (45) Google Scholar In a previous analysis from CADMIA, TB-related lesions were extensively investigated in CDA samples.6Garcia-Basteiro A. Hurtado J.C. Castillo P. et al.Unmasking the hidden tuberculosis mortality burden in a large post mortem study in Maputo Central Hospital.Eur Respir J. 2019; 54: 1900312Crossref PubMed Scopus (9) Google Scholar Microbiological methods include acid-fast bacilli smear and two molecular tools: in-house real-time polymerase chain reaction and Xpert Ultra following a pre-specified algorithm.6Garcia-Basteiro A. Hurtado J.C. Castillo P. et al.Unmasking the hidden tuberculosis mortality burden in a large post mortem study in Maputo Central Hospital.Eur Respir J. 2019; 54: 1900312Crossref PubMed Scopus (9) Google Scholar CDA diagnosis was considered the gold standard for CoD determination in CADMIA. For this specific analysis, we selected MIA samples from the lung, CNS, cerebrospinal fluid (CSF), and plasma from all the study cases with any TB finding (TB disease or Mycobacterium TB DNA detected in CDA samples). This analysis included a total of 117 patients. In 31 patients, TB was the final CoD, 31 cases had TB disease at death but had died of another CoD, and in 18 cases DNA of M TB was detected but no histological lesions compatible with TB were found. In addition, we included a subset of 37 patients with no TB findings in the CDA and with availability of the four MIA samples. MIA samples were collected in tubes containing 1 mL lysis buffer (ATL buffer, Qiagen), and stored and processed by Xpert Ultra. Lung and CNS tissue samples were processed as described previously,6Garcia-Basteiro A. Hurtado J.C. Castillo P. et al.Unmasking the hidden tuberculosis mortality burden in a large post mortem study in Maputo Central Hospital.Eur Respir J. 2019; 54: 1900312Crossref PubMed Scopus (9) Google Scholar,10García-Basteiro A.L. Ismail M.R. Carrilho C. et al.The role of Xpert MTB/RIF in diagnosing pulmonary tuberculosis in post-mortem tissues.Sci Rep. 2016; 6: 20703Crossref PubMed Scopus (14) Google Scholar and 0.5 mL plasma and CSF were mixed with the assay sample reagent buffer in a 1:3 ratio before testing. We determined the performance of Xpert Ultra for each MIA sample and for combinations of samples. We also estimated the number needed to misdiagnose (NNM) as: NNM = Total/(false positives + false negatives). The NNM is the number of patients who need to be tested for one patient to be misdiagnosed. Because specific MIA samples were not available for some patients with TB findings, a sensitivity analysis of the performance of Xpert Ultra in MIA samples was conducted among cases in which all four MIA samples were available. The Clinical Research Ethics Committee of the Hospital Clinic of Barcelona, Spain (Ref:2013/8677) and the National Bioethics Committee of Mozambique (Ref. 342/CNBS/13) approved this study. Of the 117 cases included in this analysis, 14 patients (12.0%) were children, 85 (72.7%) were adults, and 18 (15.4%) were maternal deaths. Seventy-eight patients (67.8%) were HIV positive (HIV status could not be ascertained in two cases). Table 1 shows the diagnostic performance of Xpert Ultra in different MIA samples and combinations of samples to diagnose TB as CoD. As a single organ, the highest sensitivity was observed in the lung (0.78; 95% CI, 0.58-0.91). The sensitivity of the test in plasma and CSF was high, being 0.68 (95% CI, 0.47-0.83) and 0.67 (95% CI, 0.30-0.67), respectively. For combinations of organs, the highest performance was observed for lung and CNS: sensitivity, 0.85% (95% CI, 0.66-0.96) and negative predictive value of 0.95 (95% CI, 0.88-0.99). The highest specificity in a single organ or fluid was obtained in the lung (0.98; 95% CI, 0.92-1.00).Table 1Diagnostic Performance of Xpert Ultra in Different Minimally Invasive Autopsy Samples to Diagnose TB as the Cause of DeathSample/Combination of SamplesNo. True PositiveNo. False NegativeNo. False PositiveNo. True NegativeTotal SamplesaSome MIA samples were not tested by Xpert Ultra in some cases because of lack of remaining tissue in the biobank.Sensitivity95% CISpecificity95% CIPPV95% CINPV95% CINNMLower LimitUpper LimitLower LimitUpper LimitLower LimitUpper LimitLower LimitUpper LimitLung2162811100.780.580.910.980.921.000.910.720.990.930.860.9713.8CNS21104801150.680.490.830.950.880.990.840.640.950.890.810.958.2Plasma20102801120.670.470.830.980.911.000.910.710.990.890.810.959.3CSF15163801140.480.300.670.960.900.990.830.590.960.830.740.906.0Lung and CNS2345771090.850.660.960.940.860.980.820.630.940.950.880.9912.1Lung and plasma2164751060.780.580.910.950.880.990.840.640.950.930.850.9710.6Lung and CSF2254761070.810.620.940.950.880.990.850.650.960.940.860.9811.9CNS and plasma2375751100.770.580.900.940.860.980.820.630.940.910.830.969.2CNS and CSF2386751120.740.550.880.930.850.970.790.600.920.900.820.968.0Plasma and CSF2284751090.730.540.880.950.880.990.850.650.960.900.820.969.1Lung, CNS, & plasma2346721050.850.660.960.920.840.970.790.600.920.950.870.9910.5Lung, CNS, & CSF2346731060.850.660.960.920.840.970.790.600.920.950.870.9910.6Lung, plasma, & CSF2255711030.810.620.940.930.850.980.810.620.940.930.850.9810.3CNS, plasma, & CSF2467701070.800.610.920.910.820.960.770.590.900.920.840.978.2The diagnostic values of samples or combinations of samples that may represent a significant diagnostic advantage are highlighted in bold. CSF = cerebrospinal fluid; NNM = number needed to misdiagnose; NPV = negative predictive value; PPV = positive predictive value.a Some MIA samples were not tested by Xpert Ultra in some cases because of lack of remaining tissue in the biobank. Open table in a new tab The diagnostic values of samples or combinations of samples that may represent a significant diagnostic advantage are highlighted in bold. CSF = cerebrospinal fluid; NNM = number needed to misdiagnose; NPV = negative predictive value; PPV = positive predictive value. The highest NNM was obtained using the MIA sample of the lung (13.8) and combinations including lung samples (lung and CNS, 12.1; lung and CSF, 11.9; lung and plasma, 10.6). The sensitivity analysis of only cases in which the four MIA samples were available showed similar results, although the point estimate with the highest specificity (0.99; 95% CI, 0.93-1.00) and positive predictive value (PPV) (0.94; 95% CI, 0.70-1:00) was obtained with the CSF sample (Table 2).Table 2Diagnostic Performance of Xpert Ultra in Different Minimally Invasive Autopsy Samples to Diagnose TB as the Cause of Death (Includes Only Cases in Which the Four Samples Were Available: Lung, CNS, Plasma, and CSF)Sample/Combination of SamplesNo. True PositiveNo. False NegativeNo. Fase PositiveNo. True NegativeTotal SamplesSensitivity95% CISpecificity95% CIPPV95% CINPV95% CINNMLowerLimitUpper LimitLower LimitUpper LimitLowerLimitUpper LimitLowerLimitUpper LimitLung2162731020.780.580.910.970.820.990.910.720.990.920.840.9712.8CNS2073721020.740.540.890.960.890.990.870.660.970.910.830.9610.2Plasma1982731020.700.500.860.970.911.000.900.700.990.900.810.9610.2CSF15121741020.560.350.750.990.931.000.940.701.000.860.770.937.8Lung and CNS2344711020.850.660.960.950.870.990.850.660.960.950.870.9912.8Lung and Plasma2164711020.780.580.910.950.870.990.840.640.950.920.840.9710.2Lung and CSF2253721020.810.620.930.960.890.990.880.690.970.940.850.9812.8CNS and Plasma2255701020.810.620.930.930.850.980.810.620.930.930.850.9810.2CNS and CSF2254711020.810.620.930.950.870.990.850.650.960.930.850.9811.3Plasma and CSF2163721020.780.580.910.960.890.990.880.680.970.920.840.9711.3Lung CNS Plasma2346691020.850.660.960.920.830.970.790.600.920.950.870.9810.2Lung CNS CSF2345701020.850.660.960.930.850.980.820.630.940.950.870.9911.3Lung Plasma CSF2255701020.810.620.930.930.850.980.810.620.930.930.850.9810.2CNS Plasma CSF2346691020.850.620.930.920.830.970.790.600.920.950.870.9810.2Any tissue2347681020.850.660.960.910.820.960.770.580.900.940.860.989.3The diagnostic values of samples or combinations of samples that may represent a significant diagnostic advantage are highlighted in bold. See Table 1 legend for expansion of abbreviations. Open table in a new tab The diagnostic values of samples or combinations of samples that may represent a significant diagnostic advantage are highlighted in bold. See Table 1 legend for expansion of abbreviations. This analysis shows that Xpert Ultra (a simple, rapid, and highly sensitive molecular tool) can be directly applied to specific MIA samples and achieve a reasonably high accuracy for confirming or ruling out TB as the CoD. We observed that only 15% of TB deaths would have been missed with the use of Xpert Ultra in lung and CNS MIA samples. More than two thirds of TB cases would have been diagnosed by performing Xpert Ultra in an easily accessible and homogeneous sample such as plasma, a sample with great potential for confirming TB as CoD given its high associated PPV (0.90 in this setting) and which would seldom be positive if the patient had not died of TB (specificity, 0.98). A similar high specificity and PPV are obtained with CSF, probably reflecting that most TB deaths are caused by disseminated TB, and bacilli are released and found in great quantities in peripheral blood and other compartments, such as CSF. The study includes a well-characterized sample of patients with different TB findings at death. It also includes cases from different age groups and HIV statuses. Nonetheless, it has certain limitations. First, the sample size of cases with TB as the CoD was limited. Second, we could not analyze all of the CADMIA cases in this analysis because MIA samples were not available in all of the cases. Thus, the prevalence of TB as the CoD is not real in this sample, a factor that might affect the interpretation of PPV and negative predictive value. Taking into account that we included all cases with any type of TB finding in the CDA but only a subset of cases without any finding, the specificity of the MIAs might be slightly underestimated. In conclusion, this study shows that the use of Xpert Ultra in body fluids, such as plasma or CSF, obtained at MIA, can easily and quickly diagnose or rule out TB as the CoD. This diagnostic strategy can accelerate and adequately and accurately determine CoD in settings with high TB and HIV prevalence. Nonetheless, the implications for pre-mortem patient management still need to be elucidated. Other contributions: The authors thank the families of the deceased patients included in this study. The authors are grateful to all the members of the Department of Pathology of Maputo Central Hospital, Mozambique, whose support made this study possible, and also to the staff of the Centro de Investigaçãoem Saúde de Manhiça (CISM) for their logistic support. We specifically thank Mr Bento Nhancale for his invaluable support to the study. We thank Cepheid for providing the Xpert Ultra cartridges used in the study. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript.