To our knowledge this is the first study from any developing country to use post-mortem investigations to report the causes of mortality for HIV patients on ART. The needle autopsy protocol described here was efficient and resulted in high histologic and mycobacteriologic yield. Tuberculosis was the leading cause of death regardless of ART status and was particularly high in subjects dying in the first 3 months of ART, in whom 87% had disseminated mycobacterial infection as an immediate or contributing cause of death. Multiple concurrent pathologies were common; 62% of subjects dying from mycobacterial infection had at least one additional infectious or neoplastic cause of death. IRIS contributed to 73% of early-ART mortality. Post-mortem investigations revealed clinically unrecognized causes of death in half of the subjects.
The finding that tuberculosis is the leading overall cause of death is consistent with prior autopsy studies of HIV patients from sub-Saharan Africa and India in the pre-ART era. In a 2010 meta-analysis of all autopsy studies of HIV patients from sub-Saharan Africa over the last two decades, tuberculosis was considered a cause of death in 32–45% of 593 autopsied adults. 
In a series of 236 HIV-positive, ART-naïve patients from Mumbai, tuberculosis was implicated in 63% of deaths. 
Our finding that every case of tuberculosis was disseminated beyond the lungs supports the finding by Martinson et al.
that in their largely HIV infected, ART-naïve subjects, 97% of those with tuberculosis had evidence of disseminated disease during complete autopsy. 
Our data on the importance of tuberculosis also support the conclusions of prior studies of cause-specific mortality of HIV patients on ART in low-income settings which used non-pathologic evidence from chart reviews and verbal autopsies. Tuberculosis is consistently among the leading causes of death in these studies, implicated in 16–18% of deaths in studies from Haiti, Senegal and Uganda and in 19–44% of deaths from two South African cohorts.
The higher number of deaths attributed to tuberculosis in our study is likely explained by the contribution of post-mortem investigations which revealed that a third of microbiologically and/or histologically-proven tuberculosis infections were clinically unsuspected at the time of death.
Our finding that IRIS contributed to over 70% of early-ART deaths highlights the importance and deadly potential of severe IRIS; in cohort studies based at ART clinics the condition has been described as usually self-limited and infrequently fatal. 
In cohort studies from Uganda and South Africa that have determined cause of death through chart-review and verbal autopsy, IRIS has been implicated in 7% of early-ART deaths and 17% of all ART-deaths respectively. 
Fatal IRIS has been reported, especially in central nervous system infections.
Although our study setting at a tertiary referral hospital may have influenced our findings, our high rate is supported by detailed clinicopathological data. The fatal cases described here challenge certain characteristics that have been used to define IRIS. In the consensus definition, the presence of another infection excludes IRIS 
; however case E26 in this study demonstrates convincing evidence of paradoxical central nervous system TB IRIS with a simultaneous nosocomial bacterial pneumonia. In case E33, exuberant necrotizing granulomatous inflammation of multiple organs convinced the clinico-pathologic committee to diagnose concomitant paradoxical TB IRIS and unmasking cryptococcal IRIS ().
Simultaneous C. neoformans pneumonia and paradoxical M. tuberculosis Immune Reconstitution Inflammatory Syndrome (IRIS).
This study does have a number of limitations. Small sample-size limited the ability of this study to find significant differences between pre-ART, early-ART and late-ART mortality and to pick-up low frequency causes of death. The pathology reported here is a minimum estimate as the needle autopsy was limited to sampled organs and likely missed important pathology in non-sampled organs (particularly in the abdomen/pelvis and cranium); similarly, areas of focal pathology within biopsied organs may have been missed. A study comparing results of needle and conventional autopsies found a concordance on primary cause of death in 67% of cases. 
The predominance of gram-negative organisms in pre- and post-mortem cultures may be due to the translocation of enteric organisms across HIV-damaged gut mucosa and nosocomial pneumonias, but the striking lack of gram-positive cultures is most likely the result of pre-mortem broad-spectrum antibiotics and may have caused us to underestimate the contribution of pathogens like S. aureus
and S. pneumonia
to bacterial causes of death. Measuring HIV viral load at the time of death would have been helpful in determining adherence to and effectiveness of ART. This study may have overestimated the impact of IRIS due to miscategorization of overwhelming infections as unmasking IRIS; use of a prospective study design with serial measurements of CD4 cell count and HIV viral load might have more accurately made this distinction. We did not do mycobacterial drug-susceptibility testing which would also have helped clarify whether deteriorations after ART were due to IRIS or drug-resistant disease. 
.The setting of our study in an urban tertiary referral hospital with access to advanced diagnostic procedures and intensive care facilities may limit the application of our findings to other settings.
The standardized needle autopsy described here was minimally mutilating, was efficient to perform, did not delay burial and had a very high pathological yield. While there is still a need for complete autopsies, in situations that preclude them a version of this needle autopsy protocol, perhaps modified to include brain and abdomen/pelvis sampling, has the potential to provide crucial post-mortem data.
Our findings demonstrate that tuberculosis is the major killer of HIV patients in sub-Saharan Africa, that it is frequently unrecognized and often accompanied by concurrent infections or neoplasms. Because simultaneous complex pathologies contribute to mortality, additional diagnoses should be sought for patients not clinically improving despite receiving treatment for known diagnoses. Additional study of the pathogenesis of and therapeutics for severe IRIS is needed. The development of a comprehensive response to the diagnosis and prevention of tuberculosis prior to and throughout the course of antiretroviral therapy is likely to have an enormous life-saving potential.