Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection

Summary

Background

A third of the world's population has latent infection with Mycobacterium tuberculosis, and in areas of low endemicity, most cases of active tuberculosis arise as a result of reactivation of latent bacilli. We sought to establish the cellular location of these latent organisms to facilitate their elimination.

Methods

We applied in-situ PCR to sections of macroscopically normal lung tissue from 13 individuals from Ethiopia and 34 from Mexico who had died from causes other than tuberculosis. Sections of lung tissue from six Norwegian individuals (ie, individuals from a non-endemic population) acted as negative controls, and six Ethiopian tuberculosis cases acted as positive controls.

Findings

Control necropsy samples from the Norwegian individuals were all negative by in-situ PCR and conventional PCR, whereas all samples from known Ethiopian tuberculosis cases were positive by both methods. However, in macroscopically normal lung tissue from Ethiopian and Mexican individuals without tuberculous lesions, the in-situ PCR revealed five of 13 and ten of 34 positive individuals, respectively. These results were confirmed by conventional PCR with extracted DNA. Positive cells included alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts.

Interpretation

M tuberculosis can persist intracellularly in lung tissue without histological evidence of tuberculous lesions. M tuberculosis DNA is situated not only in macrophages but also in other non-professional phagocytic cells. These findings contradict the dominant view that latent organisms exist in old classic tuberculous lesions, and have important implications for strategies aimed at the elimination of latent and persistent bacilli.

Introduction

About a third of the world's population is infected with Mycobacterium tuberculosis¹. In areas of high endemicity, the first mycobacterial infection usually occurs in childhood. In most individuals, it is kept under control by the immune system, and only in about 10% does it lead to disease.² However, in primary tuberculosis, even when successfully controlled by the immune system, not all bacteria are eliminated. Some bacilli remain in the tissues in a latent state for the rest of the individual's life.³ This latent state is important for two reasons.

First, in countries with low or moderate tuberculosis endemicity, most cases of tuberculosis result from reactivation of latent infection. 4, 5, 6 Second, the persistence of organisms that are resistant to chemotherapeutic agents requires prolonged chemotherapy of active disease, which leads to problems in terms of logistics and compliance.⁷ These persistent bacteria could exist in a physiological state similar to that of latent bacteria. Thus, new strategies for the eradication of latent organisms are required, and such strategies will arise from a better understanding of the mechanism by which the tubercle bacilli persist.

Experimental work in laboratory animals has suggested that alveolar macrophages are the first cells infected by M tuberculosis.⁸ These infected macrophages are then surrounded by newly recruited macrophages and lymphocytes that form the characteristic tuberculous granuloma.⁹ Bacterial latency is assumed to occur in these classic tuberculous lesions. However, this assumption does not explain the facts of reactivation tuberculosis—ie, that the primary lesion can occur anywhere in the lung, yet more that 90% of progressive cavitatory “post-primary” disease occurs in the upper lobes.¹⁰ Moreover, in 1927, Opie and Aronson reported that fewer than 10% of old lesions contained live bacilli, whereas such organisms could be recovered from macroscopically normal lung tissue in almost 50% of people who had died from causes other than tuberculosis.¹¹

The aims of this study were to determine whether M tuberculosis DNA is indeed present in lung tissue with no specific histopathology, and if so, to identify its cellular location.