A comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma

Several species of the family Chlamydiaceae are well-known etiological agents of acute and chronic infections in humans and animals [1, 2]. The first description of chlamydial respiratory disease in humans referred to psittacosis, also known as ornithosis, and dates back to 1879 [3]. Chlamydia (C.) psittaci, the agent responsible for this disease, has had several different names and, according to a recent proposal, should now be called Chlamydophila (Cp.) psittaci [4]. A century later, in 1986, Grayston et al. discovered another chlamydial respiratory agent, strain TWAR, which was later assigned to the species C. pneumoniae [5, 6] currently reclassified as Chlamydophila pneumoniae [4]. Meanwhile, a variety of respiratory conditions in humans has been shown to be associated with this agent. Evidence of Cp. pneumoniae infection based on serology was reported in severe cases of chronic obstructive pulmonary disease (COPD), in which emphysema is dominant [7, 8], as well as in exacerbations of COPD [9] and in persistent infections of the respiratory tract [10, 11]. The detection rate of Cp. pneumoniae by immunohistochemical staining was elevated in lung tissue from subjects with COPD, but controls were not completely negative [12]. Initially Cp. pneumoniae was thought to be virulent for humans only, but recent descriptions of isolates from horse, koala, frog and reptiles suggest a wider host spectrum and even the possibility of zoonotic transmission [13‐15].

Our previous investigations by means of immunofluorescence using a genus-specific antiserum against chlamydial LPS and scanning as well as transmission electron microscopy showed infection of the alveolar parenchyma and the bronchioles by Chlamydia spp. in patients having undergone lung volume reduction surgery for advanced pulmonary emphysema [16, 17]. Accumulation of alveolar macrophages as well as different forms of bronchiolitis and focal pneumonia accompanying emphysematic changes were found regularly [18]. In preliminary examinations using an established nested PCR with DNA hybridization [19], DNA specific of Cp. pneumoniae was detected in two out of ten cases [20]. But this detection rate was far lower than that in electron microscopy or immunofluorescence using genus-specific antibodies, which showed Chlamydia spp. in over 80% [17]. Because of this fact PCR was extended to other Chlamydiaceae. Here we report the results of a more detailed study involving a larger number of cases and samples including controls.

Strains of Cp. psittaci are known to cause infections in over 130 avian species and 32 other domestic and wild animals. Classical psittacosis represents a systemic disease in psittacine birds of acute, protracted, chronic or subclinical manifestation. Avian strains of the agent are known to be pathogenic to humans, the symptoms being mainly non-specific and influenza-like, but severe pneumonia, endocarditis and encephalitis are not uncommon [24, 25]. The possibility of persistent infection in man was first described in the 1950s [26].

In the present study and in previous investigations, transmission electron microscopy revealed elementary bodies as well as typical and aberrant reticular bodies, thus indicating active infection alongside persistent infection [10, 16, 17]. Chlamydiae could not be detected in each case, but the rate of positive findings in TEM and PCR (Table 1) was comparable to that of Cp. pneumoniae in atherosclerosis [27, 28]. Pear-shaped elementary bodies as typically found in Cp. pneumoniae infection [29] were not observed. While the findings of TEM are more indicative of Cp. psittaci infection [30], it must be noted that this method provides no clear-cut differentiation among Chlamydiaceae species, for even strains of the same species exhibit different morphology at the various developmental stages. Rather unexpectedly, Cp. pneumoniae was detected only in one sample by PCR, not indicating an important role of this agent in the cases examined here. The fact that, apart from psittacosis, Cp. psittaci has not been associated with human respiratory disorders in recent decades may be a question of sensitivity and specificity of detection. Particularly PCR with its capability to specifically identify all individual species of Chlamydiaceae at a detection limit of less than one inclusion-forming unit has opened up new possibilities in this respect.

Higher detection rates of Cp. psittaci in tissue with histological evidence of inflammation in comparison to unselected frozen tissue (6/12 = 50% vs. 7/32 = 21.9%, Fisher Yates test n.s.) indicate an association with regional activity of infection and inflammation. Besides psittacosis, Cp. psittaci has been recently associated with chronic inflammation in patients with ocular adnexal lymphomas [31]. In COPD, activated macrophages and neutrophils produce matrix metalloproteinases which are relevant in the development of emphysema [32, 33]. The release of matrix metalloproteinases was shown to be stimulated by cytokines produced in the course of chlamydial infection [34] and by chlamydial heat shock protein 60 as well [35]. These findings represent a link to the established pathogenetic concepts in pulmonary emphysema. Cp. psittaci was found at comparable and statistically significant rates in AATD and non AATD emphysema.

The fact that the chlamydial agent present in the emphysema tissue was identified by DNA sequencing as an avian serovar of Cp. psittaci provides an important indication on the source of infection. It is conceivable that the patients were infected through contact with birds, although this could not be verified for lack of relevant data on history. The detection rate of chlamydiae in cases of AATD emphysema vs. non-AATD emphysema was clearly higher, thus indicating a relevant role of Cp. psittaci infection in this disorder, or at least a higher susceptibility of AATD patients for an infection of their lungs with Cp. psittaci. Further investigations concerning smokers and non-smokers, pathogenetic relevance and zoonotic implications are required. In any circumstances, Cp. psittaci has to be considered an underestimated pathogen with considerable importance in public health, although the various facets of its specific impact have yet to be evaluated.