Chlamydia pneumoniae infections and development of lung cancer: systematic review

Background Chlamydia pneumoniae is an obligate intracellular pathogen and is a common cause of human respiratory diseases, including pneumonia. It has been already known to have a causal relationship with some chronic diseases such as chronic obstructive pulmonary disease, asthma, and atherosclerotic cardiovascular diseases. In this review, we aim to find out the association between C. pneumoniae infection and lung cancer. Methods This is a systematic review on C. pneumoniae infection and the development of lung cancer, based on published articles consolidated from PubMed and Google Scholar on the topic. Results Out of 46 articles, 27 were selected and screened through the process. Twenty-four articles positively supported the hypothesis with one animal model, while 3 of them were negatively supportive. Several proposed mechanisms explain the pathogenesis with some knowledge gaps. Conclusion Although some studies showed an association between C. pneumoniae infection and lung cancer, whether the C. pneumoniae infection is an individual risk factor for lung cancer is still debatable. And it needs further experimental studies on both humans and animals with large observational studies to better understand the association between C. pneumoniae infection and lung cancer.


Introduction
Chlamydia pneumoniae is an obligate intracellular pathogen transmitted via aerosols. C. pneumoniae was discovered after two other chlamydial species that affect humans, C. trachomatis and C. psittaci, respectively [1]. It was also known as the Taiwan acute respiratory agent (TWAR). Like all the other chlamydial species, C. pneumoniae tends to persist in tissues. It is a common cause of human respiratory diseases and most commonly manifests as pneumoniae and bronchitis. It is responsible for 10% of community-acquired pneumoniae and 5% of bronchitis, pharyngitis, and sinusitis [1]. Respiratory infections from C. pneumoniae vary in different countries and populations. It is theorized that C. pneumoniae is associated with other acute and chronic respiratory conditions such as chronic obstructive pulmonary disease, asthma, and lung cancer [2]. It is also associated with atherosclerotic cardiovascular disease, and there are multiple lines of suggestive evidence [3,4].
Lung cancer is one of the major health concerns with high morbidity and mortality [5]. About 6 out of 10 people with lung cancer die within one year after diagnosis of lung cancer. Lung cancer accounts for 11.6% (2,093,876 new cases) of new carcinoma cases and 18.4% of cancer deaths in 2018 [6]. The one and 5-year survival rates of lung cancer were 42% and 15%, respectively. Its dramatic rise in recent decades is mainly due to increased smoking among males and females, attributable to 90% of lung cancer cases.
Lung cancers are broadly divided into two main categories, non-small cell carcinoma (NSCC) and small cell carcinoma (small cell lung carcinoma, SCLC). NSCC accounts for 80% of the cases, while SCLC accounts for the remaining 20%. NSCC was further sub-classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma is the most common type of lung cancer, accounting for more than 40% of lung cancer cases. It also accounts for 60% of NSCC cases [7]. The pathophysiology of lung cancer is not yet fully understood. However, it is hypothesized that repeated exposure to particular carcinogens may lead to dysplasia of lung epithelium, giving rise to lung cancer. Most patients will show advanced disease at their presentation, so curative surgery is an option for a minority of patients.
It is being found that chronic infection may be a predisposing factor for malignant transformation and growth, and it is attributed to over 15% of malignancies worldwide. Although there is a considerable understanding in viral oncology, the role of bacteria on oncogenesis has not been thoroughly evaluated despite having much supportive evidence to appreciate the relationship between specific bacteria and cancers. Most published research addressed several factors that will induce cancer, such as different types of toxins, some medications, smoking, and obesity. However, only a few studies deal with cancer induction via bacterial infection. Etiological association between bacteria and cancer gained the attention of the researchers after discovering the carcinogenic potential of Helicobacter pylori. Studying the long-term effects of bacteria has become of great importance in cancer prevention [8].
It is hypothesized that there is a correlation between C. pneumoniae infection and the occurrence of lung cancer, and several studies have been conducted. In this review, we aim to find out the association between C. pneumoniae infection and lung cancer.

Methods
This systematic review is on C. pneumoniae infection and the development of lung cancer. Our goal was to find out the relationship between the C. pneumoniae infection and the causation of lung cancer, considering the published web data on the topic.

Literature searches
We conducted our literature search using the webbased search engines: PubMed and Google Scholar with the language restriction of English. We used the search terms: Chlamydia Pneumoniae and lung cancer, Chlamydophilia pneumoniae and lung cancer (both genus names used for the same organism), infectious ethology for lung cancer, and complications of Chlamydia Pneumoniae pneumonia. The last date of the search was on the 04 th of August, 2021. Experimental researches based on the topics, systematic reviews, meta-analysis, retrospective case-control studies, case reports, and other journal articles relevant to this topic, published from 1997 up to 2021, were considered for this review.

Data extraction and analysis
Using descriptive statistics, the data from different sources were synthesized, including medians and ranges. The searches and data extraction were conducted by two investigators using the same methodology. In cases of disagreements, results were reconciled through mutual discussion.

Results
Forty-six articles were found, and 19 articles were excluded, first considering the relevance to the title of the review than from the abstract. Among them, there was one animal study, and it was also included for the review. Selected 27 articles were screened in the process. There were 24 articles supportive of the review title, and three articles were opposive. Among them, there were three meta-analyses. We also scanned the relevant reference lists in order to identify relevant studies. In the selected case-control studies, all the cases were pathologically confirmed lung cancers, and the controls were relatively healthy people without any type of cancer. We excluded duplicate articles, presentations, textbooks, peer reviews, letters, and paper articles (Fig. 1).

Chlamydia pneumoniae infection and lung cancer
The relationship between C. pneumoniae infection and lung cancer has been vividly studied (Table 1). In 1997, Laurila and colleagues were the ones who first postulated about the association between C. pneumoniae and lung carcinogenesis. The study was designed to evaluate the association between chronic C. pneumoniae infection and the risk of lung cancer among male smokers. Two hundred and thirty cases of smoking males with lung cancer were compared with controls, analyzed for specific antibodies and complexes for C. pneumoniae infection, and found that C. pneumoniae infection was present among 52% of cases and 45% of controls. Hence it was positively associated with lung cancer [Odds ratio (OR) 1.6; 95% confidence interval (CI) 1.0-2.3] [21].
A meta-analysis conducted on 12 studies using the electronic databases in 2010, which involved 2595 lung cancer cases and 2585 controls, suggested that overall, people exposed to C. pneumoniae infection had an OR of 1.48 (95% CI 1.32-1.67) for lung cancer risk, relative to those not exposed. This study concluded that C. pneumoniae infection is associated with an increased risk of lung cancer, but a higher titer may be a better predictor of lung cancer risk [22].
A study was conducted on transforming activities of C. pneumoniae in human mesothelial cells. This study reported C. pneumoniae infection to induce transformation of human mesothelial cells. Mes1 cells infected C. pneumoniae at a multiplicity of infection of 4 inclusion forming units/cell showed many intracellular inclusion bodies. This study suggested that C. pneumoniae infection might support cellular transformation leading to an increased risk of lung cancer [8]. Another meta-analysis was conducted on the association between C. pneumoniae infection and lung cancer in 2019. It was based on 13 study articles published from 1997 to 2013, which involved 2553 lung cancer cases and 2460 controls. This study indicated that the C. pneumoniae infection IgA positive rate was significantly higher among lung carcinoma patients when compared to healthy controls [2] ( Table 2).
The only animal study was done using an experimental lung cancer model developed through a repeated intrathecal injection of C. pneumoniae into rat lungs. Among them, some of them were exposed to Benzopyrene. The antibodies (C. pneumoniae-IgA, -IgG, and -IgM) in serum were measured by microimmunofluorescence while C. pneumoniae -DNA or C. pneumoniae -Ag of rat lung cancer was detected using polymerase chain reaction (PCR) or enzyme linked immunosorbent assay (ELISA), respectively. Results showed the prevalence of C. pneumoniae infection was 72.9% in the C. pneumoniae group and 76.7% in the C. pneumoniae plus benzopyrene group, with incidences of lung cancer in the two groups of 14.6% and 44.2%, respectively (P = 0.001 and < 0.000 compared with normal controls) [24].

Pathophysiology of Chlamydia pneumoniae infection and lung cancer
Several mechanisms are proposed to explain how C. pneumoniae infection could increase the risk of lung cancer. One mechanism is through mediators of inflammation. Inflammation gives rise to reactive oxygen species that may cause damage to DNA. As well as inflammation causes cell injury, resulting in consequent cell repair, increasing the rate of cell division. Given a fixed rate of DNA damage, higher cell turnover will increase the risk of a mutation conferring a selective advantage to cells, leading to cancer. There is a possible synergistic effect with C. pneumoniae and smoking on lung cancer pathogenesis. C. pneumoniae may localize more quickly in the lungs of smokers. Superoxide oxygen free radicals, tumor necrosis factor, IL-1 h, and IL-8 are produced and secreted by activated monocytes. These inflammatory mediators will cause lung tissue and DNA damage which can result in carcinogenesis. IL-8 also acts as a promoter of tumor growth for human NSCC through its angiogenic properties [25].

Employed diagnostic methods
A case-control study investigated the role of C. pneumoniae infection in the pathogenesis of lung cancer, involving 449 lung cancer patients and 512 healthy controls. They were tested for C. pneumoniae specific IgG and IgA using micro immunofluorescence. Study results suggested that those with no evidence of serum C. pneumoniae IgA or C. pneumoniae IgG, those with both C. pneumoniae IgG plus and IgA plus had 2 times the risk (95% CI: 1.34-3.00) of developing lung cancer. The study concluded that C. pneumoniae infection is potentially associated with primary lung cancer in the Chinese Han population and has combined effects with smoking, passive smoking, and family history of cancer [9].
In an experimental study, they have investigated the association between C. pneumoniae IgG antibodies and the risk of lung cancer among non-smoking women. One hundred ninety-two cases of adult Chinese women and 90 healthy controls were considered in this study. Using an enzyme-linked immunosorbent assay, they were tested for C. pneumoniae-IgG antibodies and 61.98% of cases and 28.89% of controls were seropositive for C. pneumoniae-IgG [15].
A nested case-control study consisting of 593 lung cancer cases and 671 controls evaluated the relationship of C. pneumoniae infection and lung cancer, using traditional serological markers such as microimmunofluorescence IgG and IgA antibodies and Chlamydia heat shock protein 60 antibodies (CHSP-60), which is a marker of chronic chlamydia infection. This study showed that C. pneumoniae seropositivity by microimmunofluorescence IgG or IgA antibodies was not associated with lung cancer, while individuals seropositive for CHSP-60 IgG antibodies showed a significantly increased lung cancer risk [10].
A prospective 2-year study in Sweden assessed 210 (136 Males and 74 Females) patients diagnosed with lung cancer during those two years. Blood specimens for C. pneumoniae serology and throat specimens for C. pneumoniae DNA analysis by PCR were taken and analysed. Male lung cancer patients had significantly higher levels of IgG and IgA antibodies. Compared to controls, both male and female lung cancer patients had a significant prevalence of high antibody titers [17].

Discussion
The most common form of clinical presentation of C. pneumoniae infection is community-acquired pneumonia (CAP). Considering the global epidemiology of pneumonia, Streptococcus pneumoniae is the most common pathogen causing CAP. There is an estimated prevalence of 19.3% to 34% for S. pneumoniae in Europe [26]. Respiratory viruses cause one-third of cases of CAP. Globally 100 million cases of viral pneumonia occur annually [27]. Intracellular pathogens such as legionella pneumophilia, Mycoplasma pneumoniae, C. pneumoniae, C. psittaci, and Coxiella burnetii clinically present as 'atypical' type of pneumonia. A recent review article conducted on intracellular pathogens and occurrence of pneumonia reported that the severe cases of CAP are caused by intracellular pathogens, accounting for 1% to 7% of pneumonia cases [28]. Many reports rank C. pneumoniae among the three most common etiologic agents of CAP, and it has an incidence ranging from 6 to 25%.
Viruses probably induce cancers through their oncogenic effects on human cells. However, the exact connection between bacteria and the oncogenic effect is less understood, and it goes the same way for the C. pneumoniae infection and lung cancer pathogenesis, although there are some proposed mechanisms.
According to GLOBOCAN 2020 estimates of cancer incidence and mortality, produced by the International Agency for Research on Cancer, there are 2,206,771 new lung cancer cases with a percentage of 11.4% while having a high mortality rate [29]. Considering the incidence and high mortality of lung cancer and the epidemiology of C. pneumoniae infection, it is essential to understand the possible causal relationship in the pathogenesis of lung cancer to implement preventive methods and encourage treatments.
All the lung cancer cases included in the studies to look for the relationship between C. pneumoniae infection and lung cancer were diagnosed as primary lung carcinoma after considering the clinical symptoms, examination Page 8 of 9 Premachandra and Jayaweera Infectious Agents and Cancer (2022) 17:11 findings, radiological findings, and confirmed with histological findings. Different studies used various types of diagnosing methods to detect C. pneumoniae. Currently, there is no standard gold method to detect C. pneumoniae antigen levels. As serologic markers, the microimmunofluorescence test, IgA, and IgG antibody titres were widely used, while some studies used PCR for DNA analysis. Use of PCR is beneficial when there is a small amount of DNA, as it can be rapidly amplified [16]. However, the criteria used to define past infection of C. pneumoniae were widely varied among the studies. The lack of an exact serologic marker to identify chronic infection was a significant concern in all studies. Moreover, the reliability of those tests is still debatable. The cut-off values used in the experimental studies also varied resulting, in a comparison between studies questionable [23].
We identified nine case-control studies (9-17) and three meta-analyses [22, 25, and 30], which supported the causal association between C. pneumoniae and lung cancer. Only one animal study was done by using an experimental lung cancer model on rats [24]. Although these human studies reported statistically significant results using serological and DNA assays, they suggested that higher titres may be more predictable. Due to the lack of definitive serologic tests, a low number of cases, and matched controls, researchers highlighted the need for further investigations to conclude the hypothesis.
From five studies investigating the association between histologic specific lung cancer relations between C. pneumoniae infections, only one study reported a significant association between squamous cell cancer and small cell cancer [14]. In comparison, the two studies did not show any significant difference among histologic subtypes [13,31]. Another study observed a strong association for squamous cell cancer but a lesser association for small cell cancer and adenocarcinoma [11]. According to a study done by Kozazeybek et al. 70 patients who had small cell lung cancer, 58.5% were positive for C. pneumoniae, while 28 patients who had squamous cell cancer had 50% positivity (16). However, the small number of cases does not conclude the results.
Among the relevant literature, three case-control studies do not support the causal relationship between C. pneumoniae infection and lung cancer [18][19][20]. Out of three, one study looked for C. pneumoniae DNA by using PCR, while they failed to detect any [18]. The other two studies measured C. pneumoniae IgG and IgA titres, but there was no significant difference among cases and controls [19,20].
The causal relationship between C. pneumoniae infection and lung cancer would be varied when the environmental factors are considered. Hence stratified analyses are needed to be carried out by age, sex, active smoking, passive smoking, alcohol consumption, and family history of lung cancer.

Limitations
The insufficient number of cases, selection bias, recall bias, inadequate control for confounding, and exposure misclassification are some of the main identified limitations in the studies considered for this review. Not having a standard gold test to identify chronic infection of C. pneumoniae and poor defining of chronic C. pneumoniae infection were significant limitations of all the studies. Most studies were concluded based on serology and only two studies conducted based on molecular diagnostics.
Different study designs and methodologies suggest that there may be an actual association between the occurrence of lung cancer and C. pneumoniae infection. Although there are some proposed mechanisms concerning causation, none of them are conclusive, and still, there are some knowledge gaps.

Conclusion
Whether the C. pneumoniae infection is an independent risk factor for lung cancer is debatable, although some studies showed an association between C. pneumoniae infection and lung cancer. To better understand the association between C. pneumoniae infection and lung cancer, both experimental study designs based on animal models or large randomized controlled trials in humans and well-designed cohort studies are needed. Animal models will show a better picture of the induction and pathogenesis of lung cancer with C. pneumoniae infection.