Acute respiratory infection (ARI) includes a heterogeneous group of infections compromising one or more parts of the respiratory system, and clinically they have an evolution period of less than 15 days. The ARI includes upper respiratory tract infections (URTI) and lower respiratory tract infections (LRTI), a term that includes a number of inflammatory pathologies called according to the affected site such as rhinosinusitis, otitis, mastoiditis, pharyngitis, tonsillitis, bronchitis, pneumonia, common cold, among others [1,2,3]. According to the report of the Forum of International Respiratory Societies 2013, respiratory tract infections are the leading cause of mortality, causing 4 million of deaths per year in developed countries [4].

In United States, ARI is the leading cause of outpatient visits and prescription of antibiotics in adults, where their misuse is a causal factor of bacterial resistance, alarming phenomena today given the increased morbidity and mortality as well as the high costs associated [4]. According to the 2015 National Study of Health, welfare and aging in Bogotá, the notification of ARI cases from the emergency department, outpatients, hospitalization in general ward and in intensive care unir, showed a slight decrease with respect to the previous two years. However, the prevalence of this disease is higher in children under 5 years, people with some chronic underlying disease, immunosuppression and people over 60 years old [5].

The estimate of the Colombian older people population aged between 60 and 80 years, is around 5.2 million in an overall population of 47 million. In Bogotá, there were more than 7.5 million inhabitants, of which 11% were persons aged 60 years and older [5]. ARI incidence increases progressively with age, being pneumonia, particularly, the main cause of disease and death in the older adult and elderly. Infection in this group is characterized by a prolonged hospitalization and higher rates of risk and mortality compared to younger age groups; that can be explained by a gradual decrease in the integrity of the physical and immunological barrier against pathogens and by the variety of comorbid conditions presented in this age group [5, 6].

The etiology of ARI involves among other microorganisms the Streptococcus pneumoniae, Haemophilus influenza, Klebsiella pneumoniae, Staphylococcus aureus, and viruses such as Syncitial Respiratory Virus, influenza a and b, parainfluenza 1, 2 and 3, adenovirus, and atypical germs such as Chlamydophila pneumoniae (CP) and Mycoplasma pneumoniae (MP) [7,8,9,10]. Detection of atypical bacteria is a difficult process as they require for their diagnosis specialized techniques underused in our environment, also, there is a lack of golden tests, few commercial tests are available, and there is a lack of standardized approaches and appropriate validations for diagnostic techniques, reasons why there is diagnostics underreporting in Colombia [8,11].

MP and CP can cause severe disease in older adults consequently causing hospitalization and even death. Infection in the older adults and elderly is characterized because the hospitalization is 4 to 6 times longer than in younger populations, with risk rates around 25-30% and mortality rate 3-5 times higher than in young adults [6, 12]. Older adults often present comorbid medical conditions that favor the development of infection, sepsis and/or respiratory failure. Co-infection with other microorganisms is common and favors the development of complications. Atypical bacteria do not respond to monotherapy with b-lactam antibiotics, and additional coverage with macrolides persists as an optional therapy nowadays [13].

To contribute to the reduction of morbidity and mortality associated with atypical organisms causing ARI is necessary to perform an early and timely diagnosis to be able to choose the right treatment. In Colombia there are no unified and consistent criteria for the identification of atypical pathogens that can cause ARI, for their diagnosis, risk classification and treatment selection in older adults population. That is why it is considered pertinent to identify and determine the prevalence in our environment of M. pneumoniae and C. pneumoniae causing ARI in adults over 65 years.

In Colombia, ARIs are the fifth leading cause of death in adults older than 60 years and its impact is reflected in the high number of hospitalizations, disability of the affected population, the high degree of morbidity and mortality, the cost in the care of them and the low number of studies in the area for this age group [20]. Nationwide reports on ARI, correspond to specific studies focused on community-acquired pneumonia involving different age groups, being the most recent the studies by Caballero et al. [21]; Montúfar et al. [22]; Robledo et al. [23]; Vélez et al. [24], or correspond to research related to specific microorganisms as case studies and mortality by infection with H1N1 such as the studies from Porras et al. [25] and Rey et al. [26].

In that sense, this study provides important data, showing that from 57.7% of the isolates 18.3% were atypical organisms such as CP and MP, very significant percentage of the elderly population because these microorganisms are not identified routinely in hospitals and in some cases they might be diagnosed as viral infections being prescribed a wrong treatment.

Among the ARIs, it was found that 69% presented URTI, 31% presented LRTI, the cough was one of the most common physical findings (52.1%) and common cold was the most representative pathology in URTI (33.8%) and pneumonia in LRTI (23.9%). The data obtained are similar to those reported by other researchers in populations of older adults like Wójkowska et al. [27] showing a higher percentage of ARI-URTI; Choy et al. [28], who show the common cold as the respiratory condition that affects the most the geriatric population; Evertsen et al. [29] establishing cough as a criteria of a high predictive value in ARI and pneumonia in different studies present percentages ranging from 20% to 66% inclusive [30, 31]. The data in this age population are explained by the presence of multifactorial causes that include physiological and immune changes according to the age, immunosenescence, pulmonary changes such as mucociliary clearance and decreased protective reflexes such as cough, increased lung compliance, decreased number of alveolar macrophages and neutrophils, the presence of concomitant diseases such as COPD and the institutionalization in geriatric units among others [6, 32,33,34].

Although technological advances allow us to identify a large percentage of etiologic agents of ARI, there are still shortcomings in the identification of MP and CP and there are limitations for the proper interpretation of serological and molecular tests and their correlation to the patient's clinical symptoms [11, 32]. However, guidelines proposed by Csango et al. [18]; Paldanius et al. [16]; Chedid et al. [17], and Barbeyrac et al. [19], keep a good correlation with the clinical findings in the patients, and these guidelines were applied in the present study. Serological results for CP showed IgG reactivity of 32.4%, 2.8% IgM, and 2.8% IgA. IgG titers have been documented by different authors, who suggest that the prevalence increases with age, reaching 50% of reactivity in young adults and can reach up to 75% of reactivity in older adults and the elderly [35]. The importance of the IgG reactivity found in the study suggests that people have subclinical infections by CP or get reinfected throughout life, highlighting the problem of underdiagnosis and lack of epidemiological data on the infection [35]. In terms of IgM it is known that titers appear during the first 3 weeks and are replaced by IgG from the 6^th to 8^th week, meanwhile, in reinfection IgM is detectable in the first 2 weeks, disappears between 4 to 6 months and leads the IgG from week 1 or 2 and can last up to 3 years. To determine the pattern of infection this kinetic requires to take a second serum sample in the convalescence period or to verify the persistence of IgA or the detected Ag through molecular tests [36]. These guidelines were contemplated in the study and that is how the results could be verified with IgA; also, through qPCR two new cases that had not presented serologic reactivity, due to a higher sensitivity of the molecular technique [37], were found.

Regarding MP, it was observed that 8.4% of individuals had litres of IgA and IgM clinically associated with respiratory infection. Infections caused by MP are characterized by a slow rise of specific IgG reaching the highest values ​​at 5 weeks after the start of symptomatology. After the infection, these antibodies remain elevated for periods of up to 4 years. Therefore, low levels may indicate past infection or early stages of acute infection. In this case a second sample should be analyzed at 2 or 3 weeks to demonstrate an increase in the antibodies titer and the diagnosis requires the quadrupling of the values ​​or titers greater than or equal to 1/32 [38]. While infections by MP are common in children and young adults under 19 years and are detected in low percentages in older adults, in this age group morbidity and mortality are usually higher [6,12,39], reason why 8.4% of detected cases in this study is significant, since knowing the physiopathology, clinical and etiology of the infection by atypical microorganisms helps us improve the diagnosis, treatment and prevention of acquired pneumonia in immunocompetent adults, as established by the Colombian Association of Infectious Diseases (ACIN) [40] in its guide of clinical practice from 2013 and the institutional guidelines proposed by Forero [41]. Although serological techniques have sensitivity and specificity greater than 90%, diagnosis can be improved in its efficiency by molecular techniques. That is how Kumar and Hammerschlag [42] proposes that diagnosis of CP should include ELISA and a molecular technique such as PCR, while Waites and Talkington [43] proposes that PCR should be the test of choice for MP. This study showed that the percentage of positive cases by serology increased when molecular techniques were used. CP went from 5.6% to 8.5% and in MP from 8.4% to 9.9%. In Colombia, different authors like Robledo et al. [23], Velez et al. [24], Montufar et al. [44], and others mentioned in the guide of clinical practice from 2013 from the ACIN [40], have reported different etiologic agents associated with ARI allowing to spread the knowledge on the epidemiology of infection in some parts of the country. However, the use of molecular techniques has changed some of the epidemiological and clinical concepts on CP and MP due to the sensitivity of the techniques. For this reason, it is recommended that the persistence of MP or CP in respiratory samples require studies to standardize and define the number of copies/mL that allow the differentiation of clinical infection from residual colonization [37]. In this regard, this study used as a molecular technique a qPCR that allowed obtaining such data.

Finally, it should be emphasized the mixed infections that occurred between MP and CP, as well as MP and Kpn. Such infections have been reported in some studies and are attributable to superinfections, predisposing factors such as asthma, increasing hypersensibility to one of the infections or to behaviors that MP may have as commensal flora in immunocompetent patients [45], which is important to keep in mind when interpreting the results as well as when doing the processes of therapeutic intervention in the patient.

The work generated new knowledge on the behavior of the two microorganisms in older adults, allowing to determine that the majority of the population have immunological contact with these two microorganisms at some point; CP and MP are important etiological agents of ARI in older adults who present more aggressive clinical symptoms and the use of quantifying molecular techniques such as qPCR is important to detect microorganisms difficult to diagnose as those mentioned in the article.