Early acquisition of Pseudomonas aeruginosa in Mexican children with cystic fibrosis Infección temprana por Pseudomonas aeruginosa en niños mexicanos con fibrosis quística

INTRODUCTION: Chronic infection with Pseudomonas aeruginosa (P. aeruginosa) is associated with a greater deterioration of pulmonary function, increase in hospitalizations and shorter survival in patients with cystic fibrosis. OBJECTIVES: To evaluate the age of first detection of P. aeruginosa, effectiveness of the eradication treatment and prevalence of other pathogens in airway cultures. MATERIALS AND METHODS: A cohort of patients with CF diagnosed within first year of life, who had follow-up at University Hospital was studied. Respiratory cultures were obtained quarterly. Patients with infection by P. aeruginosa received eradication treatment. RESULTS: Thirty-five patients were included. During follow-up, 608 cultures were performed. In 34.7%, normal flora was detected, S. aureus 32.8%, P. aeruginosa 20.4%. Median age at first positive culture was 9 months. Eradication was achieved in 81.8%. CONCLUSIONS: In this cohort of patients with cystic fibrosis, the acquisition of P. aeruginosa is earlier than in other groups reported in literature.


INTRODUCTION
Cystic fibrosis is a genetic, multisystemic disease, characterized by thick airway secretion, bacterial infection, and neutrophilic chronic inflammation, which leads to progressive lung damage and chronic respiratory failure.
Pseudomonas aeruginosa (P. aeruginosa) is the most important bacteria in the airway of these patients. Reports estimated that by age eighteen 80% of patients are infected with this microorganism. It is associated with earlier deterioration of pulmonary function, increase in hospitalizations and lower survival. 1,2,3 For this reason, clinical practice guidelines recommend detection and identification of P. aeruginosa, as well as eradication treatment once detected. 4 Currently, there are no studies that evaluate the clinical course, age of first positive culture for P. aeruginosa and prevalence of other bacteria in children less than 6 years with cystic fibrosis in Mexico. The main objective of this study was to evaluate, in a cohort of pediatric patients with cystic fibrosis diagnosed within the first year of life, age of first detection of P. aeruginosa, effectiveness of eradication treatment, time to first recurrence and prevalence of different bacteria.

MATERIALS AND METHODS
A retrospective study of a cohort of patients who attended the cystic fibrosis clinic at University Hospital in Monterrey, Mexico was conducted. Patients diagnosed, within the first year of life, by neonatal screening or clinically, born between January 2004 and October 2017, who were followed regularly in the clinic until December 2018, were included. Diagnosis was established with a sweat chloride value ≥60 mmol/L obtained by quantitative pilocarpine iontophoresis. 5 Patients were followed in the cystic fibrosis clinic in accordance with international guidelines. 4 A respiratory sample was obtained by spontaneous expectoration or oropharyngeal swab (OP), at initial visit, repeated every three months and after eradication treatment. Additional samples were obtained if patients had respiratory symptoms. Age of initial infection by P. aeruginosa was defined as the age in months in which patients had the first positive culture for this bacterium. Both first detection of P. aeruginosa and recurrences during longitudinal follow-up were included in the analysis.
Family socioeconomic level was evaluated following national guidelines. Families were classified as high income (AB, C+ and C) or low income (C-, D and E). 6 Pulmonary function tests were performed by spirometry in the Pulmonary Function Laboratory of the Center for the Prevention and Rehabilitation of Chronic Pulmonary Diseases (CEPREP), following the guidelines of the American Thoracic Society (ATS). 7 The tests were performed on the Platinum Elite Series plethysmograph (MGC Diagnostics) with Breeze 7.2.B software (MGC Diagnostics). The results were expressed as a percentage of the predicted values for forced expiratory volume in the first second (FEV1) and for forced vital capacity (FVC) according to sex, age and height.
For chest tomography evaluation, the Bhalla score was used. 8

Eradication treatment of P. aeruginosa
All patients with P. aeruginosa infection received eradication treatment with inhaled tobramycin 300 mg every 12 hours for 28 days plus oral ciprofloxacin 30 mg/kg/day every 12 hours for 14 days.

Statistical Analysis
Continuous variables were reported as mean and standard deviation or median and range, as appropriate, categorical variables as absolute numbers and percentages. For the analysis of categorical variables, contingency tables and chi-square test were used. For comparison of medians, the Mann-Whitney U test was used. A value of p <0.05 was considered statistically significant. Data was analyzed using SSPS version 20.0 (IBM Corp, Armonk, NY).
The study's protocol was approved by the institutional Committee of Ethics and Research and patients and their families signed informed consent.

Microbiologic outcome
Across all clinical visits, 608 cultures were obtained. In 77 (12.6%) of them two or more bacteria were detected, with a total of 685 bacterial isolations. Bacteriological findings are summarized in Table 2.
In the study, 22 patients (62%) had their first positive culture for P. aeruginosa before the first year of life. Fourteen (40%) cultures were positive in the first appointment to the clinic.  Table 3 DISCUSSION P. aeruginosa is a microorganism that is widely distributed in nature and its optimum growth temperature is 37º C. In patients with cystic fibrosis, it is the main pathogen responsible for morbidity and mortality. 8 Studies support a relationship between the first isolation of P. aeruginosa and clinical dete-rioration in cystic fibrosis patients. Pitman et al found a strong association between early age of acquisition of P. aeruginosa and severity of lung disease. 2 Age of acquisition of P. aeruginosa is related to interactions between environmental and hostdependent factors. Host-dependent risk factors for early acquisition are genetic mutation, female sex, history of meconium ileus, among others. 9,10 In respect to environmental factors, geographic variability in the prevalence of lower airway infections in patients with cystic fibrosis has been reported. 11,12 Other factors, such as high temperature, can cause higher prevalence of P. aeruginosa in the environment, and therefore stimulating the acquisition of infection.
Epidemiological information about first detection of P. aeruginosa in Latin American countries is scarce. In Argentina mean age at first isolation has been reported at 1.18 months. 13 In a study conducted in Australia median age of first detection was 30.5 months. Also, mean duration of reported eradication was 19 months, comparable to 15 months in this study. 14 On the contrary, Psoter et al reported a mean age of 1.3 years in the USA. 15 This study showed that, compared to patients who remained without P. aeruginosa, those who acquired it were more likely to reside in places with higher average daily temperatures (13.8 vs 12.9 ° C) and higher precipitation (0.14 vs 0.12 mm). 15 In this study, one of the factors that can cause early acquisition of the bacteria is probably the high average annual temperature in the region, as the climate in Northeast Mexico is semi-arid and average annual temperature is around 24 -25 °C. 16 This association has been reported in previous studies. 14,17 Low socio-economic level has significant negative effects on cystic fibrosis, as it's associated with worse nutritional status and poorer outcomes. In Mexico it has been reported that low socioeconomic level is the most detrimental factor in the outcome of patients with cystic fibrosis, 18 although it was not statistically significant in our group.
Moreover, a study showed that Hispanic patients with cystic fibrosis residing in the United States had lower survival compared to non-Hispanic patients and one of the factors suggested by the authors to explain this ethnic difference were genetic polymorphisms not currently known. 19 In another study, after adjusting for socioeconomic status and clinical severity, Hispanic patients had 2.81 the mortality rate compared to non-Hispanic patients, which underscores the importance of intrinsic and extrinsic ethnic factors related to survival. 20 This study has certain limitations, including a small sample size and that all patients were recruited from a single center. Other factors associated with respiratory exacerbations and with early acquisition of P. aeruginosa are air pollution, in particular PM10 and PM 2.5, as well as exposure to tobacco smoke. 21 These factors were not studied in our cohort and should be investigated.
However, the same medical team followed patients longitudinally, using standardized protocols, which enhances consistency among patient management.
This is one of the scarce reports that investigates prevalence of pathogens in young cystic fibrosis patients in Mexico and demonstrates that in these patients acquisition of P. aeruginosa is earlier than in other cohorts, potentially leading to a poorer quality and length of life, thus alerting on the need of quick detection and intensive standardized eradication therapy.