Molecular detection of Acanthamoeba genotypes and Pneumocystis jirovecii in bronchoalveolar lavage specimens from immunocompetent patients with respiratory disorders

Opportunistic pathogens such as Acanthamoeba spp., Microsporidia , and Pneumocystis jirovecii are well recognized causes of pulmonary infections in immunocompromised patients. However, their prevalence and clinical significance in immunocompetent individuals with respiratory disorders remain poorly understood. This study aimed to determine the presence of Acanthamoeba spp., Microsporidia , and P. jirovecii in bronchoalveolar lavage (BAL) samples from immunocompetent patients with various respiratory disorders referred to the Iranian National Registry Center for Lophomoniasis (INRCL) at Imam Khomeini Hospital, Mazandaran province, northern Iran. This cross-sectional study analyzed 146 BAL samples collected from immunocompetent patients with respiratory disorders during 2024. DNA was extracted using a phenol-chloroform-isoamyl alcohol method. Molecular detection was performed using conventional PCR with genus-specific primers (JDP1/JDP2) for Acanthamoeba spp., and nested PCR assays targeting the ITS region for Microsporidia and the mitochondrial large subunit rRNA (mtLSU rRNA) gene for P. jirovecii . Genotype identification of Acanthamoeba isolates was achieved through bidirectional Sanger sequencing, and sequences were analyzed using BLASTn. A phylogenetic tree was constructed using MEGA-x software employing the Maximum Likelihood method with the Tamura 3-parameter model and 1,000 bootstrap replicates. Statistical analysis was performed using the Chi-square test (SPSS v20), with a P-value < 0.05 considered statistically significant. Molecular analysis identified Acanthamoeba spp. in six cases (4.11%), which were classified as genotypes T4 ( n = 5) and T5 ( n = 1). All six patients had underlying chronic respiratory conditions, with chronic cough as the predominant symptom. Additionally, P. jirovecii was detected in nine patients (6.16%), including one case of co-detection with Acanthamoeba T4. Among P. jirovecii -positive cases, the majority resided in rural areas, and all had pre-existing pulmonary diseases. Microsporidia genus were not detected in any of the samples analyzed, which may reflect their low prevalence in immunocompetent populations or possible methodological limitations. Molecular detection of Acanthamoeba (primarily genotype T4) and P. jirovecii DNA in BAL specimens from immunocompetent patients with underlying chronic lung diseases indicates the presence of these organisms in the respiratory tract. The relatively higher detection rate of P. jirovecii among rural residents warrants further epidemiological investigation. Importantly, PCR positivity alone should not be interpreted as definitive evidence of clinical disease, and the significance of these molecular findings requires careful clinical correlation. Future prospective studies with longitudinal follow-up are needed to determine whether these colonizers contribute to disease progression or exacerbation in immunocompetent individuals with pre-existing pulmonary conditions.