 patients, six samples were Acanthamoeba positive and three ones were Acanthamoeba negative by PCR method. Tables 1 present the distribution Acanthamoeba spp. in the BAL samples suspected of cancer regarding the patients' demographic characteristics. Among the patients infected with Acanthamoeba, 85% were above 65 years (p = 0.025) (Table 1). Furthermore, Fig. 1 Gel electrophoresis of the 500 bp PCR product of Acanthamoeba spp. using JDP1 and JDP2 primers, isolated from BAL culture of suspected of cancer in Kashan hospital, Iran. (NC: negative control, S1-S7 positive Acanthamoeba samples: marker 100 bp and PC: Positive control) the prevalence rates of Acanthamoeba among patients with elementary education and those with diploma were 75.6 and 60%, respectively (p = 0.035). The frequencies of pulmonary symptoms such as dyspnea, cough, tumor mass, fever, sputum, and pneumonia in the Acanthamoeba-positive patients were not significant ( Table 2).
The selection of eighteen Acanthamoeba strains for genotyping was based on pathological positive lung cancer or observing a 500 bp sharped band by PCR. Out of 18 sequenced Acanthamoeba samples, four ones were belong to cancer patients and positive Acanthamoeba and the others had sharped band of Acanthamoeba by PCR. All Acanthamoeba spp. isolates (100%) belonged to the T4 genotype. Most Acanthamoeba cysts were diagnosed with double-wall cysts, endocyst polygonal in the size of 9.5-11.5µm (Figs. 2, 3, 4) ( Table 3).

Discussion
The present research was the first research on the Acanthamoeba genotypes in a population with respiratory symptoms suspected of cancer, who were admitted to Kashan's hospitals in Iran. The findings indicated that, out of 97 BAL samples, 86.6% were positive for FLA, and Acanthamoeba was confirmed in 73.2% of the cases. The prevalence of FLA in the oral cavity of cancer patients in Kashan was reported to be 89% (Taghipour et al. 2021). Golestani et al. (2018) reported that the rates of Acanthamoeba in the dust, soil, and water samples of the Beheshti Hospital in Kashan were 52.5, 62.5, and 50%, respectively. Furthermore, the contamination rates of rural drinking water of Kashan to FLA and Acanthamoeba were 35.2% and 11.7%, respectively (Mostafaei et al. 2019). In a study conducted in Iran, Acanthamoeba infection was found to be 100% and 98.4% in the BAL samples of immunocompromised patients with respiratory disorders using culture and molecular methods, respectively (Eslamirad et al. 2020).
According to Lanocha et al. (2009), Acanthamoeba was isolated from 3.13% of the BAL samples of patients with immunodeficiency, and the T4 genotypes were identified. Nineteen cases of Acanthamoeba spp. were reported in immunodeficiency patients with pneumonia in Poland, the U.S.A, Australia, India, Japan, and France (Kot et al. 2021). In a study in Peru, Acanthamoeba spp. and the T4 and T15 genotypes were reported from the nasal swap samples of healthy individuals (Cabello Vilchez et al. 2014).
However, the prevalence rates in the present study are higher than those reported by other researchers (Lanocha et al. 2009;Kot et al. 2021) and lower than Eslamirad et al.'s reported rate (2020). The high prevalence of FLA and Acanthamoeba in this study may be due to modifications in culturing the BAL samples, which agrees with Taghipour et al.'s (2021) findings. Accordingly, the high rate of FLA and Acanthamoeba in the BAL samples in this study may probably occur due to the climatic conditions of the Kashan desert region, including dry climate, low rainfall, seasonal storms, and dust. Moreover, the high contamination rate of drinking water and dust in Kashan may also contribute to this high prevalence rate (Golestani et al. 2018;Mostafaei et al. 2019).
Since Acanthamoeba is the most common free-living amoeba, ubiquitously distributed in nature and everywhere (Siddiqui and Khan 2012), it can cause a high infection rate in patients without lung cancer. The detection of the Acanthamoeba in the BAL samples may not actually be the cause of pathology, and it may only be colonization ( Khurana et al. 2015). Golestani (2018) reported that the rate of Acanthamoeba in the dust of the Beheshti Hospital in Kashan was 52.5% (Golestani et al. 2018).
We also found that the rate of Acanthamoeba infection was higher in diabetic patients (90%); however, the difference was not significant. Diabetes has been reported as one of the risk factors for Acanthamoeba infection (Shimmura-Tomita et al. 2018).
Since Acanthamoeba is ubiquitously distributed in nature, it may be a risk factor for the transmission of the parasite to health officials and immunocompetent (Niyyati et al. 2009;Kot et al. 2021).
The high prevalence of positive Acanthamoeba in negative lung cancer may be due to pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis (CF), which cause parenchymal lesions and the colonization of Acanthamoeba in the lung (Vanspauwen et al. 2012;Colson et al. 2011).
In this study 18 Acanthamoeba genotypes were identified in the BAL specimens of pulmonary patients for the first time and deposited in the GeneBank database. Moreover, their similarity with the recorded isolates in the GeneBank was 98-100%. Other genotypes such as T1, T4, T5, T10, T11, T12, and T15 have been reported from clinical specimens worldwide (Kot et al. 2021;Cabello Vilchez et al. 2014;Walochnik et al. 2008;Booton et al. 2005;Memari et al. 2016).
Most of the Acanthamoeba genotypes in the dust of the Beheshti Hospital and the drinking water of Kashan were T4 (Golestani et al. 2018;Mostafaei et al. 2019). This finding was in a similar with the findings of the present study, as the T4 genotype was detected in all BAL samples suspected of cancer and had 98% similarity with the T4 genotype of the dust samples.
T4 is the predominant genotype in GAE and pulmonary Acanthamobiasis. Since the T4 genotype is a potential pathogen in patients with immunocompromising conditions, differential diagnosis of Acanthamoeba should be more considered in these patients (Bloch and Schuster 2005;Shin and Im 2004;Kalra et al. 2020). Accordingly, physicians and health professionals should be more attentive to this lethal infection and diagnostic options, especially in suspected encephalitis.
The results of the present study indicated the higher rates of Acanthamoeba infection in patients suspected of cancer, who aged above 66 years (85%, p = 0.025). Furthermore, the prevalence of Acanthamoeba was higher in the individuals with elementary education than in others; however, the difference was statistically significant (p = 0.035). Kot et al.'s (2021) study showed that most patients with Acanthamoeba pneumonia had weight loss and respiratory failure, which is consistent with the findings of the present study.
Some case reports have been published on Acanthamoeba pneumonia with immunodeficient status (Shin and Im 2004). The death of cancer patients from unidentified encephalitis has been reported in several studies in Iran (Edrisiian et al. 2008).
The results of the present study indicated that pulmonary symptoms such as dyspnea, cough, fever, sputum, and pneumonia in the population with Acanthamoeba positive suspected of cancer were not significant.
Because this protozoon can act as a reservoir of a variety of bacterial agents such as Legionella pneumophila, Mycobacterium tuberculosis, Helicobacter pylori, Chlamydophila pneumoniae, and Staphylococcus aureus (Dey et al. 2020;Medie et al. 2011;Siddiqui and Khan 2012), it is recommended to conduct a microbial study on these patients. However, pulmonary diseases such as asthma may cause these symptoms.

Conclusion
In general, the present findings indicated that higher frequencies of FLA and Acanthamoeba in BAL samples with pneumonia symptoms suspected of cancer compared to other previous studies. The successful cultivation of FLA and the high prevalence of Acanthamoeba in this study are due to modifications in culturing the BAL samples. The high rate of Acanthamoeba in the BAL samples may be due to parasite colonization.
Since the T4 is a pathogenic genotype, pulmonary symptoms may occur due to this parasite. Accordingly, health education and sanitation is recommended to prevent this infection.Jeff Wilpon has issued a statement regarding Mike Piazza’s snub by the Hall of Fame voters. Piazza received 57% of the vote, as he was unfairly lumped in with such players as Roger Clemens and Barry Bonds, who had evidence against them that they allegedly used steroids. There has never been any evidence that Mike Piazza used PEDs.
