High Occurrence of Emerged Lophomonas Infection among Patients Suspected of Having Pulmonary Tuberculosis: In-House PCR-Based Evidence

Objectives Lophomonas infection is a respiratory disease in humans that is associated with symptoms of cough, sputum, dyspnea, and sometimes hemoptysis, which shows the importance of differentiating this disease from tuberculosis and asthma. Methods This study was performed on 216 participants suspected of having tuberculosis who had symptoms of fever, chronic cough, or sputum and were referred to tuberculosis laboratories in three cities in Golestan Province, northeastern Iran, during 2019-2020. A sputum sample was taken from the suspected patients. DNA was extracted from the frozen samples, and an in-house polymerase chain reaction was performed to detect the Lophomonas DNA. Results Out of 216 subjects, 47 (21.75%) were infected with Lophomonas spp. Moreover, 9 patients (4.2%) were infected with tuberculosis. Also, 2 patients had a comorbidity of tuberculosis and Lophomonas infection (P=0.63). There was no significant difference in the comparison of symptoms and the rate of Lophomonas infection (P=0.84), but in the comparison of the set of symptoms of cough, sputum, and fever with those of cough and sputum, cough with fever, sputum with fever, and the rate of Lophomonas infection, there was a significant difference (P=0.012). Conclusions Lophomonas infection was relatively high in patients suspected of having tuberculosis and due to the similar clinical symptoms of Lophomonas infection and tuberculosis; it is recommended that the sputum samples of subjects suspected of having tuberculosis be examined for this parasite in order to make a correct diagnosis and the patients receive timely treatment and the appropriate medication.


Introduction
Tuberculosis (TB) is an important infectious disease and one of the top 10 causes of death in the world. In 2019, TB killed 1.4 million people, 208,000 of whom were HIV-positive. TB has been reported in all countries and age groups [1]. According to the World Health Organization (WHO) report in 2019, the incidence rate of TB was <10 cases per 100,000 subjects in Iran [1,2]. Golestan Province, in northeastern Iran, is the second most infected province in terms of cases of TB in Iran [3,4].
Te emergence of lophomoniasis, caused by the protozoan Lophomonas spp., mainly as a commensal inhabitant of cockroaches, is an unusual cause of bronchopulmonary infections [5]. Te fagellated parasite found in airway samples aids in diagnosis, though their morphology is identical to ciliated bronchial epithelium, which makes interpretation difcult. Te dust contaminated with cysts may be inhaled, initiating infection. Infections are reported mostly in adults; very few have been described in children [6].
Lophomonas infection is a respiratory disease in humans that is associated with symptoms of cough, sputum, dyspnea, and sometimes hemoptysis, which shows the importance of diferentiating this disease from TB and asthma [7,8]. So far, human Lophomonas infection has been reported from two Iranian provinces in northern (Mazandaran, Sari) and eastern (Khorasan Razavi, Mashhad) Iran [9][10][11]. According to the information published by the Iranian National Registry Center for Lophomoniasis (INRCL), Lophomonas blattarum (L. blattarum) has been confrmed by molecular and sequencing techniques in Iran [1]. Also, the parasite was isolated recently from German cockroaches (Blattella germanica) in Mazandaran, northern of Iran [6].
Till date, no study has been performed on patients suspected of having TB, and only case reports of Lophomonas and TB coinfection have been published around the world. Terefore, considering the similarity of tuberculosis with lophomoniasis in terms of clinical patterns and the lack of data about the status of this infection in patients suspected of pulmonary tuberculosis, the present study attempts to investigate the status of Lophomonas infection in these individuals using an in-house PCR test.

Study Area.
Golestan Province is one of the northern provinces of Iran. Its area is 20,367 km 3 . Te province has seven counties. Te population of the province in the general census of population in 2016 was 1,868,819 subjects. Tis province has a diverse climate due to its special geographical location. Golestan Province is divided into the following three areas in terms of unevenness: mountainous area, foothill area, and plain area. Figure 1 shows a map of Golestan Province.

Participants and Sampling
. 216 participants were randomly selected from patients with suspected tuberculosis who had symptoms of fever, chronic cough, or sputum and were referred to tuberculosis laboratories in three cities of Golestan Province, including Aq-Qala, Gonbad-e Kavus, and Gorgan in the northeastern of Iran, during November 2019-September 2020. All subjects entered the study with informed consent, and for each of them, a questionnaire containing the studied variables was considered. Te study was confrmed by the Ethical Committee of Mazandaran University of Medical Sciences (ethical code: IR.MAZUMS.REC.1399.6901).
At the tuberculosis diagnostic centers, a sputum sample was taken from the patients for testing, and we transferred a part of this sample to the laboratory for testing of the Lophomonas parasite. Since the samples were suspected of having tuberculosis, the study was conducted on them in compliance with all hygienic points. Te samples were stored at −20°C until use.

DNA Extraction.
To genomic DNA extraction, frozen sputum specimens (about 1 ml) were thawed at room temperature and then spin at 500 g for 5 min [11]. Next, the sediment was used for DNA extraction. Lysis bufer (0.93 g of EDTA, 0.5 g of SDS, 1.43 g of NaCL, and 3.15 g of Tris-HCL in 100 ml of distilled water) was added and mixed with each of the sputum sediments at an equal volume, and the microtubes were vortexed twice for 10 seconds each time, then incubated at 60°C for 30 min, centrifuged at 4,000 × g, 4°C, for 10 min, and the supernatants were transferred to the new microtubes. 15 μL of proteinase K (20 mg/ml) was added to each of the supernatants, which were incubated at 60°C for 45 min, and then 200 μL of phenol: chloroform solution (1 : 1; w/v) was added to each of the microtubes, and centrifuged at 14,000 × g, 4°C, for 15 min. 400 μL of absolute ethanol was added to the separated supernatants, vortexed once, and put into the microtubes at −20°C for 2 hours. Te samples were centrifuged at 14,000, 4°C, for 15 min and the pellets containing DNA were harvested and kept at −70 until use.

Conventional Genus-Specifc PCR (In-House PCR).
Genus-specifc PCR was performed to detect the DNA of the Lophomonas parasite in the extracted sputum samples as described by Fakhar et al. [12]. Te used primers were as follows: forward 5′-GAGAAGGCGCCTGAGAGAT-3′ and reverse 5′-ATGGGAGCAAACTCGCAGA-3′. 1 μL of each primer and 12.5 μL of mastermix were added to a microtube, and the volume was adjusted to 25 μL with ddH 2 O. Te time and temperature of the reaction were: initial denaturation at 94°C for 4 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 70°C for 30 s, and fnal extension of DNA for 5 min. Te samples after PCR were run on a 2% agarose gel in a Tris-boric acid-EDTA solution (w/v). Te gel was assessed under a UV transilluminator after staining with a SafeView TM DNA Stain. A 214 bp band was related to Lophomonas spp. In the PCR processing, L. blattarum (Accession Numbers: MZ093070) was used as positive and distilled H 2 O and DNA of Trichomonas vaginalis as a negative control.

Data Analysis.
For qualitative variables, we used percentage and frequency. A P value of less than 0.05 was regarded as statistically signifcant. Demographic data were analyzed by IBM SPSS version 26.

Positive Cases of Lophomonas Infection and Tuberculosis.
Out of 216 subjects, a total of 47 (21.75%) were infected with Lophomonas spp., (Table 1). Nine subjects (4.2%) were infected with tuberculosis. Of them, 3 (3.53%) were from Aq Qala, and 6 (5.82%) were from Gorgan districts. Also, 2 patients had a comorbidity of tuberculosis and Lophomonas infection (P � 0.63). One of them was from Aq Qala, and the other was from Gorgan City. Te most clinical symptom of the patients was fever, following sputum and cough, respectively. Tere was no signifcant diference in the comparison of clinical symptoms separately and the rate of Lophomonas infection (P � 0.84; Table 1), but in the comparison of the set of symptoms of cough, sputum, with fever, cough with sputum, cough with fever, sputum with fever, and the rate of Lophomonas infection, there was a signifcant diference (P � 0.012). Te result of PCR is shown in Figure 2

Discussion
So far, Lophomonas spp., has been mostly isolated from sputum and BAL fuid clinical samples [5]. For this reason, in the present study, we used sputum samples to examine Lophomonas infection.
Currently, the Giemsa, Trichrome, and Papanicolaou staining methods are used to identify Lophomonas parasites [13], but the identifcation of this parasite is difcult to detect based on morphology due to its high similarity to normal and/or atypical lung epithelial cells [14]. Terefore, the PCR method increases sensitivity and specifcity in the diagnosis of this parasite [12]. Te results of the present study showed that 47 out of 216 cases (21.76%) were infected with the parasite by PCR. Considering the emerging nature of this infection and the lack of evidence of the prevalence of Lophomonas infection in people suspected of tuberculosis, the results of the study cannot be compared with other studies, and the results of our study are the frst scientifc evidence in this regard.
To date, accurate data on the prevalence of this parasite in diferent countries are not available, and most studies have been performed in the form of case reports or case series of infected subjects, and most of them are from Asia [5,14,15]. However, the real global burden of Lophomonas infection is unknown; the most cases have been recorded in Iran and China [13]. Based on a systematic review regarding global Lophomonas infection the coexistence of tuberculosis with lophomoniasis was relative high (11.4%; 17/149) [14].
A study showed that the coexistence of tuberculosis and lophomoniasis was relatively common (11.4%; 17/149) [14]. According to the high prevalence (21.75%) of Lophomonas infection among the participants suspected of having tuberculosis in the present study, Golestan Province could be considered as one of the endemic areas for the emerged protozoan parasite.
Consistent with some evidence, Lophomonas infection is not afected by age or gender [5,11,16], and the fndings of our study support this (Table 1). However, the majority of cases of infection were observed in subjects over 50 years old, which could be attributed to the large sample size in this age group, the possibility of more parasite exposure for these subjects, the occurrence of clinical symptoms in old age, and more doctor visits.
Few studies have examined the association of Lophomonas infection with other respiratory diseases, including pneumonia, chronic obstructive pulmonary disease, lung cancer, allergies, interstitial lung disease, pulmonary abscess, tuberculosis, chronic cough, respiratory cysts, asthma, and bronchitis [5,11,15,16]. Of these, 21 cases of coinfection with lophomoniasis and tuberculosis have been reported so far [5,8,11,17]. However, in the present study, we showed that there is no signifcant relationship between lophomoniasis and tuberculosis (P � 0.63) and only in two cases with lophomoniasis and tuberculosis coinfection were observed. Interestingly, 47 cases of Lophomonas infection were observed in 216 patients suspected of having tuberculosis, indicating the importance of examining this parasite in subjects who exhibit the set of symptoms of cough with sputum and with or without fever.
Te most important clinical signs that suggest the possibility of Lophomonas infection include cough, sputum, and fever, which are observed in patients with asthma or tuberculosis, so researchers have shown that there is a signifcant relationship between the symptoms of asthma and Lophomonas infection [11,[18][19][20]. Recent studies have noted that asthma-like symptoms in patients with Lophomonas infection are milder than those in subjects with asthma.
According to the results of a study, the most important symptoms in children and adults with Lophomonas infection are fever and then cough [2]. However, in the present study, it was found that patients with symptoms of cough with sputum had the highest rate of Lophomonas infection, although these two symptoms may be without fever (17/47; 36.17%) or with fever (25/47; 36.17%). Terefore, fever may not be observed in all patients infected with the parasite, and this should be considered in the diagnosis.

Conclusions
To our knowledge, this is the frst evidence about Lophomonas infection in patients suspected of having pulmonary tuberculosis in an endemic area. Given that the infection was relatively high in patients suspected of having tuberculosis and also had similar clinical symptoms of Lophomonas infection with tuberculosis, it is highly recommended that the sputum samples of these patients be examined for this parasite in order to make a correct diagnosis. Also, ensure that the patients receive timely treatment and the appropriate medication. Further investigations are needed to

Data Availability
Te data are available from the corresponding author upon reasonable request.

Consent
Written informed consent was taken from the patients to include the clinical details.

Conflicts of Interest
Te authors declare that they have no conficts of interest.