Introduction

Lophomonas parasite is an extracellular flagellated protozoon found in the order Hypermastigida and suborder Lophomonadina. It is accepted as an endocommensal in the intestine of cockroaches such as Periplaneta americana (American cockroaches) and Blattella germanica (German cockroaches). Lophomonas comprise two species: Lophomonas blattarum (L. blattarum) and Lophomonas striata (L. striata) [1, 2]. The morphological characteristics are as follow: Lophomonas spp. range in size from 20 to 60 μ in length and 12–20 μ in width. Their cytoplasm is seen as granular and food phagocytosis. The nucleus is placed as a circular and dark body next to the cilium. Although both L. blattarum and L. striata have a tuft of flagella, the body of L. striata is tightly folded in its longitudinal axis by striations clearly located on its surface and resembling closely those reported for certain bacteria. Multiplication of the trophozoites is mainly by binary fission [3]. Encysted forms have been described and fully formed cysts are spherical or sometimes oblong and surrounded by a homogeneous membrane [4].

The first case of pulmonary L. blattarum infection was reported in China in 1993 [5]. Actually, the details of transmission of this neglected and emerging protozoan disease still remain a mystery. But the acceptable route of transmission is inhalation of infected air/dust with a cyst of the parasite. It can cause infections in a variety of tissues and organs, including the sinuses, lungs and the rest of the respiratory tract. These respiratory tract infections are difficult to differentiate from other common causes, e.g. viral, bacterial or fungal, by clinical manifestations only. Pulmonary lophomoniasis is a treatable infection and metronidazole is the drug of choice [6]. Several cases of human infection have been reported from Spain [7, 8], Peru [9], Turkey [10], India [11], Mexico [12] and Iran [13]. Most of the cases reported in Iran were children [13,14,15,16].

Patients with respiratory asthma, allergic rhinitis, immune deficiency, especially AIDS, organ transplantation, cancer and under treatment with corticosteroids are more susceptible to being infected by Lophomonas spp. protozoa [10, 17]. Little is known about the epidemiological aspects of Lophomonas infection.

German cockroache (Blattella germanica) as a common domestic pest has been reported from all parts of Iran [18]. Based on our recent study, Lophomonas spp. was detected in the gut of German cockroaches trapped in hospitals for the first time in Mazandaran Province, Sari, northern Iran [19]. This study indicates that at least one of the sources of infection in this region may be close contact to infected German cockroaches, which may pose a risk to patients admitted to these hospitals [19].

Considering the lack of data on the status of lophomoniasis among patients in Mazandaran Province, northern Iran, the present study aimed to determine the prevalence of Lophomonas spp. infection and survey epidemiological aspects also in patients who were referred to the Iranian National Registry Center for Lophomoniasis (INRCL), with chronic respiratory disorders, for the first time, using morphological and molecular techniques.

Subjects and Methods

Study Area and Sample Collection

This descriptive analytical study was performed in the pulmonary clinic at Imam Khomeini Hospital, Mazandaran Province, Sari, northern Iran, from December 2017 to July 2019. The province is located in the north of Iran (53°6′ E, 36°23′ N). From the geographical point of view, the province is divided into two parts, i.e. the coastal plain and the mountainous area. The central zone of the province has humid weather and also has an annual mean rainfall of 977 mm [20]. The bronchoalveolar lavage fluid (BALF) specimens of the patients submitted to the Iranian National Registry Center for lophomoniasis (INRCL) at Mazandaran University of Medical Sciences, Sari, Iran, were used for detecting Lophomonas spp. A detailed questionnaire was conducted, and physical examinations were performed. Clinical symptoms and signs, gender, age, living area, past medical history and type of specimen were recorded.

Fiberoptic Bronchoscopy (FOB)

Flexible fiberoptic bronchoscopy (FOB) examination was performed for all patients who are suitable candidates for bronchoscopy. A BALF specimen was collected by wedging the tip of the bronchoscope into the nondependent lobes, in particular, the middle lobe of the right lung and the lingula of the left lung. The lobe used for BALF collection was determined by the images of the lesion with the greatest radiologic abnormality. About 5–20 mL of sterile normal saline (with a total volume of 1 mL/kg) was instilled, divided into 5–20 aliquots, for 2–4 times. Gentle manual suction was applied to retrieve the saline. BALF was collected in sterilized containers, and 2–5 mL of BALF was submitted to the laboratory of INRCL at Imam Khomeini hospital within 1–3 h at room temperature.

Microscopic Examination

319 BALF and two nasal discharge specimens were collected and each one centrifuged at 2000 rpm for 5 min and the sedimentation was smeared onto a glass slide and examined under a light microscope for the presence of Lophomonas spp. We have also considered the Lophomonas density estimation under a light microscope. Based on our experience in INRCL, an innovative severity index (SI) for Lophomonas infection was scored as mild to severe parasite density by counting parasites per high power microscopic fields (HPF) (X400). Accordingly, the mild density was defined as 1–10 parasites/100HPF, moderate as 1–10 parasites/10HPF, and 1–10 parasites/HPF were considered as severe (Unpublished data). Also, all positive smears were stained by Papanicolaou stain to investigate more details such as flagella, nucleous, etc.

Molecular Examination

DNA Extraction

Accordingly, 200 μL of the specimen was homogenized in 200 μL of digestive buffer consisted of 50 mM Tris–HCl (pH 7.6), 1 mM EDTA and 1% Tween 20, to which 20μL of the proteinase K solution (containing 20 mg of the enzyme/mL) was added. Afterward, the homogenized solution was incubated at 45 °C overnight. Next, 200 μL of phenol: chloroform: isoamyl alcohol (25:24:1) solution was added to the latter homogenate. After being shaken vigorously, the solution was centrifuged at 14,000×g for 15 min. The supernatant was poured into a fresh microtube to which 400 μL of the cold absolute ethanol was added and kept at − 20 °C for 2 h. Afterward, 200 μL of 70% ethanol was added to the precipitate, centrifuged, and the precipitate was suspended in 50 μL of double-distilled water and stored at 4 °C until use [21].

Genus-Specific rRNA PCR Amplification

The PCRwas set up in a total volume of 25 μL, which included 12.5 μL of the Master Mix (Fermentas, Inc.), 1 μL of each primer forward (F) (5′-GAG AAG GCG CCT GAG AGA T- 3′) and reverse (R) (5′-ATG GGA GCA AACTCG CAGA- 3′) specific primers that was planned from small-subunit ribosomal RNA (SSU rRNA) [21], 5 μL of the extracted DNAand 5.5 μL of distilled water. Thirty-five cycles were performed in a thermocycler (Corbett Research, Sydney, Australia) with initial denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min, and then a final extension at 72 °C for 3 min. Subsequently, 6 μL of the PCRproducts was analyzed by electrophoresis on 1.5% (w/v) agarose gel in Tris–borate-EDTA (TBE) buffer. The electrophoresed PCR products bands were observed using UV transillumination after staining with SYBR® Safe Stain (Invitrogen®). Lophomonas DNA obtained in our previous study [5] and water without DNA were included in all test for positive and negative control respectively. The amplicons using Sanger sequencing technology was sequenced and then edited with BioEdit software (v.7.2).

Data Analysis

We used frequency and percent to describe qualitative variables, mean and standard deviation for quantitative variables. To assess relationship between each variables and positive samples odds ratio and regression test with 95% confidence interval (95%CI) were applied. The p-value less than 0.05 were regarded as statistically significant. All statistical analysis was performed in IBM SPSS version 23.

Results

In this study, 321 specimens (including 319 BALF and 2 nasal discharges) were microscopically examined. Lophomonas spp. were found in 45 (14%) (n = 44 BAL; n = 1 nasal discharge). The mean age of infected patients was 54.9 ± 17.1 years (see Table 1). The following morphological characteristics were observed in both fresh and Papanicolaou-stained smears to identify Lophomonas spp.: the trophozoite was pyriform in shape and had a tuft of several flagella on the apex (Fig. 1). Moreover, according to SI scoring, most of the (39/45) positive specimens significantly had mild density severity (P = 0.001).

Table 1 Characteristics of patients enrolled in this study
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Fig. 1
figure 1

Papanicolaou staining of bronchoalveolar lavage fluid showing L.blattarum trophozoite (black arrow) and bronchial epithelial cell (white arrow) (magnification × 40)

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A total of 45 positive subjects, 27 examined males, and 18 were female. There was a significant relationship between male and Lophomonas spp., infection (P = 0.002). Out of 18 females, 16 were housewives and 2 were others. Occupation and Lophomonas infection in females showed significant correlations (P = 0.03). There was no correlation between occupation and Lophomonas infection in males (P = 0.68). Moreover, all of the patients had a history of contact with cockroaches in indoor places. The majority of patients' symptoms were prolonged cough, hemoptysis and dyspnea. Most patients (86.7%, 39/45) were referred to the INRCL during the fall and winter seasons than other ones.

Additionally, 33.3% (3 of 9) of patients with a history of tuberculosis were also positive for Lophomonas infection (see Table 1). No correlation was found between Lophomonas infection and underlying disease (P > 0.05). In all enrolled patients, FOB findings showed mild to moderate mucosal edema and hyperemia (see Fig. 2). All patients were also properly treated with metronidazole.

Fig. 2
figure 2

Brochoscopic view of left upper division (LUD) in a patient with chief complaint of hemoptysis and ground-glass opacity in the left upper lobe on computed tomography scan images. After irrigation with cold normal saline, mild mucosal edema and hyperemia were evident

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Also, all microscopically positive specimens were confirmed with the genus-specific-SSU rRNA PCR technique (Fig. 3). Moreover, two high-quality BALF samples with sharp and non-smear bands were sequenced to confirm PCR results and deposited in the GenBank by BanKit (Accession Numbers: MN243135–36). The identity and query coverage of theses isolates in comparison with only available L. blattarum that was the first report of bronchopulmonary infection caused by L. blattarum in Thailand (Accession Numbers: JX020505.1), were 99–100% and 98.51%, respectively.

Fig. 3.
figure 3

1.5% agarose gel electrophoresis, showing the 214 bp band of PCR product, confirming Lophomonas spp. in the patient specimens. M: 100-bp DNA ladder; 1–5: PCR products of the patient specimens; N negative sample

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Discussion

Little information is known about several aspects of lophomoniasis, such as clinical features and laboratory findings worldwide. In the present study, Lophomonas infection, for the first time, was found in 14% of the sampled population in northern Iran. The high prevalence of lophomoniasis in Mazandaran Province could be attributed to the warm and humid weather, which promotes the growth of the vector of Lophomonas, specifically cockroaches and house dust mites [2, 22, 23]. In addition, contact with family members may increase the risk of transmission of Lophomonas infection. Lophomonas transmission via airborne is a possibility for this parasite. Every year, a large number of tourists from all over Iran and neighboring countries trip to Mazandaran Province, which may pose a risk to the tourists during vacation. In our study, the majority of patients were males. These results are in good agreement with other case studies. Based on previous studies, Lophomonas infection has been frequently reported in males [5, 10, 16, 24].

Despite the fact that Ghafarian et al. (2018) found that there is a high prevalence of lophomoniasis in girls. It's noted that this study was conducted only on under 18-year old children in Mashhad, eastern Iran [14]. Males have a higher susceptibility to many parasitic infections compared to women such as Plasmodium, Leishmania and Entamoeba histolytica. This can be explained by sex differences in immune function with circulating steroid, genetic and behavioral differences [25,26,27,28].

Among 45 patients, 31 (68.8%) were inhabitants in urban areas and the rest of them in rural areas. Even though our results revealed no significant difference between Lophomonas infections and inhabited areas (P = 0.63), the high population of cockroaches in urban areas can be attributed to the high prevalence of disease in these areas [29].

Surprisingly, in our study, all patients had a history of contact with cockroaches. Previous studies have reported isolation of the parasites in cockroaches and house dust mites indoor [30, 31]. Additionally, our recent study showed 1.7% of the trapped German cockroaches from different parts of hospitals in Mazandaran Province, Sari (place of the current study) were infected with Lophomonas spp. [19].

There are several possible explanations for the high prevalence of lophomoniasis in our study. For instance, a high population of cockroaches and house dust mites has been reported in the North of Iran [22, 23].

Several allergic reactions among the general human population provide evidence of the presence of cockroaches/mites. The main clinical manifestations of Lophomonas spp. infection are not based on clinical signs. Due to the similarity of clinical symptoms with other respiratory diseases, it is difficult to diagnose and/or differentiate from other conditions [3]. However, our data showed clinical findings of the patients were similar to other studies, including, hemoptysis, prolonged cough, wheeze and dyspnea [5, 6, 32, 33]. In this regard, based on available data and our population-based study in INRCL, since most patients suffer from hemoptysis, we recommend the “pink cough” term for lophomoniasis. Hence, this term can be appointed for bronchopulmonary lophomoniasis within publications regarding the disease in future.

In the present study, the majority of lophomoniasis were found during cold weather months (fall and winter). The high prevalence of the disease during these seasons may be attributed to the relatively long patent period of the infection among patients, who were frequently infected during summer, as a season with a high population of German cockroaches. On the other hand, inhaling cold air, which causes to produce proinflammatory substances and epithelial injury. Other effects of cold air contact include an increase in BAL fluid granulocytes, loss of ciliated epithelium, thickening of the lamina propria with increased concentrations of inflammatory cells, hyper responsiveness, and airway obstruction [34].

In the present study, although there was no correlation found between Lophomonas infection and underlying disease, the highest rate (33%) of co-morbidity was found among patients with tuberculosis. This report substantiates previous findings in the literature [32, 34, 35]. Consequently, since Lophomonas is an opportunistic parasite, it should be examined in respiratory secretions of patients with resistant tuberculosis treatment. Most parasitic agents in different organs that co-infect with Mycobacterium tuberculosis improve intolerance to antibacterial therapy and deteriorate the prognosis, according to a systematic review [35]. In addition, since tuberculosis and lophomoniasis have some clinical similarity, Lophomonas infection should be ruled out in patients suspected of having tuberculosis. However, Lophomonas infection may influence the patient's immune response and the course of the disease.

Given that there are no specific signs and symptoms for lophomoniasis [5, 10, 14, 16], laboratory findings, particularly PCR test, are critical for identifying this emerging disease. In this regard, microscopic examination, particularly wet mount, is routinely used as the most available method for diagnosis of Lophomonas parasite. Several morphological features are common between Lophomonas and lung epithelial cells such as creola bodies (small groups of ciliated bronchial cells) and ciliocytophthoria (detached ciliary tufts with cytoplasmic remnants). The presence of round and oval shapes in both, size similarities in the range of 15–40 µ, and motions of the cilia and flagella in the parasite have made light microscope differentiation difficult. More than 50 irregular arranged flagella with unsynchronized rhythmic, granular cytoplasm and unclear nucleous which are specific for Lophomonas. While lung epithelial cells were recognized by synchronized and rhythmic motivation of cilias, terminal bar, terminal nucleous [3, 36, 37].

In our study, Papanicolaou and Giemsa stains were used to show morphological details of Lophomonas spp. Several stains, including Hematoxylin/Eosin, Giemsa, and Papanicolaou, as well as trichrome stain, have been recommended in the literature. However, this is subject to each laboratory’s availability and/or shipment to a referral center with more preparation for parasite identification [3, 5, 14, 21, 38]. However, a study compared several staining methods to show morphological details of Lophomonas spp. on one BAL specimen. Based on the results, Trichrome stain had the best and Papanicolaou and Giemsa stains had medium quality [36].

Based on our experience at LNRC, the sensitivity and specificity of the Lophomonas wet mount examination is relatively low. One factor that can reduce the sensitivity of the wet mount is delays between specimen collection and microscopic examination. In addition, suboptimal specimen storage or transportation conditions further reduce parasite motility and thus wet mount sensitivity. Well trained microscopists for accurate identification have a great role in increasing the sensitivity of the diagnostic method. However, the advantages of this method are its available, rapid, simple and low cost [39].Consequently, the molecular-based diagnosis is at least necessary to confirm Lophomonas spp. In the present study, 45 specimens that were positive by the wet mount method were confirmed by the PCR test. Since PCR is an expensive and less accessible method further studies on microscopic examination should be undertaken.

To date, only one study of this protozoan has been diagnosed by PCR method [21]. The high sensitivity and specificity of the PCR has persuaded researchers to recommend a molecular method for the detection of lophomoniasis [3, 10, 36, 40]. It can detect organisms in latent infections, can identify organisms that cannot be cultured, avoids false positives, can detect organism before antibodies or antigens are produced, can detect organisms in tissue and vectors [39]. So, the molecular-based diagnosis was performed in the current study to confirm Lophomonas spp. It should be noted that the PCR test is currently available at INRC for detecting the parasite in different clinical samples.

Conclusion

To the best of our knowledge, this is the first registry population-based study regarding lophomoniasis. According to our study, the conventional PCR test is an available and reliable tool for confirming the Lophomonas parasite in clinical samples. Moreover, the results confirmed that L. blattarum is circulating at least in our region. Accordingly, further studies are recommended for developing a specific PCR for discriminating the parasite species such as multiplex-PCR as well as sequencing analysis of the high number of clinical samples for detecting Lophomonas species/genotyping. As a whole, our data provides unique information about different epidemiological evidence about BPL in the world.